Enhancing heat transfer at low temperatures by laser functionalization of the inner surface of metal pipes.

The latent heat transfer during vapour condensation in the condenser section of passive heat transport devices such as the two-phase closed thermosiphon is limited by film condensation. Dropwise condensation provides an increase of the heat transfer coefficient by up to one order of magnitude and can be achieved with a water-repellant surface. The inner surface of pipes made from stainless steel was functionalized by laser surface texturing with ultrashort laser pulses and subsequent storage in a liquid containing long-chained hydrocarbons. The pipes were separated into half-pipes by wire eroding to enable laser texturing of the inner surface, and were then joined by electron beam welding after laser texturing. As a result, superhydrophobic and water-repellent surfaces with a contact angle of 153° were obtained on the inner surface of the pipes with a length of up to 1 m. The functionalized pipes were used in the condenser section of a two-phase closed thermosiphon to demonstrate a heat transfer rate of 0.92 kW at 45 °C, which is approximately three times the heat transfer rate of 0.31 kW of a smooth reference pipe.

Analytical Model for the Depth Progress during Laser Micromachining of V-Shaped Grooves.

An analytical model is presented that allows predicting the progress and the final depth obtained by laser micromachining of grooves in metals with ultrashort laser pulses. The model assumes that micromachined grooves feature a V-shaped geometry and that the fluence absorbed along the walls is distributed with a linear increase from the edge to the tip of the groove. The depth progress of the processed groove is recursively calculated based on the depth increments induced by successive scans of the laser beam along the groove. The experimental validation confirms the model and its assumptions for micromachining of grooves in a Ti-alloy with femtosecond pulses and different pulse energies, repetition rates, scanning speeds and number of scans.

A Biomechanical Evaluation of a Novel Airbag Bicycle Helmet Concept for Traumatic Brain Injury Mitigation.

In this study, a novel expandable bicycle helmet, which integrates an airbag system into the conventional helmet design, was proposed to explore the potential synergetic effect of an expandable airbag and a standard commuter-type EPS helmet. The traumatic brain injury mitigation performance of the proposed expandable helmet was evaluated against that of a typical traditional bicycle helmet. A series of dynamic impact simulations on both a helmeted headform and a representative human head with different configurations were carried out in accordance with the widely recognised international bicycle helmet test standards. The impact simulations were initially performed on a ballast headform for validation and benchmarking purposes, while the subsequent ones on a biofidelic human head model were used for assessing any potential intracranial injury. It was found that the proposed expandable helmet performed admirably better when compared to a conventional helmet design-showing improvements in impact energy attenuation, as well as kinematic and biometric injury risk reduction. More importantly, this expandable helmet concept, integrating the airbag system in the conventional design, offers adequate protection to the cyclist in the unlikely case of airbag deployment failure.

Universal Toxicity Gene Signatures for Early Identification of Drug-Induced Tissue Injuries in Rats.

A new safety testing paradigm that relies on gene expression biomarker panels was developed to easily and quickly identify drug-induced injuries across tissues in rats prior to drug candidate selection. Here, we describe the development, qualification, and implementation of gene expression signatures that diagnose tissue degeneration/necrosis for use in early rat safety studies. Approximately 400 differentially expressed genes were first identified that were consistently regulated across 4 prioritized tissues (liver, kidney, heart, and skeletal muscle), following injuries induced by known toxicants. Hundred of these "universal" genes were chosen for quantitative PCR, and the most consistent and robustly responding transcripts selected, resulting in a final 22-gene set from which unique sets of 12 genes were chosen as optimal for each tissue. The approach was extended across 4 additional tissues (pancreas, gastrointestinal tract, bladder, and testes) where toxicities are less common. Mathematical algorithms were generated to convert each tissue's 12-gene expression values to a single metric, scaled between 0 and 1, and a positive threshold set. For liver, kidney, heart, and skeletal muscle, this was established using a training set of 22 compounds and performance determined by testing a set of approximately 100 additional compounds, resulting in 74%-94% sensitivity and 94%-100% specificity for liver, kidney, and skeletal muscle, and 54%-62% sensitivity and 95%-98% specificity for heart. Similar performance was observed across a set of 15 studies for pancreas, gastrointestinal tract, bladder, and testes. Bundled together, we have incorporated these tissue signatures into a 4-day rat study, providing a rapid assessment of commonly seen compound liabilities to guide selection of lead candidates without the necessity to perform time-consuming histopathologic analyses.

