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BioMed Research International 2014Western blotting is a technique that has been in practice for more than three decades that began as a means of detecting a protein target in a complex sample. Although... (Review)
Review
Western blotting is a technique that has been in practice for more than three decades that began as a means of detecting a protein target in a complex sample. Although there have been significant advances in both the imaging and reagent technologies to improve sensitivity, dynamic range of detection, and the applicability of multiplexed target detection, the basic technique has remained essentially unchanged. In the past, western blotting was used simply to detect a specific target protein in a complex mixture, but now journal editors and reviewers are requesting the quantitative interpretation of western blot data in terms of fold changes in protein expression between samples. The calculations are based on the differential densitometry of the associated chemiluminescent and/or fluorescent signals from the blots and this now requires a fundamental shift in the experimental methodology, acquisition, and interpretation of the data. We have recently published an updated approach to produce quantitative densitometric data from western blots (Taylor et al., 2013) and here we summarize the complete western blot workflow with a focus on sample preparation and data analysis for quantitative western blotting.
Topics: Blotting, Western; Densitometry; Luminescent Measurements; Proteins; Statistics as Topic
PubMed: 24738055
DOI: 10.1155/2014/361590 -
Nutrients Mar 2021Whilst the assessment of body composition is routine practice in sport, there remains considerable debate on the best tools available, with the chosen technique often... (Review)
Review
Whilst the assessment of body composition is routine practice in sport, there remains considerable debate on the best tools available, with the chosen technique often based upon convenience rather than understanding the method and its limitations. The aim of this manuscript was threefold: (1) provide an overview of the common methodologies used within sport to measure body composition, specifically hydro-densitometry, air displacement plethysmography, bioelectrical impedance analysis and spectroscopy, ultra-sound, three-dimensional scanning, dual-energy X-ray absorptiometry (DXA) and skinfold thickness; (2) compare the efficacy of what are widely believed to be the most accurate (DXA) and practical (skinfold thickness) assessment tools and (3) provide a framework to help select the most appropriate assessment in applied sports practice including insights from the authors' experiences working in elite sport. Traditionally, skinfold thickness has been the most popular method of body composition but the use of DXA has increased in recent years, with a wide held belief that it is the criterion standard. When bone mineral content needs to be assessed, and/or when it is necessary to take limb-specific estimations of fat and fat-free mass, then DXA appears to be the preferred method, although it is crucial to be aware of the logistical constraints required to produce reliable data, including controlling food intake, prior exercise and hydration status. However, given the need for simplicity and after considering the evidence across all assessment methods, skinfolds appear to be the least affected by day-to-day variability, leading to the conclusion 'come back skinfolds, all is forgiven'.
Topics: Absorptiometry, Photon; Athletes; Body Composition; Densitometry; Electric Impedance; Humans; Plethysmography; Reproducibility of Results; Skinfold Thickness; Spectrum Analysis; Sports; Ultrasonography
PubMed: 33806245
DOI: 10.3390/nu13041075 -
BioMed Research International 2019Densitometry data generated for Western blots are commonly used to compare protein abundance between samples. In the last decade, it has become apparent that assumptions...
Densitometry data generated for Western blots are commonly used to compare protein abundance between samples. In the last decade, it has become apparent that assumptions underpinning these comparisons are often violated in studies reporting Western blot data in the literature. These violations can lead to erroneous interpretations of data and may contribute to poor reproducibility of research. We assessed the reliability of Western blot data obtained to study human myometrial tissue proteins. We ran dilution series of protein lysates to explore the linearity of densitometry data. Proteins analysed included SMA, HSP27, ERK1/2, and GAPDH. While ideal densitometry data are directly proportional to protein abundance, our data confirm that densitometry data often deviate from this ideal, in which case they can fit nonproportional linear or hyperbolic mathematical models and can reach saturation. Nonlinear densitometry data were observed when Western blots were detected using infrared fluorescence or chemiluminescence, and under different SDS-PAGE conditions. We confirm that ghosting artefacts associated with overabundance of proteins of interest in Western blots can skew findings. We also confirm that when data to be normalised are not directly proportional to protein abundance, it is a mistake to use the normalisation technique of dividing densitometry data from the protein-of-interest with densitometry data from loading control protein(s), as this can cause the normalised data to be unusable for making comparisons. Using spiked proteins in a way that allowed us to control the total protein amount per lane, while only changing the amount of spiked proteins, we confirm that nonlinearity and saturation of densitometry data, and errors introduced from normalisation processes, can occur in routine assays that compare equal amounts of lysate. These findings apply to all Western blot studies, and we highlight quality control checks that should be performed to make Western blot data more quantitative.
