Poppy Seeds Signal Limitations of Urine Drug Testing Protocols.

Appropriate care for patients with chronic pain is complex, requiring a thoughtful and holistic approach to pharmacologic intervention, as well as appropriate monitoring when opioids are employed as part of a multimodal regimen. The urine drug test has become an expected standard when longterm opioids are prescribed, but it should be remembered that this test is not intended to be punitive. It is ordered to promote patient safety (Dowell et al., 2022). Recent literature and events surrounding the effect of poppy seeds on urine drug test results have drawn attention to the risks of misinterpreting this test (Bloch, 2023; Lewis et al., 2021; Reisfield et al., 2023; Temple, 2023). Misinterpretation of urine drug tests creates a potential for unfounded accusations from health care workers toward patients, thus, undermining therapeutic relationships and intensifying stigma. Such circumstances may also preclude chances to offer patients needed interventions. Therefore, a valuable opportunity exists for nurses to mitigate untoward consequences by developing a robust understanding of urine drug testing, destigmatizing chronic pain and opioid use, advocating for patients, and enacting change at both an individual and a systems-level.

An Evaluation of the Clinical Tools Used to Monitor Illicit Methamphetamine Use Among Chronic Pain Patients: A Cross Sectional Retrospective Study.

Purpose: Illicit drug use continues to be a concern for adults on opioid therapy for chronic pain. Prescribers use tools such as urine screening and confirmatory testing with mass spectrometry to monitor adherence to chronic opioid therapy contracts. Design: A cross sectional retrospective study was conducted using electronic medical records. Methods: Data was analyzed from 6558 urine samples of adult outpatients receiving opioid therapy at an urban pain specialty clinic who consented to urine drug tests. Results: From October 18, 2021, to October 21, 2022, 569 were positive amphetamine with immunoassay testing. 310 (54%) of those samples were absent amphetamine with or without methamphetamine while 259 (45.5%) were true positive amphetamine proven by confirmation testing. Analysis of confirmatory testing results identified 281 samples positive for amphetamine with or without methamphetamine. 71 samples confirmed positive for methamphetamine with or without amphetamine. 37 (52.1%) of those methamphetamine samples screened positive for amphetamine and 34 (47.9%) samples screened negative for amphetamine. 48 of the methamphetamine samples were sent for chiral confirmation testing. 45 (93.8%) samples were positive for D-methamphetamine while 3 (6.2%) samples were positive for L-methamphetamine. Only 5 (11.1%) of the 45 patients whose samples were positive for d-methamphetamine reported the use of illicit drugs before their urine sample was tested. Discussion: Confirmatory testing with mass spectrometry can detect illicit drugs, such as methamphetamine, with high sensitivity and specificity and should be used with all samples given the low sensitivity of screening immunoassay. It is of utmost importance that opioid pain medicine prescribers know the many possible interpretations of these results so that they are equipped to make appropriate clinical decisions to ensure the safety of the patient, prescriber, and practice.

Drug-Drug Interaction Between Orally Administered Hydrocodone-Acetaminophen and Inhalation of Cannabis Smoke: A Case Report.

Objective: To determine if a 2-day protocol measuring pharmacokinetic and pharmacodynamic characteristics can demonstrate drug-drug interactions when smoked cannabis is added to orally administered hydrocodone/acetaminophen combination products. Case Summary: A 51-year-old non-Hispanic white male with chronic pain diagnoses participated in a 2-day pilot protocol. The participant attended two 7-hour in-lab days where he received 10 blood draws each day and completed self-administered pain and anxiety surveys. For both days, the participant took his prescribed dose of hydrocodone/acetaminophen (1/2 tablet of 7.5 mg/325 mg combination product) with the addition of 1 smoked pre-rolled marijuana cigarette (labeled as 0.5 g; 22.17% Δ9-tetrahydrocannabinol; 0.12% cannabidiol) on Day 2. Blood specimens were analyzed using mass spectrometry to quantify the difference of plasma hydrocodone levels between Day 1 and Day 2. Results: Compared to Day 1, lower levels of pain and anxiety were reported during Day 2 with the addition of cannabis to oral hydrocodone/acetaminophen. Day 2 pharmacokinetic analysis also revealed more rapid absorption and overall lower levels of hydrocodone in plasma. Discussion: Lower hydrocodone plasma levels in Day 2 may indicate cannabis's effect on metabolism and reduce the risk of opioid toxicity. The quicker absorption rate of hydrocodone could explain lower pain and anxiety scores reported on the second day. Conclusion and Relevance: A 2-day protocol was able to capture differences across time in pharmacokinetic and pharmacodynamic measurements. Larger studies can be designed to better characterize the potential drug-drug interaction of cannabis and opioids.

