Blind spots on western blots: Assessment of common problems in western blot figures and methods reporting with recommendations to improve them

Western blotting is a standard laboratory method used to detect proteins and assess their expression levels. Unfortunately, poor western blot image display practices and a lack of detailed methods reporting can limit a reader's ability to evaluate or reproduce western blot results. While several groups have studied the prevalence of image manipulation or provided recommendations for improving western blotting, data on the prevalence of common publication practices are scarce. We systematically examined 551 articles published in the top 25% of journals in neurosciences (n = 151) and cell biology (n = 400) that contained western blot images, focusing on practices that may omit important information. Our data show that most published western blots are cropped and blot source data are not made available to readers in the supplement. Publishing blots with visible molecular weight markers is rare, and many blots additionally lack molecular weight labels. Western blot methods sections often lack information on the amount of protein loaded on the gel, blocking steps, and antibody labeling protocol. Important antibody identifiers like company or supplier, catalog number, or RRID were omitted frequently for primary antibodies and regularly for secondary antibodies. We present detailed descriptions and visual examples to help scientists, peer reviewers, and editors to publish more informative western blot figures and methods. Additional resources include a toolbox to help scientists produce more reproducible western blot data, teaching slides in English and Spanish, and an antibody reporting template

Investigating the effect of anti-inflammatory hyaluronic acid towards regenerating the annulus fibrosus

Neck and low-back pain are major causes of disability leading to a serious socio-economic burden worldwide. The pathology is strongly correlated with the intervertebral disc degeneration (IVD). Disc degenerative disease (DDD) is described as lack of cellularity and transformation in the extracellular matrix (ECM) composition, which result in adverse changes in the IVD tissue. The lack of a full understanding of the aetiology underlying the DDD in the last decades has promoted the exploration of new tissue engineering approaches to restore the disc function by identifying new inflammatory molecular markers and signalling pathways to target the disc degenerative disease. Therefore, the ultimate goal of this thesis was to identify molecular markers and signaling pathways to develop an optimal anti-inflammatory platform using an ECM- mimicking injectable hydrogel that enhances the cell survival and boosts the synthesis and the deposition of the ECM to regenerate the annulus fibrosus (AF) tissue. It was hypothesised that high molecular weight HA (HMW HA) will act as an anti-inflammatory molecule by down-regulating IFNα signalling, enhancing cell survival by decreasing Interferon-induced Protein with tetratricopeptide repeat 3 (IFIT3) and IGF-binding Protein 3 (IGFBP3) as well as Caspase 3 and as a matrix modulatory by up-regulating the production of the matrix proteins such as aggrecan, collagen I (COLI) and hyaluronan and proteoglycan link protein 1 (HAPLN1). In the first phase of this thesis, Interferon α2β (IFNα2b) was identified as the most dysregulated inflammatory signalling pathway analysing a range of downstream molecular markers comparing a microarray data from degenerative and non-degenerative human annulus fibrosus tissue. Consequently, an injectable HMW HA hydrogel was developed and evaluated as an anti-inflammatory biomolecule in the second phase. The hyaluronan hydrogel was evaluated in bovine organ culture system to investigate its capability to act as anti- inflammatory and to support extracellular matrix synthesis. In addition, this hydrogel has been further tested in an in vivo rat tail disc injury model to identify its anti-inflammatory and matrix modulatory properties in addition to its role in regulating the glyco-environment. The results obtained in this study highlighted the significance of hyaluronan in reducing inflammation by down-regulating IFNα and its signalling molecules, interferon α receptor 1 (IFNAR1) and interferon α receptor 2 (IFNAR2), signal transducer and activator of transcription 1 (STAT1), Signal transducer and activator of transcription 2 (STAT2) and janus kinase 1 (JAK1). In addition to down-regulating the anti-proliferative IGFBP3 and anti-apoptotic IFIT3 which resulted in lower caspase 3 expression. On the other hand, results have shown that annulus fibrosus ECM key components were up-regulated such as aggrecan, COLI and HAPLN1 which are major proteins contributing in the normal organization of the matrix. Hence, the glyco-environment of the IVD was evaluated as an attempt to recognise the influence of HA on the IVD glyco- environment comparing to the healthy and injured tissue. A set of specific and selective lectins were used to distinguish the switch in glycans of AF in healthy tissue, inflammation and hyaluronan treatment. The major finding in this phase of the study was that all sialylation highlighting motifs were down-regulated in healthy and hyaluronan treated tissue in contrast to injured tissue. CS composition and quantity of chondroitin sulfates (CS) revealed a change in sulfation pattern of CS on day 7 and 28 where C0S was significantly up- regulated on day 28 and similarly C4S on day 7. The results obtained from this thesis show that hyaluronan acts as an anti-inflammatory and matrix modulatory molecule in an animal model which is closer in disc dimensions and degenerative process to that of humans.2019-05-1