Intact Human Immunodeficiency Virus (HIV) Reservoir Estimated by the Intact Proviral DNA Assay Correlates With Levels of Total and Integrated DNA in the Blood During Suppressive Antiretroviral Therapy.

Accurate characterization of the human immunodeficiency virus (HIV) reservoir is imperative to develop an effective cure. HIV was measured in antiretroviral therapy-suppressed individuals using the intact proviral DNA assay (IPDA), along with assays for total or integrated HIV DNA, and inducible HIV RNA or p24. Intact provirus correlated with total and integrated HIV.

High-quality high-throughput silicon laser milling using a 1 kW sub-picosecond laser.

We report on high-quality high-throughput laser milling of silicon with a sub-ps laser delivering more than 1 kW of average laser power on the workpiece. In order to avoid heat accumulation effects, the processing strategy for high-quality laser milling was adapted to the available average power by using five-pulse bursts, a large beam diameter of 372 µm to limit the peak fluence per pulse to approximately 0.7J/cm2, and a high feed rate of 24 m/s. As a result, smooth surfaces with a low roughness of Sa≤0.6µm were achieved up to the investigated milling depth of 313 µm while maintaining a high material removal rate of 230mm3/min.

NRL-/- gene edited human embryonic stem cells generate rod-deficient retinal organoids enriched in S-cone-like photoreceptors.

Organoid cultures represent a unique tool to investigate the developmental complexity of tissues like the human retina. NRL is a transcription factor required for the specification and homeostasis of mammalian rod photoreceptors. In Nrl-deficient mice, photoreceptor precursor cells do not differentiate into rods, and instead follow a default photoreceptor specification pathway to generate S-cone-like cells. To investigate whether this genetic switch mechanism is conserved in humans, we used CRISPR/Cas9 gene editing to engineer an NRL-deficient embryonic stem cell (ESC) line (NRL-/- ), and differentiated it into retinal organoids. Retinal organoids self-organize and resemble embryonic optic vesicles (OVs) that recapitulate the natural histogenesis of rods and cone photoreceptors. NRL-/- OVs develop comparably to controls, and exhibit a laminated, organized retinal structure with markers of photoreceptor synaptogenesis. Using immunohistochemistry and quantitative polymerase chain reaction (qPCR), we observed that NRL-/- OVs do not express NRL, or other rod photoreceptor markers directly or indirectly regulated by NRL. On the contrary, they show an abnormal number of photoreceptors positive for S-OPSIN, which define a primordial subtype of cone, and overexpress other cone genes indicating a conserved molecular switch in mammals. This study represents the first evidence in a human in vitro ESC-derived organoid system that NRL is required to define rod identity, and that in its absence S-cone-like cells develop as the default photoreceptor cell type. It shows how gene edited retinal organoids provide a useful system to investigate human photoreceptor specification, relevant for efforts to generate cells for transplantation in retinal degenerative diseases.

Letermovir Resistance Analysis in a Clinical Trial of Cytomegalovirus Prophylaxis for Hematopoietic Stem Cell Transplant Recipients.

BACKGROUND: Letermovir (LET), a cytomegalovirus (CMV) deoxyribonucleic acid (DNA) terminase inhibitor, was recently approved for prophylaxis of CMV infection in adult CMV-seropositive recipients of allogeneic hematopoietic stem cell transplantation. Cytomegalovirus genotyping was performed to identify LET-resistance-associated variants (RAVs) among subjects in a Phase 3 trial. METHODS: The CMV UL56 and UL89 genes, encoding subunits of CMV DNA terminase, were sequenced from plasma collected from subjects with clinically significant CMV infection (CS-CMVi). Novel variants were evaluated by recombinant phenotyping to assess their potential to confer resistance to LET. RESULTS: Genotyping was successful for 50 of 79 LET subjects with CS-CMVi. Resistance-associated variants (encoding pUL56 V236M and C325W) were detected independently in subjects 1 and 3 who experienced CS-CMVi while receiving LET prophylaxis, and 2 other variants (encoding pUL56 E237G and R369T) were detected >3 weeks after subjects 2 and 3, respectively, had discontinued LET prophylaxis and received preemptive therapy with ganciclovir. CONCLUSIONS: The detected incidence of CMV resistance among subjects who received LET as prophylaxis in this Phase 3 trial was low. The LET RAVs that were detected mapped to the CMV UL56 gene at positions associated with reduced susceptibility to LET based on resistance selections in cell culture.