Project Vienna: A Novel Precell Mass Filter for Collision/Reaction Cell MC-ICPMS/MS.

The last decade has seen widespread adoption of triple quadrupole-based inductively coupled plasma-tandem mass spectrometry (ICPMS/MS) technique using a collision/reaction cell in combination with a precell bandpass mass analyzer to measure isotopes otherwise masked by spectral interferences. High-precision isotope ratio analysis containing such isotopes would benefit from a similar capability on a multicollector inductively coupled plasma mass spectrometry (MC-ICPMS) platform, but using a quadrupole-based precell mass analyzer for MC-ICPMS/MS has several limitations. To overcome these limitations, we developed a novel precell mass analyzer for MC-ICPMS/MS using sector field technology. The new precell mass analyzer, comprising two Wien filters and a selection aperture, and a hexapole collision/reaction cell were integrated together in a single module and added to the commercially available Thermo Scientific Neptune XT MC-ICPMS to create a prototype MC-ICPMS/MS we named Vienna. Vienna was proven to retain the same performance of the base MC-ICPMS in terms of sensitivity, accuracy, and precision. Using the Vienna mass filter to eliminate Ar-based species, the abundance sensitivity achievable was equivalent to TIMS at mass 237.05, which was used to accurately determine the low 236U/238U isotope ratio of the uranium reference material IRMM184 (certified value, 1.2446 × 10-7). The performance of Vienna was then tested for a variety of geoscience applications that were expected to benefit from MC-ICPMS/MS technique, including Ca, K, Si, and in situ Rb/Sr dating by laser ablation.

In situ Rb-Sr dating by collision cell, multicollection inductively-coupled plasma mass-spectrometry with pre-cell mass-filter, (CC-MC-ICPMS/MS).

We document the utility for in situ Rb-Sr dating of a one-of-a-kind tribrid mass spectrometer, 'Proteus', coupled to a UV laser ablation system. Proteus combines quadrupole mass-filter, collision cell and sector magnet with a multicollection inductively-coupled plasma mass spectrometer (CC-MC-ICPMS/MS). Compared to commercial, single collector, tribrid inductively-coupled plasma mass spectrometers (CC-ICPMS/MS) Proteus has enhanced ion transmission and offers simultaneous collection of all Sr isotopes using an array of Faraday cups. These features yield improved precision in measured 87Sr/86Sr ratios, for a given mass of Sr analysed, approximately a factor of 25 in comparison to the Thermo Scientific™ iCAP TQ™ operated under similar conditions. Using SF6 as a reaction gas on Proteus, measurements of Rb-doped NIST SRM (standard reference material) 987 solutions, with Rb/Sr ratios from 0.01-100, yield 87Sr/86Sr that are indistinguishable from un-doped NIST SRM 987, demonstrating quantitative 'chemical resolution' of Rb from Sr. We highlight the importance of mass-filtering before the collision cell for laser ablation 87Sr/86Sr analysis, using an in-house feldspar standard and a range of glass reference materials. By transmitting only those ions with mass-to-charge ratios 82-92 u/e into the collision cell, we achieve accurate 87Sr/86Sr measurements without any corrections for atomic or polyatomic isobaric interferences. Without the pre-cell mass-filtering, measured in situ 87Sr/86Sr ratios are inaccurate. Combining in situ measurements of Rb/Sr and radiogenic Sr isotope ratios we obtain mineral isochrons. We utilise a sample from the well-dated Dartmoor granite (285 ± 1 Ma) as a calibrant for our in situ ages and, using the same conditions, produce accurate Rb-Sr isochron ages for samples of the Fish Canyon tuff (28 ± 2 Ma) and Shap granite pluton (397 ± 1 Ma). Analysing the same Dartmoor granite sample using identical laser conditions and number of spot analyses using the Thermo Scientific™ iCAP TQ™ yielded an isochron slope 5× less precise than Proteus. We use an uncertainty model to illustrate the advantage of using Proteus over single collector CC-ICPMS/MS for in situ Rb-Sr dating. The results of this model show that the improvement is most marked for samples that have low Rb/Sr (<10) or are young (<100 Ma). We also report the first example of an in situ, internal Rb-Sr isochron from a single potassium-feldspar grain. Using a sample from the Shap granite, we obtained accurate age and initial 87Sr/86Sr with 95% confidence intervals of ±1.5% and ±0.03% respectively. Such capabilities offer new opportunities in geochronological studies.