Topics: Blotting, Western; Densitometry; Electrophoresis, Polyacrylamide Gel; Humans; Proteins; Reproducibility of Results
PubMed: 30800670
DOI: 10.1155/2019/5214821 -
Nutrients Apr 2022The historical 1975 Reference Man is a ‘model’ that had been used as a basis for the calculation of radiation doses, metabolism, pharmacokinetics, sizes for organ... (Review)
Review
The historical 1975 Reference Man is a ‘model’ that had been used as a basis for the calculation of radiation doses, metabolism, pharmacokinetics, sizes for organ transplantation and ergonomic optimizations in the industry, e.g., to plan dimensions of seats and other formats. The 1975 Reference Man was not an average individual of a population; it was based on the multiple characteristics of body compositions that at that time were available, i.e., mainly from autopsy data. Faced with recent technological advances, new mathematical models and socio-demographic changes within populations characterized by an increase in elderly and overweight subjects a timely ‘state-of-the-art’ 2021 Reference Body are needed. To perform this, in vivo human body composition data bases in Kiel, Baton Rouge, San Francisco and Honolulu were analyzed and detailed 2021 Reference Bodies, and they were built for both sexes and two age groups (≤40 yrs and >40 yrs) at BMIs of 20, 25, 30 and 40 kg/m2. We have taken an integrative approach to address ‘structure−structure’ and ‘structure−function’ relationships at the whole-body level using in depth body composition analyses as assessed by gold standard methods, i.e., whole body Magnetic Resonance Imaging (MRI) and the 4-compartment (4C-) model (based on deuterium dilution, dual-energy X-ray absorptiometry and body densitometry). In addition, data obtained by a three-dimensional optical scanner were used to assess body shape. The future applications of the 2021 Reference Body relate to mathematical modeling to address complex metabolic processes and pharmacokinetics using a multi-level/multi-scale approach defining health within the contexts of neurohumoral and metabolic control.
Topics: Absorptiometry, Photon; Adipose Tissue; Adult; Aged; Body Composition; Body Water; Female; Humans; Magnetic Resonance Imaging; Male; Whole Body Imaging
PubMed: 35406138
DOI: 10.3390/nu14071526 -
Canadian Association of Radiologists... Aug 2017
Review
Topics: Absorptiometry, Photon; Bone Density; Female; Gender Identity; Humans; Male; Osteoporosis; Transgender Persons
PubMed: 28396004
DOI: 10.1016/j.carj.2016.10.006 -
International Ophthalmology Aug 2021To evaluate corneal and crystalline lens densitometry in patients with vernal keratoconjunctivitis (VKC).
AIM
To evaluate corneal and crystalline lens densitometry in patients with vernal keratoconjunctivitis (VKC).
METHODS
This is a case-control study. Patients with VKC and age-gender-matched healthy controls underwent a complete ophthalmologic examination with corneal and crystalline lens densitometry measurements. Additionally, the anterior chamber parameters comprise anterior and posterior K and astigmatism, anterior chamber depth (ACD), anterior chamber volume (ACV), anterior chamber angle (ACA), pachymeter, and corneal volume (CV). Patients who had clinically grade 0 or grade 1 VKC and with only tarsal conjunctiva involvement during the conductance of the study were included. The variables were compared statistically.
RESULTS
One hundred and nine eyes were included in the study, in which fifty-one were in the VKC group. There were 25 males in the VKC group (26 female) and 22 males in the control group (36 female). A statistically significant difference was found between the groups in terms of posterior corneal astigmatism (p = 0.02). The mean corneal pachymeter, CV, ACD, ACA, and ACV were similar in both groups (p = 0.63, p = 0.26, p = 0.60, p = 0.41, and p = 0.32, respectively). The total mean corneal densitometry in the zones extending from 6 to 10 mm and 10 to 12 mm was increased in the VKC group compared to the control group (p = 0.04 and p = 0.012, respectively). The mean crystalline lens was found to be denser in the VKC group compared to the control group (8.96 ± 1.6 vs. 8.5 ± 0.57, respectively, p = 0.04).
