Methods and Protocols

Osteonecrosis of the femoral head (ONFH) occurs frequently in adolescents and young adults and causes progressive deformation and destruction of the hip joint and impairs standing and walking, resulting in a significant decrease in the quality of life of patients. In addition, studies have shown that a history of corticosteroid administration and heavy alcohol consumption are closely related to the occurrence of ONFH. However, the detailed mechanism by which steroid administration and alcohol consumption are associated with the development of the disease is still unknown. With many researches still ongoing and without a clear biological pathway for osteonecrosis, effective preventive measures cannot be taken. Therefore, the current focus of ONFH treatment is to establish an early diagnosis and treatment strategy. We obtained the femoral heads of four patients with steroidal ONFH and three patients with alcoholic ONFH. We then compared the femoral heads of steroidal and alcoholic osteonecrosis by analyzing them at the molecular level by Raman spectroscopy. Crystallographic changes (deformations) in the mineral phase and fraction of organic material respect to the total mass were then plotted as a function. We found that changes in bone composition in ONFH were different in steroidal and alcoholic ONFH. We conclude that this suggests that the developmental mechanisms of steroidal and alcoholic ONFH may follow different paths. We also noticed that while steroid seem to lead to a more marked degradation of the tissue, alcohol seem to affect also the quality of the healthy tissue. Full article

Background: The most used types of retention of implant-supported prostheses are screw-retained or cement-retained restorations. The advantages and disadvantages of both have been identified by various authors over the years. However, cement-retained implant crowns and fixed partial dentures are among the most used types of restorations in implant prostheses, due to their aesthetic and clinical advantages. When cemented prostheses are made on implants, the problem of cement residues is important and often associated with biological implant pathologies. The objective of this research was to establish to what extent the techniques to reduce excess cement really affect the volume of cement residues. Materials and Methods: This review was written following the PRISMA statement; a detailed search was carried out in three different electronic databases—PubMed, Scopus, and Cochrane Library. The inclusion criteria were prospective clinical studies, with at least 10 participants per group, and with at least 6 months of the follow-up period. Results: There have been many proposals for techniques supposed to reduce the amount of excess cement in the peri-implant sulcus and on the prosthetic components, but of these, which are exceptional in their in vitro capabilities, very few have been clinically validated, and this represents the real limitation and a great lack of knowledge regarding this topic. Three articles met the inclusion criteria, which were analyzed and compared, to obtain the information necessary for the purposes of the systematic review. Discussion: Extraoral cementation can reduce the excess cement, which, after a normal excess removal procedure, is, nevertheless, of such size that it does not affect the possibility of peri-implant pathologies developing. All these studies concluded that a small amount of cement residue is found in the gingival sulcus, and using eugenol-free oxide cements, the residues were only deposited on the metal surfaces, with a better peri-implant tissues health. Conclusion: Despite the limitations of this study, it was possible to carefully analyze these characteristics and obtain valuable suggestions for daily clinical practice. Resinous cements are considered, due to the free monomers present in them, toxic for the soft tissues. The provisional zinc-oxide cements, also eugenol-free, represent the ideal choice. The different grades of retentive forces provided by these cements do not seem to have clinical effects on the decementation of restorations. Full article

New biomaterials and scaffolds for bone tissue engineering (BTE) applications require to be tested in a bone microenvironment reliable model. On this assumption, the in vitro laboratory protocols with bone cells represent worthy experimental systems improving our knowledge about bone homeostasis, reducing the costs of experimentation. To this day, several models of the bone microenvironment are reported in the literature, but few delineate a protocol for testing new biomaterials using bone cells. Herein we propose a clear protocol to set up an indirect co-culture system of human-derived osteoblasts and osteoclast precursors, providing well-defined criteria such as the cell seeding density, cell:cell ratio, the culture medium, and the proofs of differentiation. The material to be tested may be easily introduced in the system and the cell response analyzed. The physical separation of osteoblasts and osteoclasts allows distinguishing the effects of the material onto the two cell types and to evaluate the correlation between material and cell behavior, cell morphology, and adhesion. The whole protocol requires about 4 to 6 weeks with an intermediate level of expertise. The system is an in vitro model of the bone remodeling system useful in testing innovative materials for bone regeneration, and potentially exploitable in different application fields. The use of human primary cells represents a close replica of the bone cell cooperation in vivo and may be employed as a feasible system to test materials and scaffolds for bone substitution and regeneration. Full article