A review of the biomarkers and in vivo models for the diagnosis and treatment of heterotopic ossification following blast and trauma-induced injuries

Heterotopic ossification (HO) is the process of de novo bone formation in non-osseous tissues. HO can occur following trauma and burns and over 60% of military personnel with blast-associated amputations develop HO. This rate is far higher than in other trauma-induced HO development. This suggests that the blast effect itself is a major contributing factor, but the pathway triggering HO following blast injury specifically is not yet fully identified. Also, because of the difficulty of studying the disease using clinical data, the only sources remain the relevant in vivo models. The aim of this paper is first to review the key biomarkers and signalling pathways identified in trauma and blast induced HO in order to summarize the molecular mechanisms underlying HO development, and second to review the blast injury in vivo models developed. The literature derived from trauma-induced HO suggests that inflammatory cytokines play a key role directing different progenitor cells to transform into an osteogenic class contributing to the development of the disease. This highlights the importance of identifying the downstream biomarkers under specific signalling pathways which might trigger similar stimuli in blast to those of trauma induced formation of ectopic bone in the tissues surrounding the site of the injury. The lack of information in the literature regarding the exact biomarkers leading to blast associated HO is hampering the design of specific therapeutics. The majority of existing blast injury in vivo models do not fully replicate the combat scenario in terms of blast, fracture and amputation; these three usually happen in one insult. Hence, this paper highlights the need to replicate the full effect of the blast in preclinical models to better understand the mechanism of blast induced HO development and to enable the design of a specific therapeutic to supress the formation of ectopic bone

The role of hyaluronic acid in intervertebral disc regeneration

Intervertebral disc (IVD) degeneration is a leading cause of low back pain worldwide, incurring a significant burden on the healthcare system and society. IVD degeneration is characterized by an abnormal cell-mediated response leading to the stimulation of different catabolic biomarkers and activation of signalling pathways. In the last few decades, hyaluronic acid (HA), which has been broadly used in tissue-engineering, has popularised due to its anti-inflammatory, analgesic and extracellular matrix enhancing properties. Hence, there is expressed interest in treating the IVD using different HA compositions. An ideal HA-based biomaterial needs to be compatible and supportive of the disc microenvironment in general and inhibit inflammation and downstream cascades leading to the innervation, vascularisation and pain sensation in particular. High molecular weight hyaluronic acid (HMW HA) and HA-based biomaterials used as therapeutic delivery platforms have been trialled in preclinical models and clinical trials. In this paper, we reviewed a series of studies focused on assessing the effect of different compositions of HA as a therapeutic, targeting IVD degeneration. Overall, tremendous advances have been made towards an optimal form of a HA biomaterial to target specific biomarkers associated with IVD degeneration, but further optimization is necessary to address regeneration

Replacing bar graphs of continuous data with more informative graphics: Are we making progress?

Barzooka is an automated screening tool designed to detect 9 graph types in scientific publications: 1) bar graphs of counts and proportions, 2) bar graphs of continuous data, 3) bar graphs combined with dot plots, 4) dot plots, 5) box plots, 6) violin plots, 7) histograms, 8) pie charts, and 9) study flow charts. This project contains data and code for two external datasets used to validate Barzooka, and one dataset used to examine changes in graph use from 2010 to 2020 in 23 fields

Development of a rodent high energy blast injury model for investigating conditions associated with traumatic amputations

In recent conflicts, most injuries to the extremities are due to blast resulting in a large number of lower limb amputations. These lead to heterotopic ossification (HO), phantom limb pain (PLP), and functional deficit. The mechanism of blast loading produces a combined facture and amputation. Therefore, to study these conditions, in vivo models that replicate this combined effect are required. The aim of this study is to develop a preclinical model of blast-induced lower limb amputation

Autologous protein solution - a promising solution for osteoarthritis?

