Meta-Analytic Methodology for Basic Research: A Practical Guide

Basic life science literature is rich with information, however methodically quantitative attempts to organize this information are rare. Unlike clinical research, where consolidation efforts are facilitated by systematic review and meta-analysis, the basic sciences seldom use such rigorous quantitative methods. The goal of this study is to present a brief theoretical foundation, computational resources and workflow outline along with a working example for performing systematic or rapid reviews of basic research followed by meta-analysis. Conventional meta-analytic techniques are extended to accommodate methods and practices found in basic research. Emphasis is placed on handling heterogeneity that is inherently prevalent in studies that use diverse experimental designs and models. We introduce MetaLab, a meta-analytic toolbox developed in MATLAB R2016b which implements the methods described in this methodology and is provided for researchers and statisticians at Git repository (https://github.com/NMikolajewicz/MetaLab). Through the course of the manuscript, a rapid review of intracellular ATP concentrations in osteoblasts is used as an example to demonstrate workflow, intermediate and final outcomes of basic research meta-analyses. In addition, the features pertaining to larger datasets are illustrated with a systematic review of mechanically-stimulated ATP release kinetics in mammalian cells. We discuss the criteria required to ensure outcome validity, as well as exploratory methods to identify influential experimental and biological factors. Thus, meta-analyses provide informed estimates for biological outcomes and the range of their variability, which are critical for the hypothesis generation and evidence-driven design of translational studies, as well as development of computational models

Exosomal release of L-plastin by breast cancer cells facilitates metastatic bone osteolysis

Bone metastasis from breast and prostate carcinomas is facilitated by activation of bone-resorbing osteoclasts. Using proteomics approaches, we have identified peroxiredoxin-4 (PRDX4) as a cancer-secreted mediator of osteoclastogenesis. We now report characterization of L-plastin in the conditioned media (CM) of MDA-MB-231 human breast cancer cells using immunoblotting and mass spectrometry. The osteoclastogenic potential of MDAMB-231 CM with siRNA-silenced L-plastin was significantly reduced. L-plastin was detected in cancer-derived exosomes, and inhibition of exosomal release significantly decreased the osteoclastogenic capacity of MDA-MB-231 CM. When added to osteoclast precursors primed with RANKL for 2 days, recombinant L-plastin induced calcium/NFATc1-mediated osteoclastogenesis to the levels similar to continuous treatment with RANKL. Using shRNA, we generated MDA-MB-231 cells lacking L-plastin, PRDX4, or both and injected these cell populations intratibially in CD-1 immunodeficient mice. Micro-CT and histomorphometric analysis demonstrated a complete loss of osteolysis when MDA-MB-231 cells lacking both L-plastin and PRDX4 were injected. A meta-analysis established an increase in L-plastin and PRDX4 mRNA expression in numerous human cancers, including breast and prostate carcinomas. This study demonstrates that secreted L-plastin and PRDX4 mediate osteoclast activation by human breast cancer cells