Background: Population studies gathering measured data on fitness and physical behavior, covering physical activity, standing, sedentary behavior, and time in bed, are scarce. This article describes the protocol of the FINFIT 2021 study that measures fitness and physical behavior in a population-based sample of adults and analyzes their associations and dose–response relationships with several health indicators. Methods: The study comprises a stratified random sample of 20–69-year-old men and women (n = 16,500) from seven city-centered regions in Finland. Physical behavior is measured 24/7 by tri-axial accelerometry and analyzed with validated MAD-APE algorithms. Health and fitness examinations include fasting blood samples, measurements of blood pressure, anthropometry, and health-related fitness. Domains of health, functioning, well-being, and socio-demographics are assessed by a questionnaire. The data are being collected between September 2021 and February 2022. Discussion: The study provides population data on physical fitness and physical behavior 24/7. Physical behavior patterns by intensity and duration on an hour-by-hour basis will be provided. In the future, the baseline data will be assessed against prospective register-based data on incident diseases, healthcare utilization, sickness absence, premature retirement, and death. A similar study will be conducted every fourth year with a new random population sample. Full article

Human cytomegalovirus is a herpesvirus that has a worldwide seroprevalence of more than 60% of adults in developed countries and 90% in developing countries. Severe disabilities in newborns are characteristic of the human cytomegalovirus congenital infection, and this virus is implicated in graft rejection in transplant patients. To treat and follow-up the infection, the CMVPCR viral loads are required, and the DNA extraction step remains very important; however, the quantity, quality, and purity of extracted DNA from different biological fluids influence the results of PCR amplification, that is why for reliable results, the choice of nucleic acid extraction methods requires careful attention. Materials and methods: In this study, we compare 4 protocols, I (EZ1 DSP Virus kit), II (EZ1 Virus mini kit), III (QIAamp DSP virus kit), and IV (heating); the extractions are made from plasma collected on EDTA tubes, and the concentration of extracted DNA was measured on NanoDrop Lite followed by real-time CMVPCR using an Artus CMV QS-RGQ kit. All protocols are performed following the manufacturer’s instructions. Results: This study is conducted on the samples of 135 transplant patients whose follow-up medical tests related to human cytomegalovirus infection; since most of the CMVPCR results are negative, we have chosen the 10 CMVPCR positive samples and 2 negative samples as controls to conduct this comparison study. By using NanoDrop Lite to evaluate the DNA concentration, the yield of extracted DNA is higher in our heating protocol than other protocols, the EZ1 DSP virus kit and EZ1 Virus mini kit show homogeneous quantities, and the QIAamp DSP virus kit shows very low DNA yields. Comparing cycle threshold and viral loads by real-time PCR, all these protocols identified negative samples (100%), and the previously positive samples used were as follows: protocol IV (90%), protocol II (60%), and protocol I (40%). QIAamp DSP virus kit results were not real-time PCR applicable and were non-conclusive because of the low DNA yields. Conclusion: Our developed heating method (protocol IV) is very effective, reliable, simple, fast, and cheap compared to the other protocols in our study. Full article

Murine hepatitis virus (MHV) is a non-human pathogen betacoronavirus that is evolutionarily and structurally related to the human pathogenic viruses SARS-CoV, MERS-CoV, and SARS-CoV-2. However, unlike the human SARS and MERS viruses, MHV requires a biosafety level 2 laboratory for propagating and safe handling, making it a potentially suitable surrogate virus. Despite this utility, few papers discussed the propagation and quantification of MHV using cell lines readily available in biorepositories making their implementations not easily reproducible. This article provides protocols for propagating and quantifying MHV-A59 using the recommended NCTC clone 1469 and clone 929 cell lines from American Type Culture Collection (ATCC). More specifically, the methods detail reviving cells, routine cell passaging, preparing freeze stocks, infection of NCTC clone 1469 with MHV and subsequent harvesting, and plaque assay quantification of MHV using NCTC clone 929 cells. Using these protocols, a BSL-2 laboratory equipped for cell culture work would generate at least 6.0 log plaque-forming units (PFU) per mL of MHV lysate and provide an optimized overlay assay using either methylcellulose or agarose as overlays for the titration of infectious virus particles. The protocols described here are intended to be utilized for persistence and inactivation studies of coronaviruses. Full article