Use of a Bile Salt Export Pump Knockdown Rat Susceptibility Model to Interrogate Mechanism of Drug-Induced Liver Toxicity.

Inhibition of the bile salt export pump (BSEP) may be associated with clinical drug-induced liver injury, but is poorly predicted by preclinical animal models. Here we present the development of a novel rat model using siRNA knockdown (KD) of Bsep that displayed differentially enhanced hepatotoxicity to 8 Bsep inhibitors and not to 3 Bsep noninhibitors when administered at maximally tolerated doses for 7 days. Bsep KD alone resulted in 3- and 4.5-fold increases in liver and plasma levels, respectively, of the sum of the 3 most prevalent taurine conjugated bile acids (T3-BA), approximately 90% decrease in plasma and liver glycocholic acid, and a distinct bile acid regulating gene expression pattern, without resulting in hepatotoxicity. Among the Bsep inhibitors, only asunaprevir and TAK-875 resulted in serum transaminase and total bilirubin increases associated with increases in plasma T3-BA that were enhanced by Bsep KD. Benzbromarone, lopinavir, and simeprevir caused smaller increases in plasma T3-BA, but did not result in hepatotoxicity in Bsep KD rats. Bosentan, cyclosporine A, and ritonavir, however, showed no enhancement of T3-BA in plasma in Bsep KD rats, as well as Bsep noninhibitors acetaminophen, MK-0974, or clarithromycin. T3-BA findings were further strengthened through monitoring TCA-d4 converted from cholic acid-d4 overcoming interanimal variability in endogenous bile acids. Bsep KD also altered liver and/or plasma levels of asunaprevir, TAK-875, TAK-875 acyl-glucuronide, benzbromarone, and bosentan. The Bsep KD rat model has revealed differences in the effects on bile acid homeostasis among Bsep inhibitors that can best be monitored using measures of T3-BA and TCA-d4 in plasma. However, the phenotype caused by Bsep inhibition is complex due to the involvement of several compensatory mechanisms.

Conditional Dicer1 depletion using Chrnb4-Cre leads to cone cell death and impaired photopic vision.

Irreversible photoreceptor cell death is a major cause of blindness in many retinal dystrophies. A better understanding of the molecular mechanisms underlying the progressive loss of photoreceptor cells remains therefore crucial. Abnormal expression of microRNAs (miRNAs) has been linked with the aetiology of a number of retinal dystrophies. However, their role during the degenerative process remains poorly understood. Loss of cone photoreceptors in the human macula has the greatest impact on sight as these cells provide high acuity vision. Using a Chrnb4-cre; Dicerflox/flox conditional knockout mouse (Dicer CKO) to delete Dicer1 from cone cells, we show that cone photoreceptor cells degenerate and die in the Dicer-deleted retina. Embryonic eye morphogenesis appeared normal in Dicer CKO mice. Cone photoreceptor abnormalities were apparent by 3 weeks of age, displaying either very short or absent outer segments. By 4 months 50% of cones were lost and cone function was impaired as assessed by electroretinography (ERG). RNAseq analysis of the Dicer CKO retina revealed altered expression of genes involved in the visual perception pathway. These data show that loss of Dicer1 leads to early-onset cone cell degeneration and suggest that Dicer1 is essential for cone photoreceptor survival and homeostasis.

Estimation of the depth limit for percussion drilling with picosecond laser pulses.

We present a model to predict the final depth of percussion-drilled holes that are produced with picosecond laser pulses in metals. It is based on the assumption that boreholes always have conical geometries when the drilling process terminates. We show that the model is valid for various process parameters when drilling in stainless steel. This was even confirmed by drilling with 3 mJ pulses, which resulted in a 10 mm deep borehole without thermal damage.

Chronic Verubecestat Treatment Suppresses Amyloid Accumulation in Advanced Aged Tg2576-AßPPswe Mice Without Inducing Microhemorrhage.