Purinergic mechanotransduction in bone

Athletes' skeletons get stronger with training, while bones weaken in people who cannot move or in astronauts experiencing weightlessness. These changes are mediated by bone cells which can perceive mechanical forces, convert them to biochemical signals and induce changes in bone mass and strength. Among the first detectable signals following mechanical stimulation is the release of the energy-rich molecule ATP. Extracellular ATP was proposed as a mechanotransductive signaling molecular over 20 years ago and has been shown to signal through 15 different purinergic receptors (P2). However, the exact contribution of ATP release and P2 signaling to mechanically-induced bone adaptation remains unclear. The primary aims of this dissertation were to investigate the mechanisms of mechanically-stimulated ATP release from bone cells and to determine how the purinergic signal transmits information about the nature of the mechanical stimulus to neighbouring cells.Many studies examining the mechanisms of mechanically-stimulated ATP release have been conducted to date. Since large-scale quantitative synthesis of basic research has not been attempted, I developed the theoretical foundation, computational resources and workflow required to conduct a meta-analysis in the basic sciences. This enabled consolidation and synthesis of quantitative data from 278 studies that investigated the amount, kinetics and mechanisms of ATP release, as well as the influence of pathologies on ATP release. I demonstrated that mechanically-stimulated ATP release is a conserved phenomenon across mammalian cells, and that mechanically-stimulated cells release 38.6 (95% CI: 18.2 to 81.8) amoles ATP/cell on average with a characteristic time constant of 32 s (95% CI: 16 to 66). Importantly, vesicles and voltage sensitive calcium channels were implicated in the release of ATP from mechanically-stimulated osteoblasts and osteocytes. I next investigated the mechanisms of ATP release from osteoblasts experimentally. I determined that mechanical stimulation of a single murine osteoblast led to the release of 70 ± 24 amole ATP. Osteoblasts were found to contain ATP-rich vesicles that were released upon mechanical stimulation, however, pharmacological interventions that promoted vesicular exocytosis reduced ATP release, while inhibitors of vesicular release potentiated ATP release. In search of an alternative route of ATP release, I found that mechanical stresses induced reversible cell membrane injury in vitro and in vivo. Calcium/PKC-dependent vesicular exocytosis facilitated membrane repair, thereby ensuring cell viability and reducing ATP release. Thus, I propose that exocytosis of ATP-containing vesicles limits the efflux of intracellular ATP through reversibly damaged membranes by facilitating membrane repair. Overall, the studies presented here demonstrate that (i) bone cell injury is critical for purinergic mechanotransduction, (ii) membrane repair occurs through calcium/PKC-dependent vesicular exocytosis, and (iii) information about the severity of injury and cellular mechano-adaptive status is integrated at the level of the purinergic signal. Importantly, identification of molecular mediators, including PKCµ, may represent future therapeutic targets in prevention of disuse-related bone loss.Les squelettes des athlètes deviennent plus forts à l'entraînement, tandis que les os s'affaiblissent chez les personnes qui ne peuvent pas bouger ou chez les astronautes en apesanteur. Ces changements sont médiés par les cellules osseuses qui peuvent percevoir les forces mécaniques, les convertir en signaux biochimiques et induire des changements dans la masse et la force osseuses. Parmi les premiers signaux détectables à la suite d'une stimulation mécanique figure la libération d'une molécule riche en énergie, l'ATP. L'ATP extracellulaire a été proposé comme molécule de signalisation mécanotransductive il y a plus de 20 ans et il a été démontré qu'elle est signalée par 15 récepteurs purinergiques différents (P2). Cependant, la contribution exacte de la libération d'ATP et de la signalisation P2 à l'adaptation osseuse induite mécaniquement demeure incertaine. Les principaux objectifs de cette thèse étaient d'étudier les mécanismes de la libération d'ATP stimulée mécaniquement par les cellules osseuses et de déterminer comment le signal purinergique transmet l'information sur la nature du stimulus mécanique aux cellules voisines.De nombreuses études portant sur les mécanismes de libération d'ATP stimulée mécaniquement ont été menées à ce jour. Comme aucune synthèse quantitative à grande échelle de la recherche fondamentale n'a été entreprise, j'ai mis au point les bases théoriques, les ressources informatiques et le déroulement du travail nécessaires pour effectuer une méta-analyse en sciences fondamentales. Cela a permis de consolider et de synthétiser les données quantitatives de 278 études portant sur la quantité, la cinétique et les mécanismes de libération de l'ATP, ainsi que l'influence des pathologies sur la libération de l'ATP. J'ai démontré que la libération d'ATP stimulée mécaniquement est un phénomène conservé dans les cellules de mammifères et que les cellules stimulées mécaniquement libèrent en moyenne 38,6 (IC 95 % : 18,2 à 81,8) amoles ATP/cellule avec une constante de temps caractéristique de 32 s (IC 95 % : 16 à 66). Il est important de noter que les vésicules et les canaux calciques sensibles au voltage ont été impliqués dans la libération d'ATP des ostéoblastes et ostéocytes stimulés mécaniquement. J'ai ensuite étudié expérimentalement les mécanismes de libération de l'ATP par les ostéoblastes. J'ai déterminé que la stimulation mécanique d'un seul ostéoblaste murin a entraîné la libération de 70 ± 24 amole ATP. Les ostéoblastes contiennent des vésicules riches en ATP qui sont libérées par stimulation mécanique, mais les interventions pharmacologiques qui favorisent l'exocytose vésiculaire réduisent la libération d'ATP, tandis que les inhibiteurs de la libération vésiculaire potentialisent la libération d'ATP. À la recherche d'une autre voie de libération de l'ATP, j'ai découvert que les contraintes mécaniques provoquaient des lésions réversibles de la membrane cellulaire in vitro et in vivo. L'exocytose vésiculaire dépendante du calcium et du PKC a facilité la réparation de la membrane, assurant ainsi la viabilité cellulaire et réduisant la libération d'ATP. Ainsi, je propose que l'exocytose des vésicules contenant de l'ATP limite le flux d'ATP intracellulaire à travers les membranes endommagées de façon réversible en facilitant la réparation des membranes. Dans l'ensemble, les études présentées ici démontrent que (i) la lésion des cellules osseuses est critique pour la mécanotransduction purinergique, (ii) la réparation membranaire se produit par exocytose vésiculaire dépendante du calcium/PKC, et (iii) l'information sur la gravité de la lésion et le statut mécano-adaptatif cellulaire est intégrée au niveau du signal purinergique. Il est important de noter que l'identification des médiateurs moléculaires, y compris la PKCµ, pourrait représenter des cibles thérapeutiques futures dans la prévention de la perte osseuse liée à l'utilisation