Biological sex is one of the more critically important physiological parameters needed for managing threatened animal species because it is crucial for informing several of the management decisions surrounding conservation breeding programs. Near-infrared spectroscopy (NIRS) is a non-invasive technology that has been recently applied in the field of wildlife science to evaluate various aspects of animal physiology and may have potential as an in vivo technique for determining biological sex in live amphibian species. This study investigated whether NIRS could be used as a rapid and non-invasive method for discriminating biological sex in the endangered Houston toad (Anaxyrus houstonensis). NIR spectra (N = 396) were collected from live A. houstonensis individuals (N = 132), and distinct spectral patterns between males and females were identified using chemometrics. Linear discriminant analysis (PCA-LDA) classified the spectra from each biological sex with accuracy ≥ 98% in the calibration and internal validation datasets and 94% in the external validation process. Through the use of NIRS, we have determined that unique spectral signatures can be holistically captured in the skin of male and female anurans, bringing to light the possibility of further application of this technique for juveniles and sexually monomorphic species, whose sex designation is important for breeding-related decisions. Full article

After a DNA double-strand break, cells utilize either non-homologous end joining or homologous recombination to repair the broken DNA ends. Homologous recombination requires extensive nucleolytic processing of one of the DNA strands, resulting in long stretches of 3′ single-strand DNA overhangs. Typically, single-stranded DNA is measured using immunofluorescence microscopy to image the foci of replication protein A, a single-stranded DNA-binding protein. Microscopy analysis of bromodeoxyuridine foci under nondenaturing conditions has also been used to measure single-stranded DNA. Here, we describe a proximity ligation assay which uses genome-wide bromodeoxyuridine incorporation to label single-stranded DNA in order to measure the association of a protein of interest with single-stranded DNA. This method is advantageous over traditional foci analysis because it is more direct and specific than traditional foci co-localization microscopy methods, uses only one color channel, and can reveal protein-single-stranded DNA interactions that are rare and potentially undetectable using traditional microscopy methods. We show here the association of replication protein A and bromodeoxyuridine as proof-of-concept. Full article

Physical activity is beneficial for improving health and reducing sick leave absences. This article describes a protocol for an intervention using an interactive accelerometer smartphone application, telephone counselling, and physical activity recordings to increase the physical activity of workers in the military and improve their health. Under the protocol, employees from six military brigades in Finland will be randomly assigned to intervention and control groups. The intervention group’s participants will use accelerometers to measure their daily physical activities and their quality of sleep for six months. They will receive feedback based on these measurements via a smartphone application. The intervention group’s participants will be encouraged to exercise for two hours per week during working hours, and to participate in telephone counselling. The control group’s participants will continue with their normal exercise routines, without the accelerometer or feedback. The participants of both groups will be measured at the baseline, after the intervention period, and six months after the end of the intervention. The measurements will include accelerometer recordings, biochemical laboratory tests, body composition measurements, physical fitness tests, and questionnaires on sociodemographic factors, physical activities, and health. The primary outcomes will indicate changes in physical activity, physical fitness, and sick leave absences. The findings will help to develop a straightforward and cost-effective model for supporting the health and working capabilities of employees in the military and other workplaces. Full article

Empathy is positively related to healthcare workers and patients’ wellbeing. There is, however, limited research on the effects of empathy education delivered in acute clinical settings and its impact on healthcare consumers. This research tests the feasibility and the potential efficacy outcomes of an immersive education programme developed by the research team in collaboration with clinical partners and a multidisciplinary advisory group. Healthcare worker participants in the intervention ward will receive an 8-week immersive empathy education. The primary outcome (feasibility) will be assessed by evaluating the acceptability of the intervention and the estimated resources. The secondary outcome (efficacy) will be assessed using a quasi-experimental study design. Non-parametric tests will be used to test healthcare worker participants’ empathy, burnout, and organisational satisfaction (within-group and across groups), and healthcare consumer participants’ satisfaction (between-group) over time. Despite growing interest in the importance of empathy in professional relationships, to our knowledge, the present pilot study is the first to explore the feasibility and efficacy of an immersive empathy education in New Zealand. Our findings will provide critical evidence to support the development of a randomised cluster trial and potentially provide preliminary evidence for the effectiveness of this type of empathy education. Full article

