Retrospective harm benefit analysis of pre-clinical animal research for six treatment interventions

The harm benefit analysis (HBA) is the cornerstone of animal research regulation and is considered to be a key ethical safeguard for animals. The HBA involves weighing the anticipated benefits of animal research against its predicted harms to animals but there are doubts about how objective and accountable this process is.i. To explore the harms to animals involved in pre-clinical animal studies and to assess these against the benefits for humans accruing from these studies; ii. To test the feasibility of conducting this type of retrospective HBA.Data on harms were systematically extracted from a sample of pre-clinical animal studies whose clinical relevance had already been investigated by comparing systematic reviews of the animal studies with systematic reviews of human studies for the same interventions (antifibrinolytics for haemorrhage, bisphosphonates for osteoporosis, corticosteroids for brain injury, Tirilazad for stroke, antenatal corticosteroids for neonatal respiratory distress and thrombolytics for stroke). Clinical relevance was also explored in terms of current clinical practice. Harms were categorised for severity using an expert panel. The quality of the research and its impact were considered. Bateson's Cube was used to conduct the HBA.The most common assessment of animal harms by the expert panel was 'severe'. Reported use of analgesia was rare and some animals (including most neonates) endured significant procedures with no, or only light, anaesthesia reported. Some animals suffered iatrogenic harms. Many were kept alive for long periods post-experimentally but only 1% of studies reported post-operative care. A third of studies reported that some animals died prior to endpoints. All the studies were of poor quality. Having weighed the actual harms to animals against the actual clinical benefits accruing from these studies, and taking into account the quality of the research and its impact, less than 7% of the studies were permissible according to Bateson's Cube: only the moderate bisphosphonate studies appeared to minimise harms to animals whilst being associated with benefit for humans.This is the first time the accountability of the HBA has been systematically explored across a range of pre-clinical animal studies. The regulatory systems in place when these studies were conducted failed to safeguard animals from severe suffering or to ensure that only beneficial, scientifically rigorous research was conducted. Our findings indicate a pressing need to: i. review regulations, particularly those that permit animals to suffer severe harms; ii. reform the processes of prospectively assessing pre-clinical animal studies to make them fit for purpose; and iii. systematically evaluate the benefits of pre-clinical animal research to permit a more realistic assessment of its likely future benefits

Seasonal mixed layer depth shapes phytoplankton physiology, viral production, and accumulation in the North Atlantic

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Diaz, B. P., Knowles, B., Johns, C. T., Laber, C. P., Bondoc, K. G. V., Haramaty, L., Natale, F., Harvey, E. L., Kramer, S. J., Bolaños, L. M., Lowenstein, D. P., Fredricks, H. F., Graff, J., Westberry, T. K., Mojica, K. D. A., Haëntjens, N., Baetge, N., Gaube, P., Boss, E., Carlson, C. A., Behrenfeld, M. J., Van Mooy, B. A. S., Bidle, K. D. Seasonal mixed layer depth shapes phytoplankton physiology, viral production, and accumulation in the North Atlantic. Nature Communications, 12(1), (2021): 6634, https://doi.org/10.1038/s41467-021-26836-1.Seasonal shifts in phytoplankton accumulation and loss largely follow changes in mixed layer depth, but the impact of mixed layer depth on cell physiology remains unexplored. Here, we investigate the physiological state of phytoplankton populations associated with distinct bloom phases and mixing regimes in the North Atlantic. Stratification and deep mixing alter community physiology and viral production, effectively shaping accumulation rates. Communities in relatively deep, early-spring mixed layers are characterized by low levels of stress and high accumulation rates, while those in the recently shallowed mixed layers in late-spring have high levels of oxidative stress. Prolonged stratification into early autumn manifests in negative accumulation rates, along with pronounced signatures of compromised membranes, death-related protease activity, virus production, nutrient drawdown, and lipid markers indicative of nutrient stress. Positive accumulation renews during mixed layer deepening with transition into winter, concomitant with enhanced nutrient supply and lessened viral pressure.This work was made possible by NASA’s Earth Science Program in support of the North Atlantic Aerosol and Marine Ecosystem Study (15-RRNES15-0011 and 0NSSC18K1563 to K.D.B.; NNX15AF30G to M.J.B.), as well as with support from the National Science Foundation (OIA-2021032 to K.D.B., OCE-157943 to C.A.C., and OCE-1756254 to B.A.S.V.M.), the Gordon and Betty Moore Foundation (Award# 3789 to K.G.V.B.), and NASA’s Future Investigators in Space Science and Technology program (FINESST; grant #826380 to K.D.B.; graduate support to BD)

Seasonal Mixed Layer Depth Shapes Phytoplankton Physiology, Viral Production, and Accumulation In the North Atlantic