Effects of Long-Term Sclerostin Deficiency on Trabecular Bone Mass and Adaption to Limb Loading Differ in Male and Female Mice

A new therapeutic option to treat osteoporosis is focused on Wnt signaling and its inhibitor sclerostin, a product of the Sost gene. In this work, we study the effect of sclerostin deficiency on trabecular bone formation and resorption in male and female mice and whether it affects mechano-responsiveness. Male and female 10- and 26-week-old Sost knockout (KO) and littermate controls (LCs) were subjected to in vivo mechanical loading of the left tibia for 2 weeks. The right tibia served as internal control. The mice were imaged using in vivo micro-computed tomography at days 0, 5, 10, and 15 and tibiae were collected for histomorphometric analyses after euthanasia. Histomorphometry and micro-CT-based 3D time-lapse morphometry revealed an anabolic and anti-catabolic effect of Sost deficiency although increased trabecular bone resorption accompanied by diminished trabecular bone formation occurred with age. Loading led to diminished resorption in adult female but not in male mice. A net gain in bone volume could be achieved with mechanical loading in Sost KO adult female mice, which occurred through a further reduction in resorbed bone volume. Our data show that sclerostin deficiency has a particularly positive effect in adult female mice. Sclerostin antibodies are approved to treat postmenopausal women with high risk of osteoporotic fractures. Further studies are required to clarify whether both sexes benefit equally from sclerostin inhibition

Systematic Review of Cerebrospinal Fluid Biomarker Discovery in Neuro-Oncology: A Roadmap to Standardization and Clinical Application

Effective diagnosis, prognostication, and management of CNS malignancies traditionally involves invasive brain biopsies that pose significant risk to the patient. Sampling and molecular profiling of cerebrospinal fluid (CSF) is a safer, rapid, and noninvasive alternative that offers a snapshot of the intracranial milieu while overcoming the challenge of sampling error that plagues conventional brain biopsy. Although numerous biomarkers have been identified, translational challenges remain, and standardization of protocols is necessary. Here, we systematically reviewed 141 studies (Medline, SCOPUS, and Biosis databases; between January 2000 and September 29, 2022) that molecularly profiled CSF from adults with brain malignancies including glioma, brain metastasis, and primary and secondary CNS lymphomas. We provide an overview of promising CSF biomarkers, propose CSF reporting guidelines, and discuss the various considerations that go into biomarker discovery, including the influence of blood-brain barrier disruption, cell of origin, and site of CSF acquisition (eg, lumbar and ventricular). We also performed a meta-analysis of proteomic data sets, identifying biomarkers in CNS malignancies and establishing a resource for the research community

HR-pQCT measures of bone microarchitecture predict fracture:systematic review and meta-analysis