Verubecestat is a potent BACE1 enzyme inhibitor currently being investigated in Phase III trials for the treatment of mild-to-moderate and prodromal Alzheimer's disease. Multiple anti-amyloid immunotherapies have been dose-limited by adverse amyloid related imaging abnormalities such as vasogenic edema (ARIA-E) and microhemorrhage (ARIA-H) observed in human trials and mice. Verubecestat was tested in a 12-week nonclinical study for the potential to exacerbate microhemorrhage (ARIA-H) profiles in 18-22-month-old post-plaque Tg2576-AßPPswe mice. Animals were treated with verubecestat or controls including the anti-Aß antibody analog of bapineuzumab (3D6) as a positive control for ARIA induction. ARIA-H was measured using in-life longitudinal T2*-MRI and Prussian blue histochemistry at study end. Verubecestat reduced plasma and cerebrospinal fluid Aß40 and Aß42 by >90% and 62% to 68%, respectively. The ARIA-H profile of verubecestat-treated mice was not significantly different than controls. Anti-Aß treatment significantly increased ARIA-H detected by Prussian blue staining; however, anti-Aß antibody treatment did not impact plaque status. Verubecestat treatment significantly suppressed the accumulation of total levels of brain Aß40 and Aß42 and Thioflavin S positive plaque load. Stereological analysis of cortex and hippocampus plaque load similarly revealed significantly reduced area of Aß immunoreactivity and reduced plaque number in verubecestat-treated animals compared to controls. The absence of elevated ARIA events in verubecestat-treated mice was associated with a significant reduction in the level of accumulated CNS amyloid pathology and brain Aß peptides; effects consistent with the desired therapeutic mechanism of verubecestat in AD patients. These data will be compared with longitudinal MRI profiles from ongoing clinical trials.

Logical Experimental Design and Execution in the Biomedical Sciences.

Lack of reproducibility has been highlighted as a significant problem in biomedical research. The present unit is devoted to describing ways to help ensure that research findings can be replicated by others, with a focus on the design and execution of laboratory experiments. Essential components for this include clearly defining the question being asked, using available information or information from pilot studies to aid in the design the experiment, and choosing manipulations under a logical framework based on Mill's "methods of knowing" to build confidence in putative causal links. Final experimental design requires systematic attention to detail, including the choice of controls, sample selection, blinding to avoid bias, and the use of power analysis to determine the sample size. Execution of the experiment is done with care to ensure that the independent variables are controlled and the measurements of the dependent variables are accurate. While there are always differences among laboratories with respect to technical expertise, equipment, and suppliers, execution of the steps itemized in this unit will ensure well-designed and well-executed experiments to answer any question in biomedical research. © 2017 by John Wiley & Sons, Inc.

A multinational study distinguishing Alzheimer's and healthy patients using cerebrospinal fluid tau/Aß42 cutoff with concordance to amyloid positron emission tomography imaging.

INTRODUCTION: Changes in cerebrospinal fluid (CSF) tau and amyloid ß (Aß)42 accompany development of Alzheimer's brain pathology. Robust tau and Aß42 immunoassays were developed to establish a tau/Aß42 cutoff distinguishing mild-to-moderate Alzheimer's disease (AD) subjects from healthy elderly control (HC) subjects. METHODS: A CSF tau/Aß42 cutoff criteria was chosen, which distinguished the groups and maximized concordance with amyloid PET. Performance was assessed using an independent validation cohort. RESULTS: A tau/Aß42 = 0.215 cutoff provided 94.8% sensitivity and 77.7% specificity. Concordance with PET visual reads was estimated at 86.9% in a ∼50% PET positive population. In the validation cohort, the cutoff demonstrated 78.4% sensitivity and 84.9% specificity to distinguish the AD and HC populations. DISCUSSION: A tau/Aß42 cutoff with acceptable sensitivity and specificity distinguished HC from mild-to-moderate AD subjects and maximized concordance to brain amyloidosis. The defined cutoff demonstrated that CSF analysis may be useful as a surrogate to imaging assessment of AD pathology.

Antibiotic-Induced Elevations of Plasma Bile Acids in Rats Independent of Bsep Inhibition.