Seasonal shifts in phytoplankton accumulation and loss largely follow changes in mixed layer depth, but the impact of mixed layer depth on cell physiology remains unexplored. Here, we investigate the physiological state of phytoplankton populations associated with distinct bloom phases and mixing regimes in the North Atlantic. Stratification and deep mixing alter community physiology and viral production, effectively shaping accumulation rates. Communities in relatively deep, early-spring mixed layers are characterized by low levels of stress and high accumulation rates, while those in the recently shallowed mixed layers in late-spring have high levels of oxidative stress. Prolonged stratification into early autumn manifests in negative accumulation rates, along with pronounced signatures of compromised membranes, death-related protease activity, virus production, nutrient drawdown, and lipid markers indicative of nutrient stress. Positive accumulation renews during mixed layer deepening with transition into winter, concomitant with enhanced nutrient supply and lessened viral pressure

Linking the FTO obesity rs1421085 variant circuitry to cellular, metabolic, and organismal phenotypes in vivo

Variants in FTO have the strongest association with obesity; however, it is still unclear how those noncoding variants mechanistically affect whole-body physiology. We engineered a deletion of the rs1421085 conserved cis-regulatory module (CRM) in mice and confirmed in vivo that the CRM modulates Irx3 and Irx5 gene expression and mitochondrial function in adipocytes. The CRM affects molecular and cellular phenotypes in an adipose depot–dependent manner and affects organismal phenotypes that are relevant for obesity, including decreased high-fat diet–induced weight gain, decreased whole-body fat mass, and decreased skin fat thickness. Last, we connected the CRM to a genetically determined effect on steroid patterns in males that was dependent on nutritional challenge and conserved across mice and humans. Together, our data establish cross-species conservation of the rs1421085 regulatory circuitry at the molecular, cellular, metabolic, and organismal level, revealing previously unknown contextual dependence of the variant’s action

Machine Learning based histology phenotyping to investigate the epidemiologic and genetic basis of adipocyte morphology and cardiometabolic traits

Genetic studies have recently highlighted the importance of fat distribution, as well as overall adiposity, in the pathogenesis of obesity-associated diseases. Using a large study (n = 1,288) from 4 independent cohorts, we aimed to investigate the relationship between mean adipocyte area and obesity-related traits, and identify genetic factors associated with adipocyte cell size. To perform the first large-scale study of automatic adipocyte phenotyping using both histological and genetic data, we developed a deep learning-based method, the Adipocyte U-Net, to rapidly derive mean adipocyte area estimates from histology images. We validate our method using three state-of-the-art approaches; CellProfiler, Adiposoft and floating adipocytes fractions, all run blindly on two external cohorts. We observe high concordance between our method and the state-of-the-art approaches (Adipocyte U-net vs. CellProfiler: R2visceral = 0.94, P < 2.2 × 10-16, R2subcutaneous = 0.91, P < 2.2 × 10-16), and faster run times (10,000 images: 6mins vs 3.5hrs). We applied the Adipocyte U-Net to 4 cohorts with histology, genetic, and phenotypic data (total N = 820). After meta-analysis, we found that mean adipocyte area positively correlated with body mass index (BMI) (Psubq = 8.13 × 10-69, βsubq = 0.45; Pvisc = 2.5 × 10-55, βvisc = 0.49; average R2 across cohorts = 0.49) and that adipocytes in subcutaneous depots are larger than their visceral counterparts (Pmeta = 9.8 × 10-7). Lastly, we performed the largest GWAS and subsequent meta-analysis of mean adipocyte area and intra-individual adipocyte variation (N = 820). Despite having twice the number of samples than any similar study, we found no genome-wide significant associations, suggesting that larger sample sizes and a homogenous collection of adipose tissue are likely needed to identify robust genetic associations.This article is freely available via Open Access. Click on the Publisher URL to access it via the publisher's site.C.A.G received a pump priming grant from Novo Nordisk to carry out this work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.published version, accepted versio

Update: cohort mortality study of workers highly exposed to polychlorinated biphenyls (PCBs) during the manufacture of electrical capacitors, 1940-1998

BACKGROUND: The National Institute for Occupational Safety and Health previously reported mortality for a cohort of workers considered highly exposed to polychlorinated biphenyls (PCBs) between 1939 and 1977 at two electrical capacitor manufacturing plants. The current study updated vital status, examined liver and rectal cancer mortality previously reported in excess in this cohort and evaluated mortality from non-Hodgkin's lymphoma (NHL) and cancers of the stomach, intestine, breast, prostate, skin (melanoma) and brain reported to be in excess in other cohort and case-control studies of PCB-exposed persons. METHODS: Mortality was updated through 1998 for 2572 workers. Age-, gender-, race- and calendar year-adjusted standardized mortality ratios (SMRs) and 95% confidence intervals (CI) were calculated using U.S., state and county referent rates. SMRs using U.S. referent rates are reported. Duration of employment was used as a surrogate for exposure. RESULTS: Consistent with the previous follow-up, mortality from biliary passage, liver and gall bladder cancer was significantly elevated (11 deaths, SMR 2.11, CI 1.05 – 3.77), but mortality from rectal cancer was not (6 deaths, SMR 1.47, CI 0.54 – 3.21). Among women, mortality from intestinal cancer (24 deaths, SMR 1.89, CI 1.21 – 2.82) and from "other diseases of the nervous system and sense organs", which include Parkinson's disease and amyotrophic lateral sclerosis, (15 deaths, SMR 2.07, CI 1.16 – 3.42) were elevated. There were four ALS deaths, all women (SMR 4.35, CI 1.19–11.14). Mortality was elevated for myeloma (7 deaths, SMR 2.11, CI 0.84 – 4.34), particularly among workers employed 10 years or more (5 deaths, SMR 2.80, CI 0.91 – 6.54). No linear associations between mortality and duration of employment were observed for the cancers of interest. CONCLUSION: This update found that the earlier reported excess in this cohort for biliary, liver and gall bladder cancer persisted with longer follow-up. Excess mortality for intestinal cancer among women was elevated across categories of duration of employment; myeloma mortality was highest among those working 10 years or more. The small numbers of deaths from liver and intestinal cancers, myeloma and nervous system diseases coupled with the lack of an exposure-response relationship with duration of employment preclude drawing definitive conclusions regarding PCB exposure and these causes of death