To sustain energy-demanding developmental processes, oocytes must accumulate adequate stores of metabolic substrates and mitochondrial numbers prior to the initiation of maturation. In the past, researchers have utilized pooled samples to study oocyte metabolism, and studies that related multiple metabolic outcomes in single oocytes, such as ATP concentration and mitochondrial DNA copy number, were not possible. Such scenarios decreased sensitivity to intraoocyte metabolic relationships and made it difficult to obtain adequate sample numbers during studies with limited oocyte availability. Therefore, we developed and validated procedures to measure both mitochondrial DNA (mtDNA) copy number and ATP quantity in single oocytes. Validation of our procedures revealed that we could successfully divide oocyte lysates into quarters and measure consistent results from each of the aliquots for both ATP and mtDNA copy number. Coefficient of variation between the values retrieved for mtDNA copy number and ATP quantity quadruplicates were 4.72 ± 0.98 and 1.61 ± 1.19, respectively. We then utilized our methodology to concurrently measure mtDNA copy number and ATP quantity in germinal vesicle (GV) and metaphase two (MII) stage oocytes. Our methods revealed a significant increase in ATP levels (GV = 628.02 ± 199.53 pg, MII = 1326.24 ± 199.86 pg, p < 0.001) and mtDNA copy number (GV = 490,799.4 ± 544,745.9 copies, MII = 1,087,126.9 ± 902,202.8 copies, p = 0.035) in MII compared to GV stage oocytes. This finding is consistent with published literature and provides further validation of the accuracy of our methods. The ability to produce consistent readings and expected results from aliquots of the lysate from a single oocyte reveals the sensitivity and feasibility of using this method. Full article

Ichthyoplankton is the cluster of planktonic organisms that consists of fish eggs and larvae. These planktonic stages belong to the temporary zooplankton, representing future exploitable stocks. The study of the early ontogenesis of fish plays a key role in the understanding and evaluation of these populations through the study of their abundance and their spatio-temporal distribution. To better understand and protect these fisheries resources, it is essential to identify the different stages of fish embryonic development. This identification is usually performed using the classical method, based on morphological criteria under a binocular magnifying glass; however, this methodology is not always sufficient and is time consuming and, therefore, it is necessary to rely increasingly on molecular tools. The major problem with these tools is the yield and quality of the nucleic acids extracted from ichthyoplankton, especially in the case of eggs, which are small. Several methods have been used for DNA extraction from ichthyoplankton, either automated or manual, but very often from larvae or adults. In the present work, five fish egg DNA extraction protocols were compared based on their DNA yield and extraction quality, verified by agarose gel electrophoresis and quantitative PCR amplification. The results showed that extraction by our heat-protocol for direct PCR (Hp-dPCR) presents the simplest and cheapest protocol of all the kits used in this study, providing a sufficient quantity and quality of nucleic acids to be used for PCR amplification, and being within the reach of third world laboratories that often do not have sufficiently large budgets to obtain automated kits. Full article

Microglia, the resident brain immune effectors cells, show dynamic activation level changes for most neuropsychiatric diseases, reflecting their complex regulatory function and potential as a therapeutic target. Emerging single-cell molecular biology studies are used to investigate the genetic modification of individual cells to better understand complex gene regulatory pathways. Although multiple protocols for microglia isolation from adult mice are available, it is always challenging to get sufficient purified microglia from a single brain for simultaneous DNA and RNA extraction for subsequent downstream analysis. Moreover, for data comparison between treated and untreated groups, standardized cell isolation techniques are essential to decrease variability. Here, we present a combined method of microglia isolation from a single adult mouse brain, using a magnetic bead-based column separation technique, and a column-based extraction of purified DNA-RNA from the isolated microglia for downstream application. Our current method provides step-by-step instructions accompanied by visual explanations of important steps for isolating DNA-RNA simultaneously from a highly purified microglia population. Full article