Drug-induced liver injury (DILI) is a common toxicity observed in drug development and can lead to withdrawal of approved drugs from the market. To better understand the numerous mechanisms of DILI, recent efforts have focused on transporter inhibition, specifically liver canalicular bile salt export pump (Bsep) as one mechanism of DILI, and on the potential use of plasma bile acids as monitorable mechanism-based biomarkers of Bsep inhibition. To explore alternative mechanisms of bile acid increases in plasma, 6 antibiotic and 2 nonantibiotic drugs unlikely to be Bsep inhibitors were evaluated in rat studies. Surprisingly, all 6 antibiotics demonstrated 2- to 14-fold increases of plasma taurocholic acid (TCA). Also, unconjugated primary bile acids and secondary bile acids (both taurine-conjugated and unconjugated) were decreased in rat plasma after antibiotic treatments, but not with the nonantibiotic drugs. These results suggest alternative mechanisms of bile acids regulation such as attenuation of bacterial deconjugation of bile acids following reduction of gut microflora by antibiotics. Measurements of TCA transport in rat hepatocytes and Bsep-containing membrane vesicles suggest that inhibition of uptake into hepatocytes could also contribute to increases in plasma bile acid concentrations, while excluding inhibition of Bsep as a mechanism. These studies further demonstrate that there are several mechanisms that can lead to conjugated bile acid increases in plasma. By carefully considering the time course and magnitude of changes of individual bile acids relative to any changes seen in transaminases and bilirubin, interpretations and conclusions of the involvement of Bsep inhibition are enabled.

Net Reclassification Index and Integrated Discrimination Index Are Not Appropriate for Testing Whether a Biomarker Improves Predictive Performance.

One of the goals of the Critical Path Institute's Predictive Safety Testing Consortium (PSTC) is to promote best practices for evaluating novel markers of drug induced injury. This includes the use of sound statistical methods. For rat studies, these practices have centered around comparing the area under the receiver-operator characteristic curve for each novel injury biomarker to those for the standard markers. In addition, the PSTC has previously used the net reclassification index (NRI) and integrated discrimination index (IDI) to assess the increased certainty provided by each novel injury biomarker when added to the information already provided by the standard markers. Due to their relatively simple interpretations, NRI and IDI have generally been popular measures of predictive performance. However recent literature suggests that significance tests for NRI and IDI can have inflated false positive rates and thus, tests based on these metrics should not be relied upon. Instead, when parametric models are employed to assess the added predictive value of a new marker, following (Pepe, M. S., Kerr, K. F., Longton, G., and Wang, Z. (2013). Testing for improvement in prediction model performance. Stat. Med. 32, 1467-1482), the PSTC recommends that likelihood based methods be used for significance testing.

Impact of glycoprotein B genotype and naturally occurring ORF UL56 polymorphisms upon susceptibility of clinical human cytomegalovirus isolates to letermovir.

Letermovir is a novel anti-HCMV drug in Phase III development that targets the UL56 subunit of the viral terminase complex. In immunocompromised patients four major glycoprotein B (gB) subtypes are known and may influence pathogenesis and thus disease outcomes. Using a panel of 74 letermovir-naïve, low-passage, clinical HCMV isolates, we examined the potential impact of i) gB genotype and ii) naturally occurring UL56 sequence variations upon susceptibility to letermovir. Our data show that letermovir's potency is independent of gB subtype and show that naturally-occurring letermovir-resistance is rare or possibly absent.

Rat Urinary Osteopontin and Neutrophil Gelatinase-Associated Lipocalin Improve Certainty of Detecting Drug-Induced Kidney Injury.