Nitric oxide production and antioxidant function during viral infection of the coccolithophore Emiliania huxleyi

Emiliania huxleyi is a globally important marine phytoplankton that is routinely infected by viruses. Understanding the controls on the growth and demise of E. huxleyi blooms is essential for predicting the biogeochemical fate of their organic carbon and nutrients. In this study, we show that the production of nitric oxide (NO), a gaseous, membrane-permeable free radical, is a hallmark of early-stage lytic infection in E. huxleyi by Coccolithoviruses, both in culture and in natural populations in the North Atlantic. Enhanced NO production was detected both intra- and extra-cellularly in laboratory cultures, and treatment of cells with an NO scavenger significantly reduced viral production. Pre-treatment of exponentially growing E. huxleyi cultures with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) prior to challenge with hydrogen peroxide (H2O2) led to greater cell survival, suggesting that NO may have a cellular antioxidant function. Indeed, cell lysates generated from cultures treated with SNAP and undergoing infection displayed enhanced ability to detoxify H2O2. Lastly, we show that fluorescent indicators of cellular ROS, NO, and death, in combination with classic DNA- and lipid-based biomarkers of infection, can function as real-time diagnostic tools to identify and contextualize viral infection in natural E. huxleyi blooms

Met-Independent Hepatocyte Growth Factor-mediated regulation of cell adhesion in human prostate cancer cells

BACKGROUND: Prostate cancer cells communicate reciprocally with the stromal cells surrounding them, inside the prostate, and after metastasis, within the bone. Each tissue secretes factors for interpretation by the other. One stromally-derived factor, Hepatocyte Growth Factor (HGF), was found twenty years ago to regulate invasion and growth of carcinoma cells. Working with the LNCaP prostate cancer progression model, we found that these cells could respond to HGF stimulation, even in the absence of Met, the only known HGF receptor. The new HGF binding partner we find on the cell surface may help to clarify conflicts in the past literature about Met expression and HGF response in cancer cells. METHODS: We searched for Met or any HGF binding partner on the cells of the PC3 and LNCaP prostate cancer cell models, using HGF immobilized on agarose beads. By using mass spectrometry analyses and sequencing we have identified nucleolin protein as a novel HGF binding partner. Antibodies against nucleolin (or HGF) were able to ameliorate the stimulatory effects of HGF on met-negative prostate cancer cells. Western blots, RT-PCR, and immunohistochemistry were used to assess nucleolin levels during prostate cancer progression in both LNCaP and PC3 models. RESULTS: We have identified HGF as a major signaling component of prostate stromal-conditioned media (SCM) and have implicated the protein nucleolin in HGF signal reception by the LNCaP model prostate cancer cells. Antibodies that silence either HGF (in SCM) or nucleolin (on the cell surfaces) eliminate the adhesion-stimulatory effects of the SCM. Likewise, addition of purified HGF to control media mimics the action of SCM. C4-2, an LNCaP lineage-derived, androgen-independent human prostate cancer cell line, responds to HGF in a concentration-dependent manner by increasing its adhesion and reducing its migration on laminin substratum. These HGF effects are not due to shifts in the expression levels of laminin-binding integrins, nor can they be linked to expression of the known HGF receptor Met, as neither LNCaP nor clonally-derived C4-2 sub-line contain any detectable Met protein. Even in the absence of Met, small GTPases are activated, linking HGF stimulation to membrane protrusion and integrin activation. Membrane-localized nucelolin levels increase during cancer progression, as modeled by both the PC3 and LNCaP prostate cancer progression cell lines. CONCLUSION: We propose that cell surface localized nucleolin protein may function in these cells as a novel HGF receptor. Membrane localized nucleolin binds heparin-bound growth factors (including HGF) and appears upregulated during prostate cancer progression. Antibodies against nucleolin are able to ameliorate the stimulatory effects of HGF on met-negative prostate cancer cells. HGF-nucleolin interactions could be partially responsible for the complexity of HGF responses and met expression reported in the literature