The use of ROI overlays and a semi-automated method for measuring cortical area in ImageJ for histological analysis.

A major part of histologic studies is the use of high resolution imaging for data collection and analysis. ImageJ, a freely available software from the NIH designed for image analysis, has many features that are not well-known among bone histologists and can be incredibly beneficial in terms of stream-lining data collection and maximizing limited resources. The aims of this technical note are twofold: (a) to describe methods for image annotation and measurement using region of interest overlays in ImageJ, and (b) to present a new code for a semi-automated method of measuring cortical bone areas from high resolution cross-sectional images also using ImageJ.

A survey on practices for active surveillance of carriage of multidrug-resistant bacteria in hospitals in the Autonomous Community of Valencia, Spain.

A questionnaire-based cross-sectional study was conducted to gather information on current microbiological practices for active surveillance of carriage of multidrug-resistant (MDR) bacteria in hospitals from 14 health departments of the Autonomous Community of Valencia (ACV), Spain, which together provided medical attention to 3,271,077 inhabitants in 2017, approximately 70% of the population of the ACV. The survey consisted of 35 questions on MDR bacteria screening policies, surveillance approach chosen (universal vs. targeted), and microbiological methods and processes in use for routine detection and reporting of colonization by MDR bacteria, including the anatomical sites scheduled to be sampled for each MDR bacterial species, and the methodology employed (culture-based, molecular-based, or both). Our study revealed striking differences across centers, likely attributable to the lack of consensus on optimal protocols for sampling, body sites for screening, and microbiological testing, thus underscoring the need for consensus guidelines on these issues.

Prevalence of drifting osteons distinguishes human bone.

The histological, or microscopic, appearance of bone tissue has long been studied to identify species-specific traits. There are several known histological characteristics to discriminate animal bone from human, but currently no histological characteristic that has been consistently identified in human bone exclusive to other mammals. The drifting osteon is a rare morphotype found in human long bones and observationally is typically absent from common mammalian domesticates. We surveyed previously prepared undecalcified histological sections from 25 species (human n = 221; nonhuman primate n = 24; nonprimate n = 169) to see if 1) drifting osteons were indeed more common in humans and 2) this could be a discriminating factor to identify human bone histologically. We conclude that drifting osteons are indeed more prevalent in human and nonhuman primate bone relative to nonprimate mammalian bone. Two criteria identify a rib or long bone fragment as human, assuming the fragment is unlikely to be from a nonhuman primate given the archaeological context: 1) at least two drifting osteons are present in the cross-section and 2) a drifting osteon prevalence (or as a percentage of total secondary osteons) of ≥ 1%. We present a quantitative histological method that can positively discriminate human bone from nonprimate mammalian bone in archaeological contexts.

Scaling of Haversian canal surface area to secondary osteon bone volume in ribs and limb bones.

Studies of secondary osteons in ribs have provided a great deal of what is known about remodeling dynamics. Compared with limb bones, ribs are metabolically more active and sensitive to hormonal changes, and receive frequent low-strain loading. Optimization for calcium exchange in rib osteons might be achieved without incurring a significant reduction in safety factor by disproportionally increasing central canal size with increased osteon size (positive allometry). By contrast, greater mechanical loads on limb bones might favor reducing deleterious consequences of intracortical porosity by decreasing osteon canal size with increased osteon size (negative allometry). Evidence of this metabolic/mechanical dichotomy between ribs and limb bones was sought by examining relationships between Haversian canal surface area (BS, osteon Haversian canal perimeter, HC.Pm) and bone volume (BV, osteonal wall area, B.Ar) in a broad size range of mature (quiescent) osteons from adult human limb bones and ribs (modern and medieval) and various adult and subadult non-human limb bones and ribs. Reduced major axis (RMA) and least-squares (LS) regressions of HC.Pm/B.Ar data show that rib and limb osteons cannot be distinguished by dimensional allometry of these parameters. Although four of the five rib groups showed positive allometry in terms of the RMA slopes, nearly 50% of the adult limb bone groups also showed positive allometry when negative allometry was expected. Consequently, our results fail to provide clear evidence that BS/BV scaling reflects a rib versus limb bone dichotomy whereby calcium exchange might be preferentially enhanced in rib osteons.

The utility of osteon shape and circularity for differentiating human and non-human Haversian bone.