Bioinformatics: indispensable, yet hidden in plain sight?

BACKGROUND: Bioinformatics has multitudinous identities, organisational alignments and disciplinary links. This variety allows bioinformaticians and bioinformatic work to contribute to much (if not most) of life science research in profound ways. The multitude of bioinformatic work also translates into a multitude of credit-distribution arrangements, apparently dismissing that work. RESULTS: We report on the epistemic and social arrangements that characterise the relationship between bioinformatics and life science. We describe, in sociological terms, the character, power and future of bioinformatic work. The character of bioinformatic work is such that its cultural, institutional and technical structures allow for it to be black-boxed easily. The result is that bioinformatic expertise and contributions travel easily and quickly, yet remain largely uncredited. The power of bioinformatic work is shaped by its dependency on life science work, which combined with the black-boxed character of bioinformatic expertise further contributes to situating bioinformatics on the periphery of the life sciences. Finally, the imagined futures of bioinformatic work suggest that bioinformatics will become ever more indispensable without necessarily becoming more visible, forcing bioinformaticians into difficult professional and career choices. CONCLUSIONS: Bioinformatic expertise and labour is epistemically central but often institutionally peripheral. In part, this is a result of the ways in which the character, power distribution and potential futures of bioinformatics are constituted. However, alternative paths can be imagined.

Oxidative Stress Attenuates Lipid Synthesis and Increases Mitochondrial Fatty Acid Oxidation in Hepatoma Cells Infected with Hepatitis C Virus.

Cytopathic effects are currently believed to contribute to hepatitis C virus (HCV)-induced liver injury and are readily observed in Huh7.5 cells infected with the JFH-1 HCV strain, manifesting as apoptosis highly correlated with growth arrest. Reactive oxygen species, which are induced by HCV infection, have recently emerged as activators of AMP-activated protein kinase. The net effect is ATP conservation via on/off switching of metabolic pathways that produce/consume ATP. Depending on the scenario, this can have either pro-survival or pro-apoptotic effects. We demonstrate reactive oxygen species-mediated activation of AMP-activated kinase in Huh7.5 cells during HCV (JFH-1)-induced growth arrest. Metabolic labeling experiments provided direct evidence that lipid synthesis is attenuated, and ß-oxidation is enhanced in these cells. A striking increase in nuclear peroxisome proliferator-activated receptor α, which plays a dominant role in the expression of ß-oxidation genes after ligand-induced activation, was also observed, and we provide evidence that peroxisome proliferator-activated receptor α is constitutively activated in these cells. The combination of attenuated lipid synthesis and enhanced ß-oxidation is not conducive to lipid accumulation, yet cellular lipids still accumulated during this stage of infection. Notably, the serum in the culture media was the only available source for polyunsaturated fatty acids, which were elevated (2-fold) in the infected cells, implicating altered lipid import/export pathways in these cells. This study also provided the first in vivo evidence for enhanced ß-oxidation during HCV infection because HCV-infected SCID/Alb-uPA mice accumulated higher plasma ketones while fasting than did control mice. Overall, this study highlights the reprogramming of hepatocellular lipid metabolism and bioenergetics during HCV infection, which are predicted to impact both the HCV life cycle and pathogenesis.

STAT1 is phosphorylated and downregulated by the oncogenic tyrosine kinase NPM-ALK in ALK-positive anaplastic large-cell lymphoma.