Osteoarthritis (OA) is a global health issue with myriad pathophysiological factors and is one of the most common causes of chronic disability in adults due to pain and altered joint function. The end stage of OA develops from a destructive inflammatory cycle, driven by the pro-inflammatory cytokines interleukin-1β (IL-1β) and tumour necrosis factor alpha (TNFα). Owing to the less predictable results of total knee arthroplasty (TKA) in younger patients presenting with knee OA, there has been a surge in research evaluating less invasive biological treatment options, one of which is autologous protein solution (APS). APS is an autologous blood derivative obtained by using a proprietary device, made of APS separator, which isolates white blood cells (WBCs) and platelets in a small volume of plasma, and APS concentrator, which further concentrates platelets, WBCs and plasma proteins, resulting in a concentrated solution with high levels of growth factors including the anti-inflammatory mediators against IL-1β and TNFα. A single intraarticular injection of APS appears to be a promising solution for treatment of early-stage OA from current evidence, the majority of which comes from preclinical studies. More clinical studies are needed before APS can be widely accepted as a treatment modality for OA. Cite this article: EFORT Open Rev 2021;6:716-726. DOI: 10.1302/2058-5241.6.20004

Development of a blast injury model for investigating conditions associated with traumatic amputations

INTRODUCTION: Most injuries in recent conflicts are due to blast, 70% of which are to the extremities resulting in a large number of lower limb amputations. Functional deficits due to blast induced amputation include difficulty in weight bearing and associated normal gait abnormali-ties. Significant complications following traumatic amputation are pain in the residual limb, and phantom limb pain. Heterotopic Ossification (HO) - ectopic bone formation in the soft tissues - is also highly prevalent (64%) among blast-related military amputations. The existing non-specific treatments include non-steroidal anti-inflammatory drugs (NSAID)s and low-dose radiation therapy which remain unsatisfactory leav-ing surgical bone excision the only possible curative treatment. While the prevention of HO in military amputees is the ultimate choice of treat-ment, it is yet to be identified, as the initial cause of triggering the disease is not understood. For this reason, and because studying amputation complications in humans is difficult, novel in vivo models need to be developed for further understanding of the disease mechanisms. There-fore, we hypothesised that developing a preclinical blast injury model in the hindlimb of rats which better represents the IED detonation in en-closed spaces could answer questions regarding the exact mechanism of HO and phantom limb pain. Current in vivo models exist, but none of these incorporate all blast features, that is, the blast, and the fracture in one insult. This research aims to develop a novel translational blast injury model in rats to better understand the mechanisms of phantom limb pain and HO. METHODS: This study was performed under institutional and departmental license from the Home Office UK. In line with the 3Rs principle, optimisation of the blast pressure was achieved using 34 male cadaveric Sprague-Dawley rats weighing between 285-481g to refine the experi-ments without using live animals to achieve a trans-tibial fracture at the left hindlimb utilizing different burst pressures (7-13bar). The rats were placed on a special harness which supported and protected the rest of the body exposing only the left hind limb to different blast waves gener-ated by a shock tube. Both blast and blast associated fracture were induced in one experiment. The tibial fracture was evaluated by x-ray and confirmed by dissection. RESULTS: Different blast pressure parameters showed that the blast waves followed a normal pattern including peak pressure, positive pres-sure duration followed by a negative under-pressure and consequent return to the ambient pressure (Figure 1A). The peak pressure, impulse and the positive duration decreased predictably (Figure 1B,1C, 1D) by reducing the blast pressure. 13 and 12 bar pressures produced multiple fractures in the hip, femur and tibia as well as above and below knee fractures on both limbs (Figure 2) rendering these pressures inappropriate for translation into a survivable model. Experiments at 11 and 10 bar caused consistent above (undesirable) and below (desirable) knee frac-tures on both (undesirable) limbs. Reducing the pressure to 9 bar resulted in 75% reduction in the injuries to the right limb and 62.5% reduction on the left limb, producing highly consistent isolated unilateral tibial fractures in most cases. The anomalies were in the smaller animals (285-300 g) that resulted in 25% and 37.5% undesired injuries in the right and left limbs, respectively. 7 bar pressure induced no fractures. DISCUSSION: In this study we developed a blast injury model in the left rat hindlimb which can be translated into a survivable model to study conditions following blast-induced amputation. Animal size was a significant factor and thus, small animals should be excluded from in vivo studies. This study recommends 9 bar pressure in medium-sized male Sprague-Dawley rats (320-450g) within a custom shock tube and har-ness to achieve consistent blast-induce isolated unilateral fractures of the tibia. This is the first model that combines the blast and the fracture in a single insult without using a drop weight to separately induce the fracture, therefore, better simulating the battlefield scenario. The translation of this model opens up new avenues to explore the systemic serum biomarkers and to identify novel signalling pathways associated with HO initiation and progression following blast injuries and could potentially be used to analyse other blast-related conditions. SIGNIFICANCE: Developing a preclinical blast injury model is key for understanding the mechanism of HO suffered by military personnel following blast injuries. This will lead to novel diagnostic tools in the clinic and specific therapeutic design that will prevent HO, thus improving veterans’ quality of life