Distinguishing human from non-human bone fragments is usually accomplished by observation of gross morphology. When macroscopic analysis is insufficient, histological approaches can be applied. Microscopic features, like plexiform bone or osteon banding, are characteristic of non-humans. In the absence of such features, distinguishing Haversian bone as either human or non-human proves problematic. This study proposes a histomorphometric approach for classifying species from Haversian bone. Two variables, osteon area (On.Ar.) and circularity (On.Cr.), are examined. Measurements were collected from three species (deer, dog, human) represented by various skeletal elements; only ribs were available for humans (ribs: deer n = 6, dog n = 6, human n = 26; humeri: deer n = 6, dog n = 6; femora: deer n = 6, dog n = 6). Qualitative analysis comparing human to non-human On.Ar. demonstrated that human ribs have larger mean On.Ar. (0.036 mm(2)) than non-human ribs (deer = 0.017 mm(2) , dog = 0.013 mm(2)). On.Cr. in the ribs showed minor differences between species (deer = 0.877; dog = 0.885; human = 0.898). Results demonstrated no significant difference across long bone quadrants in long bones. Discriminant analyses run on the means for each sample demonstrated overlap in deer and dog samples, clustering the non-human and human groups apart from each other. Mean On.Cr. proved a poor criterion (ribs only: 76.3%, pooled elements: 66.1%), while mean On.Ar. proved useful in identifying human from non-human samples (ribs only: 92.1%, pooled elements: 93.5%). When variables were combined, accuracy increased to 100% correct classification for rib data and 98.4% when considering data from all elements. These results indicate that On.Ar. and On.Cr. are valuable histomorphometric tools for distinguishing human from non-human Haversian bone.

Analysis of histomorphometric variables: Proposal and validation of osteon definitions.

Histological analysis of bone tissue has been used to explore a variety of questions relating to age-at-death, habitual behaviors, health, and nutritional stress. Identification of intact and fragmentary osteons is of key interest to many researchers in these studies, yet the definitions of these features vary between researchers making cross-study comparisons problematic. Furthermore, histological variable definitions are often ambiguous or require subjective classifications by the observer. As a result, and as indicated by previous studies, observer error and misclassification of certain variables, namely intact and fragmentary osteons, can be significant. This study proposes new definitions for intact and fragmentary osteons that are designed to limit observer subjectivity and also explore efficacy of combining osteon types into one variable. A sample of 30 6th rib cross-sections from a modern forensic population was used to test the validity of the proposed definitions. Observations of intact osteon population density (OPD(I)) and fragmentary osteon population density (OPD(F)) were made by three observers for each cross-section. These observations were used to explore the interobserver error associated with the proposed definitions and determine if combining variables into one variable (OPD) mitigates persisting classification difficulties. Results indicate that the proposed definitions significantly reduce interobserver error and misclassification of intact and fragmentary osteons. However, the interobserver error associated with fragmentary osteons is still high. Evaluation of the variables independently indicates that combining variables has potential to reduce the predictive strength of an age estimation model and the ability to interpret age-related bone remodeling.

Rib Variation at Multiple Locations and Implications for Histological Age Estimation.

Existing histological age estimation methods using the rib were developed mainly from the midshaft; however, in forensic practice, uncertainty of sampling location often arises due to fragmented or previously sampled ribs. The potential for error increases when sampling location is uncertain and utilizing a section beyond the midshaft (either anterior or posterior) may result in erroneous age estimates. Additionally, there is debate within the field regarding the minimum number of sections needed for accurate age estimation. The aim of this research is to determine the importance of the midshaft distinction for age-at-death assessment and the necessity of analyzing serial sections by evaluating histological variables at sampling locations along the length of the rib. Three seriated histological sections at three sampling locations (anterior, midshaft, and posterior) were obtained from sixth ribs of ten postmortem human subjects. Cortical area (Ct.Ar) and osteon population density (OPD) were collected from each section (n = 90). Significant differences were determined in Ct.Ar between sampling locations, demonstrating the variation present along the length of the rib. A comparison of OPD at sampling locations revealed significant differences, suggesting that sampling site is critical to accurate age estimates. When sampling location is uncertain, a more anterior section should be taken. Analysis of serial sections within locations revealed no significant differences in OPD or Ct.Ar, supporting the practice of collecting data from one section for age estimation. While an age estimate can be achieved through the analysis of one section, best practice suggests reading two sections to capture intraindividual variation.

Hunting down fungal secretomes using liquid-phase IEF prior to high resolution 2-DE.

The secreted proteins (secretome) of fungi play a key role in interactions of pathogenic and symbiotic fungi with plants. Using the plant pathogenic fungus Leptosphaeria maculans and symbiont Laccaria bicolor grown in culture, we have established a proteomic protocol for extraction, concentration and resolution of the fungal secretome. As no proteomic data were available on mycelium tissues from both L. maculans and L. bicolor, mycelial proteins were studied; they also helped verifying the purity of secretome samples. The quality of protein extracts was initially assessed by both 1-DE and 2-DE using first a broad pH range for IEF, and then narrower acidic and basic pH ranges, prior to 2-DE. Compared with the previously published protocols for which only dozens of 2-D spots were recovered from fungal secretome samples, up to approximately 2000 2-D spots were resolved by our method. MS identification of proteins along several pH gradients confirmed this high resolution, as well as the presence of major secretome markers such as endopolygalacturonases, beta-glucanosyltransferases, pectate lyases and endoglucanases. Shotgun proteomic experiments evidenced the enrichment of secreted protein within the liquid medium. This is the first description of the proteome of L. maculans and L. bicolor, and the first application of liquid-phase IEF to any fungal extracts.