The tumorigenicity of most cases of ALK-positive anaplastic large-cell lymphoma (ALK+ ALCL) is driven by the oncogenic fusion protein NPM-ALK in a STAT3-dependent manner. Because it has been shown that STAT3 can be inhibited by STAT1 in some experimental models, we hypothesized that the STAT1 signaling pathway is defective in ALK+ ALCL, thereby leaving the STAT3 signaling unchecked. Compared with normal T cells, ALK+ ALCL tumors consistently expressed a low level of STAT1. Inhibition of the ubiquitin-proteasome pathway appreciably increased STAT1 expression in ALK+ ALCL cells. Furthermore, we found evidence that NPM-ALK binds to and phosphorylates STAT1, thereby promoting its proteasomal degradation in a STAT3-dependent manner. If restored, STAT1 is functionally intact in ALK+ ALCL cells, because it effectively upregulated interferon-γ, induced apoptosis/cell-cycle arrest, potentiated the inhibitory effects of doxorubicin, and suppressed tumor growth in vivo. STAT1 interfered with the STAT3 signaling by decreasing STAT3 transcriptional activity/DNA binding and its homodimerization. The importance of the STAT1/STAT3 functional interaction was further highlighted by the observation that short interfering RNA knockdown of STAT1 significantly decreased apoptosis induced by STAT3 inhibition. Thus, STAT1 is a tumor suppressor in ALK+ ALCL. Phosphorylation and downregulation of STAT1 by NPM-ALK represent other mechanisms by which this oncogenic tyrosine kinase promotes tumorigenesis.

Acute Responses to the 7-Minute Workout.

Riegler, M, Stotz, G, Fitzgerald, K, Munoz, CK, Lewis, J, Ring, S, and Astorino, TA. Acute responses to the 7-minute workout. J Strength Cond Res 31(9): 2572-2578, 2017-A popular smartphone application called the 7-Minute Workout (7Min) claims to be scientifically proven to promote weight loss and improve cardiovascular function. The application has 10 million downloads and has been reviewed by 350,000 users. However, changes in metabolic and perceptual variables in response to 7Min are unknown. Our study compared acute responses between 7Min and a time-matched session of cycling-based high-intensity interval exercise (HIIE). Fourteen active men and women (age and V[Combining Dot Above]O2max = 25.4 ± 8.3 years and 40.5 ± 6.4 ml·kg·min) initially underwent V[Combining Dot Above]O2max testing. During 2 subsequent sessions separated by ≥48 hours, they completed 7Min or HIIE. During exercise, oxygen uptake (V[Combining Dot Above]O2), heart rate (HR), blood lactate concentration (BLa), and rating of perceived exertion were measured. Peak V[Combining Dot Above]O2 was higher (p < 0.001) in HIIE vs. 7Min, and HIIE yielded greater (p < 0.001) mean V[Combining Dot Above]O2 (1.83 ± 0.41 L·min vs. 1.44 ± 0.32 L·min) and HR (159.0 ± 10.7 b·min vs. 140.7 ± 18.3 b·min, p < 0.001) vs. 7Min. Blood lactate concentration increased (p < 0.001) during exercise but was similar between bouts (p = 0.07). Rating of perceived exertion was higher (p = 0.008) in response to HIIE vs. 7Min. Although 7Min yields lower peak V[Combining Dot Above]O2 and HR than HIIE, it is characterized by bursts approaching 90 %HRmax and causes significant BLa accumulation, representing vigorous exercise. Nevertheless, 7Min is on the low end of the intensity spectrum, which questions whether it represents true HIIE and will confer similar benefits if performed long term.

Doing laboratory ethnography: reflections on method in scientific workplaces.

Laboratory ethnography extended the social scientist's gaze into the day-to-day accomplishment of scientific practice. Here we reflect upon our own ethnographies of biomedical scientific workspaces to provoke methodological discussion on the doing of laboratory ethnography. What we provide is less a 'how to' guide and more a commentary on what to look for and what to look at. We draw upon our empirical research with stem cell laboratories and animal houses, teams producing robotic surgical tools, musicians sonifying data science, a psychiatric genetics laboratory, and scientists developing laboratory grown meat. We use these cases to example a set of potential ethnographic themes worthy of pursuit: science epistemics and the extended laboratory, the interaction order of scientific work, sensory realms and the rending of science as sensible, conferences as performative sites, and the spaces, places and temporalities of scientific work.

Strengthening laboratory capacity through the surveillance of rotavirus gastroenteritis in Central Africa: the Surveillance Épidémiologique en Afrique Centrale (SURVAC) Project.