Traditional kidney biomarkers are insensitive indicators of acute kidney injury, with meaningful changes occurring late in the course of injury. The aim of this work was to demonstrate the diagnostic potential of urinary osteopontin (OPN) and neutrophil gelatinase-associated lipocalin (NGAL) for drug-induced kidney injury (DIKI) in rats using data from a recent regulatory qualification submission of translational DIKI biomarkers and to compare performance of NGAL and OPN to five previously qualified DIKI urinary biomarkers. Data were compiled from 15 studies of 11 different pharmaceuticals contributed by Critical Path Institute's Predictive Safety Testing Consortium (PSTC) Nephrotoxicity Working Group (NWG). Rats were given doses known to cause DIKI or other target organ toxicity, and urinary levels of the candidate biomarkers were assessed relative to kidney histopathology and serum creatinine (sCr) and blood urea nitrogen (BUN).OPN and NGAL outperformed sCr and BUN in identifying DIKI manifested as renal tubular epithelial degeneration or necrosis. In addition, urinary OPN and NGAL, when used with sCr and BUN, increased the ability to detect renal tubular epithelial degeneration or necrosis. NGAL and OPN had comparable or improved performance relative to Kim-1, clusterin, albumin, total protein, and beta-2 microglobulin. Given these data, both urinary OPN and NGAL are appropriate for use with current methods for assessing nephrotoxicity to identify and monitor DIKI in regulatory toxicology studies in rats. These data also support exploratory use of urinary OPN and NGAL in safety monitoring strategies of early clinical trials to aid in the assurance of patient safety.

Effect of Tween-20 on Core Biomarkers Measured in Cerebrospinal Fluid from Patients with Alzheimer's Disease, Mild Cognitive Impairment, or Healthy Control Individuals.

BACKGROUND: There is substantial variation caused by preanalytical procedures in the measurement of cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease (AD) reported in the literature. OBJECTIVE: Determine whether the detergent Tween-20 improves diagnostic accuracy. METHODS: CSF proteins (Aß42, Aß40, total tau, and phosphorylated tau) were measured by standard ELISA, in uncentrifuged CSF with or without 0.05% Tween-20 from patients with AD or amnestic mild cognitive impairment, and healthy elderly controls. In the main study, collection tubes containing Tween-20 (Sarstedt 15 mL) were filled with 5 mL CSF to ensure consistent detergent concentration across subsequent aliquots into Corning 2 mL tubes. These latter were also the primary collection vessel for samples without Tween-20. The effect of centrifugation, and extra tube transfer of samples with Tween-20 were also examined. RESULTS: 0.05% Tween-20 significantly increased mean measured CSF concentration of Aß42 (30% ), Aß40 (23% ), and total tau (4% ), but not phosphorylated tau. Generally, these increases were similar in all groups, although for Aß42, the mean percentage increase with Tween-20 was slightly larger for AD. Areas under receiver-operator characteristic curves were similar whether Tween-20 was present or not. Centrifuged CSF without Tween-20 significantly reduced the measured concentration of Aß42 versus non-centrifuged samples, a difference not seen when detergent was added. Similar CSF Aß42 levels were found whether Tween-20 was added at collection in an extra tube or directly to the main collection tube. CONCLUSION: Addition of Tween-20 to CSF did not improve differentiation of patients from controls.

Soluble BACE-1 Activity and sAßPPß Concentrations in Alzheimer's Disease and Age-Matched Healthy Control Cerebrospinal Fluid from the Alzheimer's Disease Neuroimaging Initiative-1 Baseline Cohort.

ß-site amyloid precursor protein-cleaving enzyme 1 (BACE1) plays an important role in the development of Alzheimer's disease (AD), freeing the amyloid-ß (Aß) N-terminus from the amyloid-ß protein precursor (AßPP), the first step in Aß formation. Increased BACE1 activity in AD brain or cerebrospinal fluid (CSF) has been reported. Other studies, however, found either no change or a decrease with AD diagnosis in either BACE1 activity or sAßPPß, the N-terminal secreted product of BACE1 (sBACE1) activity on AßPP. Here, sBACE1 enzymatic activity and secreted AßPPß (sAßPPß) were measured in Alzheimer's Disease Neuroimaging Initiative-1 (ADNI-1) baseline CSF samples and no statistically significant changes were found in either measure comparing healthy control, mild cognitively impaired, or AD individual samples. While CSF sBACE1 activity and sAßPPß demonstrated a moderate yet significant degree of correlation with each other, there was no correlation of either analyte to CSF Aß peptide ending at residue 42. Surprisingly, a stronger correlation was demonstrated between CSF sBACE1 activity and tau, which was comparable to that between CSF Aß42 and tau. Unlike for these latter two analytes, receiver-operator characteristic curves demonstrate that neither CSF sBACE1 activity nor sAßPPß concentrations can be used to differentiate between healthy elderly and AD individuals.