\u3cp\u3eHigh-resolution peripheral quantitative computed tomography (HR-pQCT) is a noninvasive imaging modality for assessing volumetric bone mineral density (vBMD) and microarchitecture of cancellous and cortical bone. The objective was to (1) assess fracture-associated differences in HR-pQCT bone parameters; and (2) to determine if HR-pQCT is sufficiently precise to reliably detect these differences in individuals. We systematically identified 40 studies that used HR-pQCT (39/40 used XtremeCT scanners) to assess 1291 to 3253 and 3389 to 10,687 individuals with and without fractures, respectively, ranging in age from 10.9 to 84.7 years with no comorbid conditions. Parameters describing radial and tibial bone density, microarchitecture, and strength were extracted and percentage differences between fracture and control subjects were estimated using a random effects meta-analysis. An additional meta-analysis of short-term in vivo reproducibility of bone parameters assessed by XtremeCT was conducted to determine whether fracture-associated differences exceeded the least significant change (LSC) required to discern measured differences from precision error. Radial and tibial HR-pQCT parameters, including failure load, were significantly altered in fracture subjects, with differences ranging from −2.6% (95% confidence interval [CI] −3.4 to −1.9) in radial cortical vBMD to −12.6% (95% CI −15.0 to −10.3) in radial trabecular vBMD. Fracture-associated differences reported by prospective studies were consistent with those from retrospective studies, indicating that HR-pQCT can predict incident fracture. Assessment of study quality, heterogeneity, and publication biases verified the validity of these findings. Finally, we demonstrated that fracture-associated deficits in total and trabecular vBMD and certain tibial cortical parameters can be reliably discerned from HR-pQCT-related precision error and can be used to detect fracture-associated differences in individual patients. Although differences in other HR-pQCT measures, including failure load, were significantly associated with fracture, improved reproducibility is needed to ensure reliable individual cross-sectional screening and longitudinal monitoring. In conclusion, our study supports the use of HR-pQCT in clinical fracture prediction.\u3c/p\u3

The proteomic landscape of glioblastoma recurrence reveals novel and targetable immunoregulatory drivers

Glioblastoma (GBM) is characterized by extensive cellular and genetic heterogeneity. Its initial presentation as primary disease (pGBM) has been subject to exhaustive molecular and cellular profiling. By contrast, our understanding of how GBM evolves to evade the selective pressure of therapy is starkly limited. The proteomic landscape of recurrent GBM (rGBM), which is refractory to most treatments used for pGBM, are poorly known. We, therefore, quantified the transcriptome and proteome of 134 patient-derived pGBM and rGBM samples, including 40 matched pGBM-rGBM pairs. GBM subtypes transition from pGBM to rGBM towards a preferentially mesenchymal state at recurrence, consistent with the increasingly invasive nature of rGBM. We identified immune regulatory/suppressive genes as important drivers of rGBM and in particular 2-5-oligoadenylate synthase 2 (OAS2) as an essential gene in recurrent disease. Our data identify a new class of therapeutic targets that emerge from the adaptive response of pGBM to therapy, emerging specifically in recurrent disease and may provide new therapeutic opportunities absent at pGBM diagnosis

Estimates of anthropogenic carbon uptake from four three-dimensional global ocean models

We have compared simulations of anthropogenic CO$_2$ in the four threedimensional ocean models that participated in the first phase of the Ocean Carbon-Cycle Model Intercomparison Project (OCMIP), as a means to identify their major differences.Simulated global uptake agrees to within $\pm$19%, giving a range of 1.85$\pm$0.35 PgC yr$^{-1}$ for the 1980-1989 average. Regionally, the Southern Ocean dominates the present-day air-sea flux of anthropogenic CO$_2$ in all models, with one third to one half of the global uptake occurring south of 30°S. The highest simulated total uptake in the Southern Ocean was 70% larger than the lowest. Comparison with recent data-based estimates of anthropogenic CO$_2$ suggesthat most of the models substantially overestimate storage in the Southern Ocean; elsewhere they generally underestimate storage by less than 20%. Globally, the OCMIP models appear to bracket the real ocean's present uptake, based on comparison of regional data-basedstimates of anthropogenic CO$_2$ and bomb $^{14}$C. Column inventories of bomb $^{14}$C have become more similar to those for anthropogenic CO$_2$ with the time that has elapsed between the Geochemical Ocean Sections Study (1970s) and Word Ocean Circulation Experiment (1990s) global sampling campaigns. Our ability to evaluate simulated anthropogenic CO$_2$ would improve if systematic errors associated with the data-based estimates could be provided regionally