OBJECTIVES: The goal of the SURVAC pilot project was to strengthen disease surveillance and response in three countries; Cameroon (CAE), Democratic Republic of the Congo (DRC) and Central African Republic (CAR). METHODS: Seven laboratories involved in rotavirus surveillance were provided with equipment, reagents and supplies. CDC and WHO staff provided on-site classroom and bench training in biosafety, quality assurance, quality control (QC), rotavirus diagnosis using Enzyme Immunoassay (EIA) and genotyping of rotavirus strains using the Reverse Transcription Polymerase-chain reaction (RT-PCR). All laboratory data were reported through WHO/AFRO. RESULTS: Twenty-three staff members were trained on RT-PCR for rotavirus genotyping which was introduced for the first time in all three countries. In CAE, the number of samples analysed by EIA and RT-PCR increased tenfold between 2007 and 2013. In DRC, this number increased fivefold, from 2009 to 2013 whereas in CAR, it increased fourfold between 2011 and 2013. All laboratories passed WHO proficiency testing in 2014. CONCLUSION: Laboratory capacity was strengthened through equipping laboratories and strengthening a subregional laboratory workforce for surveillance of rotavirus gastroenteritis. Each of the three countries generated rotavirus surveillance and genotyping data enabling the mapping of circulating genotypes. These results will help monitor the impact of rotavirus vaccination in these countries.

Characterization of lipid and lipoprotein metabolism in primary human hepatocytes.

Primary rodent hepatocytes and hepatoma cell lines are commonly used as model systems to elucidate and study potential drug targets for metabolic diseases such as obesity and atherosclerosis. However, if therapies are to be developed, it is essential that our knowledge gained from these systems is translatable to that of human. Here, we have characterized lipid and lipoprotein metabolism in primary human hepatocytes for comparison to rodent primary hepatocytes and human hepatoma cell lines. Primary human hepatocytes were maintained in collagen coated dishes in confluent monolayers for up to 3 days. We found primary human hepatocytes were viable, underwent lipid synthesis, and were able to secret lipoproteins up to 3 days in culture. Furthermore, the lipoprotein profile secreted by primary human hepatocytes was comparable to that found in human plasma; this contrasts with primary rodent hepatocytes and human hepatoma cells. We also investigated the pharmacological effects of nicotinic acid (niacin, NA), a potent dyslipidemic drug, on hepatic lipid synthesis and lipoprotein secretion. We found NA increased the expression of ATP-binding cassette transporter A1 in primary human hepatocytes, which may potentially explain how NA increases plasma high-density lipoproteins in humans. In conclusion, primary human hepatocytes are a more relevant model to study lipid synthesis and lipoprotein secretion than hepatoma cells or rodent primary hepatocyte models. Further research needs to be done to maintain liver specific functions of primary human hepatocytes in prolonged cultures for these cells to be a viable model.

Comparison of custom to standard TKA instrumentation with computed tomography.

PURPOSE: There is conflicting evidence whether custom instrumentation for total knee arthroplasty (TKA) improves component position compared to standard instrumentation. Studies have relied on long-limb radiographs limited to two-dimensional (2D) analysis and subjected to rotational inaccuracy. We used postoperative computed tomography (CT) to evaluate preoperative three-dimensional templating and CI to facilitate accurate and efficient implantation of TKA femoral and tibial components. METHODS: We prospectively evaluated a single-surgeon cohort of 78 TKA patients (51 custom, 27 standard) with postoperative CT scans using 3D reconstruction and contour-matching technology to preoperative imaging. Component alignment was measured in coronal, sagittal and axial planes. RESULTS: Preoperative templating for custom instrumentation was 87 and 79 % accurate for femoral and tibial component size. All custom components were within 1 size except for the tibial component in one patient (2 sizes). Tourniquet time was 5 min longer for custom (30 min) than standard (25 min). In no case was custom instrumentation aborted in favour of standard instrumentation nor was original alignment of custom instrumentation required to be adjusted intraoperatively. There were more outliers greater than 2° from intended alignment with standard instrumentation than custom for both components in all three planes. Custom instrumentation was more accurate in component position for tibial coronal alignment (custom: 1.5° ± 1.2°; standard: 3° ± 1.9°; p = 0.0001) and both tibial (custom: 1.4° ± 1.1°; standard: 16.9° ± 6.8°; p < 0.0001) and femoral (custom: 1.2° ± 0.9°; standard: 3.1° ± 2.1°; p < 0.0001) rotational alignment, and was similar to standard instrumentation in other measurements. CONCLUSIONS: When evaluated with CT, custom instrumentation performs similar or better to standard instrumentation in component alignment and accurately templates component size. Tourniquet time was mildly increased for custom compared to standard.