CONCLUSION
Posterior corneal astigmatism is increased in VKC cases in comparison with age- and gender-matched controls. The peripheral anterior 6-12 mm annular corneal zone showed increased corneal densitometry in VKC cases compared to the healthy subjects. Additionally, the lens clarity is found to be decreased subclinically in VKC cases compared to control cases.
Topics: Case-Control Studies; Conjunctivitis, Allergic; Cornea; Densitometry; Female; Humans; Lens, Crystalline; Male
PubMed: 33754236
DOI: 10.1007/s10792-021-01822-0 -
Reumatologia Clinica 2020One of the missions of the Spanish Society of Rheumatology is to provide the necessary tools for excellence in health care. Currently, there is no reference point to...
INTRODUCTION
One of the missions of the Spanish Society of Rheumatology is to provide the necessary tools for excellence in health care. Currently, there is no reference point to quantify medical actions in this specialty, and this is imperative.
MATERIAL AND METHOD
A list of actions was drawn up and a hierarchical classification system was established by developing a complexity index, calculated based on the completion time and difficulty level of each action.
RESULTS
The results of the Delphi method tended to the consensus opinion within a group (mean σ2 - σ1=0.75-1.43=-0.68, mean IQR2 - IQR1=0.8-1.9=-1.1). The values of the complexity index ranged between 48 and 465 points. Among consultation actions, those reaching the highest scores were the first inpatient visit (366) and visits to the patient's home (369). Among diagnostic techniques, biopsies were prominent, those with the highest score were: bone biopsy (465), sural nerve biopsy (416) and synovial biopsy (380). Ultrasound scan scored 204, capillaroscopy 113 and densitometry 112. Among therapeutic techniques, infiltration/ arthrocentesis/articular injection in children reached the highest difficulty (388). The score for ultrasound-guided articular injection was 163. The score for clinical report on disability was 323 and expert report 370.
CONCLUSIONS
A nomenclature of 54 actions in Rheumatology was compiled. Biopsies (bone, sural nerve, synovial), inpatient visits, visits to the patient's home, infiltrations in children, and the preparation of the expert report were identified as the most complex actions. Musculoskeletal ultrasound is twice as complex as subsequent visits, capillaroscopy or bone densitometry.
Topics: Arthrocentesis; Biopsy; Bone and Bones; Delphi Technique; Densitometry; House Calls; Humans; Injections, Intra-Articular; Inpatients; Microscopic Angioscopy; Rheumatology; Sural Nerve; Synovial Membrane; Time Factors; Ultrasonography
PubMed: 30745278
DOI: 10.1016/j.reuma.2018.11.010 -
Clinical & Experimental Optometry Mar 2021Corneal and lens densitometry measurements provide clinically important information for the evaluation and monitoring of corneal and lens health in patients with vernal...
CLINICAL RELEVANCE
Corneal and lens densitometry measurements provide clinically important information for the evaluation and monitoring of corneal and lens health in patients with vernal keratoconjunctivitis.
BACKGROUND
To compare the corneal and lens densitometry values between paediatric patients with vernal keratoconjunctivitis (VKC) and healthy individuals.
METHODS
This study included 72 eyes of 72 patients with VKC (25 with mild VKC [Group 1], 22 with moderate VKC [Group 2], and 25 with severe VKC [Group 3]), and 25 eyes of 25 healthy subjects (Group 4). Corneal and lens densitometry values were measured using Pentacam HR as follows: for corneal densitometry in two different corneal zones (0-2 and 2-6-mm) and four different corneal depths (at the total thickness, anterior, central, and posterior layers), and lens densitometry in three different lens zones (Zone 1: 2.0-mm, Zone 2: 4.0-mm, and Zone 3: 6.0-mm).
RESULTS
In the 0-2-mm corneal zone for the total thickness and all three layers, corneal densitometry values in Group 3 were significantly higher than those in Groups 1, 2, and 4 (for all values p < 0.012). There was no significant difference in the mean corneal densitometry values between Groups 1 and 2 (for all values p > 0.05). In these groups, the mean corneal densitometry values were significantly higher than those in Group 4 for the anterior layer in the 0-2 and 2-6-mm corneal zones (for all values p < 0.012). The mean values for Zone 3 and average lens densitometry values in Groups 2 and 3 were significantly higher than those in Group 4 (p = 0.001 and p = 0.001, respectively).