Drug counterfeits have been an international issue for almost two decades, and the latest statistics show that fake medications will continue to penetrate legitimate pharmaceutical supply chains (PSCs). Therefore, identifying the issues faced by PSCs is essential to combat the counterfeit drug problem, which will require the implementation of technologies in various phases of the PSC to gain better visibility. In this regard, a literature review was conducted to fulfill the following objectives: (i) review the application of traceability technologies in various PSC phases to detect counterfeits; (ii) analyze the various barriers affecting the establishment of a safe PSC and the critical success factors used to overcome those barriers; and (iii) develop a conceptual framework and guidelines to demonstrate the influence of traceability technologies and success factors on overcoming the various barriers in different phases of the PSC. The major finding of this review was that traceability technologies and the critical success factors have a significant influence on overcoming the barriers to establishing a safe PSC. Full article

Plant leaves present an intricate array of layers providing a robust barrier against pathogens and abiotic stressors. However, these layers may also constitute an obstacle for the assessment of intracellular processes, especially when using fluorescence microscopy approaches. Current methods for leaf mitochondrial membrane potential determinations have been traditionally performed in thin mesophyll sections, in isolated protoplasts or in fluorescent protein-expressing transgenic plants. This may limit the amount of information obtained about overall mitochondrial morphology in intact leaves. Here, we detail a fast and straightforward protocol to assess changes in leaf mitochondrial membrane potential associated with mitochondrial dysfunction in the model plant Arabidopsis thaliana. This protocol also permits mitochondrial shape, dynamics and polarity assessment in leaves subjected to diverse stress conditions. Full article

To explore the biological and immunological basis of human rheumatoid arthritis and human atherosclerosis, we planned and reported a detailed design and rationale for Orencia Atherosclerosis and Rheumatoid Arthritis Study (ORACLE Arthritis Study) using highly sensitive, high-throughput, human autoantibody measurement methods with cell-free protein synthesis technologies. Our previous study revealed that subjects with atherosclerosis had various autoantibodies in their sera, and the titers of anti-Th2 cytokine antibodies were correlated with the severity of atherosclerosis. Because rheumatoid arthritis is a representative autoimmune disease, we hypothesized that both rheumatoid arthritis and atherosclerosis are commonly developed by autoantibody-mediated autoimmune processes, leading to incessant inflammatory changes in both articular joint tissues and vessel walls. We planned a detailed examination involving carotid artery ultrasonography, measurements of adhesion molecules, such as ICAM-1 (intercellular adhesion molecule 1) and VCAM-1 (vascular cell adhesion molecule 1) for the evaluation of atherosclerosis progression, and high-throughput, high-sensitivity, autoantibody analyses using cell-free technologies, with detailed examinations of the disease activity of rheumatoid arthritis. Analyses of correlations and associations between biological markers and degrees of carotid atherosclerosis over time under consistent conditions may enable us to understand the biological and humoral immunity background of human atherosclerosis and autoimmune diseases. Full article

Internal derangement (ID) in the temporomandibular joint (TMJ) is defined as a mechanical problem of the joint that interferes with its function. It is attributed to an abnormal interaction among the articular disc, condyle, and joint eminence. The aim of this study is to evaluate diagnostic efficacy of non-invasive hand-carried ultrasonography instrumentation (US) to provide high-level images for a correct diagnosis of ID. Twenty-eight ID patients, 15 female and 13 males, were examined both clinically and by MRI images in order to achieve a diagnosis of ID (using Helkimo index). Then, they were submitted to US examination with a 12 MHz transducer by using hand-carried instrumentation by a clinician that was blind to their diagnosis and clinical data. TMJ US examination was performed with the mouth closed and mouth open, with proper technique. Each position was then evaluated with two different orientations of the transducer. US showed acceptable results in identifying bone structures. Lower values of diagnostic efficacy were obtained for disc position during joint movements with respect to MRI images. MRI still represents the gold standard for the identification of joint structures. If not corroborated by clinical and anamnestic data, the diagnostic efficacy of US in identifying the position of the disc during opening and closing jaw movements appears limited than compared to MRI. Full article