Arylacetamide deacetylase: a novel host factor with important roles in the lipolysis of cellular triacylglycerol stores, VLDL assembly and HCV production.

BACKGROUND & AIMS: Very low density lipoproteins (VLDLs) are triacylglycerol (TG)-rich lipoproteins produced by the human liver. VLDLs derive the majority of their TG cargo from the lipolysis of TG stored in hepatocellular lipid droplets (LDs). Important roles for LDs and the VLDL secretory pathway in the cell culture production of infectious hepatitis C virus (HCV) have been established. We hypothesized that TG lipolysis and VLDL production are impaired during HCV infection so that these cellular processes can be diverted towards HCV production. METHODS: We used an HCV permissive cell culture system (JFH-1/HuH7.5 cells) to examine the relationship between TG lipolysis, VLDL assembly, and the HCV lifecycle using standard biochemical approaches. RESULTS: Lipolysis of cellular TG and VLDL production were impaired in HCV infected cells during the early peak of viral infection. This was partially explained by an apparent deficiency of a putative TG lipase, arylacetamide deacetylase (AADAC). The re-introduction of AADAC to infected cells restored cellular TG lipolysis, indicating a role for HCV-mediated downregulation of AADAC in this process. Defective lipolysis of cellular TG stores and VLDL production were also observed in HuH7.5 cells stably expressing a short hairpin RNA targeting AADAC expression, proving AADAC deficiency contributes to these defective pathways. Finally, impaired production of HCV was observed with AADAC knockdown cells, demonstrating a role for AADAC in the HCV lifecycle. CONCLUSIONS: This insight into the biology of HCV infection and possibly pathogenesis identifies AADAC as a novel and translationally relevant therapeutic target.

Sensitive and specific quantitative detection of rotavirus A by one-step real-time reverse transcription-PCR assay without antecedent double-stranded-RNA denaturation.

A real-time quantitative reverse transcription-PCR (qRT-PCR) assay using the recombinant thermostable Thermus thermophilus (rTth) enzyme was developed to detect and quantify rotavirus A (RVA). By using rTth polymerase, significant improvement was achieved over the existing real-time RT-PCR assays, which require denaturation of the RVA double-stranded RNA (dsRNA) prior to assay setup. Using a dsRNA transcript for segment 7, which encodes the assay target NSP3 gene, the limit of detection for the improved assay was calculated to be approximately 1 genome copy per reaction. The NSP3 qRT-PCR assay was validated using a panel of 1,906 stool samples, 23 reference RVA strains, and 14 nontarget enteric virus samples. The assay detected a diverse number of RVA genotypes and did not detect other enteric viruses, demonstrating analytical sensitivity and specificity for RVA in testing stool samples. A XenoRNA internal process control was introduced and detected in a multiplexed qRT-PCR format. Because it does not require an antecedent dsRNA denaturation step, this assay reduces the possibility of sample cross-contamination and requires less hands-on time than other published qRT-PCR protocols for RVA detection.

Hepatitis C virus envelope glycoprotein fitness defines virus population composition following transmission to a new host.

Genetic variability is a hallmark of RNA virus populations. However, transmission to a new host often results in a marked decrease in population diversity. This genetic bottlenecking is observed during hepatitis C virus (HCV) transmission and can arise via a selective sweep or through the founder effect. To model HCV transmission, we utilized chimeric SCID/Alb-uPA mice with transplanted human hepatocytes and infected them with a human serum HCV inoculum. E1E2 glycoprotein gene sequences in the donor inoculum and recipient mice were determined following single-genome amplification (SGA). In independent experiments, using mice with liver cells grafted from different sources, an E1E2 variant undetectable in the source inoculum was selected for during transmission. Bayesian coalescent analyses indicated that this variant arose in the inoculum pretransmission. Transmitted variants that established initial infection harbored key substitutions in E1E2 outside HVR1. Notably, all posttransmission E1E2s had lost a potential N-linked glycosylation site (PNGS) in E2. In lentiviral pseudoparticle assays, the major posttransmission E1E2 variant conferred an increased capacity for entry compared to the major variant present in the inoculum. Together, these data demonstrate that increased envelope glycoprotein fitness can drive selective outgrowth of minor variants posttransmission and that loss of a PNGS is integral to this improved phenotype. Mathematical modeling of the dynamics of competing HCV variants indicated that relatively modest differences in glycoprotein fitness can result in marked shifts in virus population composition. Overall, these data provide important insights into the dynamics and selection of HCV populations during transmission.