CONCLUSION
The current study showed corneal clarity changes in patients with mild, moderate, and especially severe VKC. An increase in the lens densitometry values was also observed in patients with moderate and severe VKC than in healthy individuals.
Topics: Child; Conjunctivitis, Allergic; Cornea; Densitometry; Humans; Lens, Crystalline
PubMed: 32945010
DOI: 10.1111/cxo.13144 -
Ophthalmic & Physiological Optics : the... Sep 2022To investigate whether Pentacam densitometry readings are affected by corneal tilt.
PURPOSE
To investigate whether Pentacam densitometry readings are affected by corneal tilt.
METHODS
In a prospective study, the right eyes of 86 healthy participants aged 42.8 ± 20.0 years (range 18-79 years) were imaged using Scheimpflug tomography. Elevation maps were exported to calculate corneal tilt using custom-made software, and densitometry readings were acquired directly from the corneal densitometry analysis add-on to the standard software Oculus Pentacam HR. Simple mediation analysis was applied to study age as a confounding factor in the correlation between corneal tilt and corneal densitometry.
RESULTS
Corneal tilt and corneal densitometry are not independent from one another because age is significantly correlated with both corneal tilt (r = 0.50, p < 0.001) and corneal densitometry (r = 0.91, p < 0.001). Only 3.8% of the correlation between tilt and densitometry operates directly, while the remaining 96.2% depends on age.
CONCLUSIONS
Corneal tilt plays a role in corneal densitometry readings, even though the interaction is strongly influenced by age. Age is a well-known factor in densitometry readings that should be taken into consideration when interpreting Scheimpflug densitometry.
Topics: Cornea; Corneal Topography; Densitometry; Healthy Volunteers; Humans; Prospective Studies
PubMed: 35708180
DOI: 10.1111/opo.13020 -
Acta Reumatologica Portuguesa 2011To assess the utility of quantitative ultrasound (QUS) of the calcaneus for diagnosing osteoporosis compared to the gold standard, bone densitometry using dual-emission... (Review)
Review
OBJECTIVE
To assess the utility of quantitative ultrasound (QUS) of the calcaneus for diagnosing osteoporosis compared to the gold standard, bone densitometry using dual-emission X-ray absorptiometry (DXA), according to published reports.
DESIGN
In this systematic review, the Medline/PUBMED, Medline Ovid and Journals@Ovid, and Wilson General Sciences Full Text database were used. The search strategy involved use of the following MeSH descriptors: [osteoporosis AND (densitometry OR ultrasonography)], and 39 articles published between 2001 and April 2010 were assessed. However, only six articles met the inclusion criteria: sensitivity and specificity of QUS, sample (women or men with no treatment or other disease likely to change bone mass index), devices used, comparative T-score between QUS of the calcaneus and DXA. The GE-Lunar Achilles and Hologic Sahara devices were used in most of the tests reported and were effective.
RESULTS
All studies assessed compared QUS of the calcaneus to DXA of the lumbar spine or femoral neck, as the gold standard. QUS sensitivity ranged from 79% to 93% and specificity ranged from 28% to 90% when at the lower threshold. It is a controversial parameter, because the gold-standard threshold (T-score < -2.5, DXA) could not be used for QUS without errors in osteoporosis diagnosis. All studies had a threshold determined by the authors’ criteria, with a variability of -1.7 (pDXA T--score) and -2.4 for QUS, leading to the same prevalence of osteoporosis, and a T-score of < -3.65 for QUS was equivalent to a T-score < -2.5 for DXA.
CONCLUSIONS
Based on the analysis of seven studies, we conclude that QUS of the calcaneus still cannot be used to confirm diagnosis of osteoporosis by comparing the results to those of patients who had already received such a diagnosis based on DXA. However, further research should be conducted in this area, because it is possible to improve the number diagnoses by varying the cutoff T-score. Furthermore, using QUS of the calcaneus was a helpful tool for assessing pathological fractures, whether or not they were associated with osteoporosis.
Topics: Absorptiometry, Photon; Bone Density; Calcaneus; Humans; Osteoporosis; Ultrasonography
PubMed: 22472924
DOI: No ID Found