Additive effect of sirolimus and anti-death receptor 5 agonistic antibody against hepatocellular carcinoma.

BACKGROUND & AIMS: Despite careful patient selection, hepatocellular carcinoma (HCC) recurs in 10-20% of cases after liver transplantation, and the use of potent adjuvant anticancer drugs would be welcome. The aim of this study was to evaluate the efficiency of a combined therapy of rapamycin (sirolimus) and anti-death receptor (DR)5 monoclonal antibody (mAb) on HCC. METHODS: We first assessed the side effects of anti-DR5 mAb administration in vivo by giving various doses of anti-DR5 mAb. Cell proliferation assays were then performed using mouse Hepa1-6 cells or human Huh7 cells to quantify the relative cell viability under various concentrations of sirolimus, anti-DR5 mAb or a combination. Next, one million Hepa1-6 cells were transplanted into C.B17-SCID-beige mice subcutaneously, and four groups were created: (1) untreated, (2) anti-DR5 mAb alone, (3) sirolimus alone and (4) anti-DR5 mAb + sirolimus. RESULTS: Anti-DR5 mAb (200 and 300 µg/day) induced liver dysfunction with partial necrosis of the liver, but 100 µg/day was well tolerated with transaminitis, but normal bilirubin and only minor histological liver damage. In vitro, anti-DR5 mAb lysed Hepa1-6 and Huh7 cells in a dose-dependent manner, and combinations of sirolimus and anti-DR5 mAb demonstrated an additive effect. In vivo studies demonstrated that tumour sizes were significantly smaller in the combined therapy group than in the monotherapy groups. CONCLUSIONS: Combining sirolimus and low-dose anti-DR5 mAb has a significant effect against HCC. This strategy represents a potential novel approach for the management of HCC.

Human cytomegalovirus infection in humanized liver chimeric mice.

AIM: Cytomegalovirus is a common viral pathogen that influences the outcome of organ transplantation. To date, there is no established method to evaluate the effects of human CMV (HCMV) treatments in vivo except for human clinical trials. In the current study, we describe the development of a mouse model that supports the in vivo propagation of HCMV. METHODS: One million viable human hepatocytes, purified from human livers, were injected into the spleens of severe combined immunodeficient/albumin linked-urokinase type plasminogen activator transgenic mice. A clinical strain of HCMV was inoculated in mice with confirmed human hepatocyte engraftment or in non-chimeric controls. Infection was monitored through HCMV titers in the plasma. Mice were administrated ganciclovir (50 mg/kg per day, i.p.) beginning at 2 days post-HCMV inoculation, or human liver natural killer (NK) cells (20 × 10(6) cells/mouse, i.v.) 1 day prior to HCMV inoculation. RESULTS: Chimeric mice that received HCMV showed high plasma titers of HCMV DNA on days 1 and 6 that became undetectable by day 11 post-inoculation. In contrast, non-transplanted mice had only residual plasma inoculum detection at day 1 and no detectable viremia thereafter. The levels of HCMV DNA were reduced by ganciclovir treatment or by human liver NK cell adoptive transfer, while HCMV-infected chimeric mice that were not treated sustained viremia during the follow up. CONCLUSION: Human liver chimeric mice provide an in vivo model for the study of acute HCMV infection of hepatocytes.

Closing the regulatory regress: GMP accreditation in stem cell laboratories.

Contemporary biomedical research is conducted amidst regimes of national and transnational regulation. Regulation, like rules generally, cannot specify all the practicalities of their application. Regulations for biomedical research impose considerable constraints on laboratories and others. In principle, there is a never-ending regress whereby scientists have to provide increasingly more guarantees that protocols have been followed, standards reached and maintained, and rules adhered to. In practice, regulatory regress is not the actual outcome, as actors find ways of establishing closure for all practical purposes. Based on ethnographic case studies of two sites of biomedical work--the UK Stem Cell Bank and an anonymous laboratory working with primary human foetal material--this article documents the possibility of regulatory regress and strategies aimed at its closure.