Microdamage as a Bone Quality Component: Practical Guidelines for the Two-Dimensional Analysis of Linear Microcracks in Human Cortical Bone.

Microdamage is a component of bone quality believed to play an integral role in bone health. However, comparability between existing studies is fraught with issues due to highly variable methods of sample preparation and poorly defined quantification criteria. To address these issues, this article has two aims. First, detailed methods for preparation and analysis of linear microcracks in human ribs, specifically addressing troubleshooting issues cited in previous studies, are laid out. Second, new, partially validated criteria are proposed in an effort to reduce subjective differences in microcrack counts and measures, ensuring more comparable results between studies. Revised definitions based on current literature in conjunction with a digital atlas to reduce observer inaccuracy and bias are presented. The goal is to provide a practical methodology for bone biologists and biomechanists to collect and analyze linear microcracks for basic science research. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Light Field Image Dataset of Skin Lesions.

Light field imaging technology has been attracting increasing interest because it enables capturing enriched visual information and expands the processing capabilities of traditional 2D imaging systems. Dense multiview, accurate depth maps and multiple focus planes are examples of different types of visual information enabled by light fields. This technology is also emerging in medical imaging research, like dermatology, allowing to find new features and improve classification algorithms, namely those based on machine learning approaches. This paper presents a contribution for the research community, in the form of a publicly available light field image dataset of skin lesions (named SKINL2 v1.0). This dataset contains 250 light fields, captured with a focused plenoptic camera and classified into eight clinical categories, according to the type of lesion. Each light field is comprised of 81 different views of the same lesion. The database also includes the dermatoscopic image of each lesion. A representative subset of 17 central view images of the light fields is further characterised in terms of spatial information (SI), colourfulness (CF) and compressibility. This dataset has high potential for advancing medical imaging research and development of new classification algorithms based on light fields, as well as in clinically-oriented dermatology studies.

Sources of Variability in Structural Bending Response of Pediatric and Adult Human Ribs in Dynamic Frontal Impacts.

Despite safety advances, thoracic injuries in motor vehicle crashes remain a significant source of morbidity and mortality, and rib fractures are the most prevalent of thoracic injuries. The objective of this study was to explore sources of variation in rib structural properties in order to identify sources of differential risk of rib fracture between vehicle occupants. A hierarchical model was employed to quantify the effects of demographic differences and rib geometry on structural properties including stiffness, force, displacement, and energy at failure and yield. Three-hundred forty-seven mid-level ribs from 182 individual anatomical donors were dynamically (~2 m/s) tested to failure in a simplified bending scenario mimicking a frontal thoracic impact. Individuals ranged in age from 4 - 108 years (mean 53 ± 23 years) and included 59 females and 123 males of diverse body sizes. Age, sex, body size, aBMD, whole rib geometry and cross-sectional geometry were explored as predictors of rib structural properties. Measures of cross-sectional rib size (Tt.Ar), bone quantity (Ct.Ar), and bone distribution (Z) generally explained more variation than any other predictors, and were further improved when normalized by rib length (e.g., robustness and WBSI). Cortical thickness (Ct.Th) was not found to be a useful predictor. Rib level predictors performed better than individual level predictors. These findings moderately explain differential risk for rib fracture and with additional exploration of the rib's role in thoracic response, may be able contribute to ATD and HBM development and alterations in addition to improvements to thoracic injury criteria and scaling methods.

Variability of in vivo linear microcrack accumulation in the cortex of elderly human ribs.

Excessive accumulation of microdamage in the skeleton in vivo is believed to contribute to fragility and risk of fracture, particularly in the elderly. Current knowledge of how much in vivo damage accrual varies between individuals, if at all, is lacking. In this study, paired sixth ribs from five male and five female elderly individuals (76-92 years, mean age = 84.7 years) were examined using en bloc staining and fluorescent microcopy to quantify linear microcracks present at the time of death (i.e. in vivo microdamage). Crack number, crack length, crack density, and crack surface density were measured for each complete cross-section, with densities calculated using the variable of bone area (which accounts for the influence of porosity on the cortex, unlike the more frequently used cortical area), and analyzed using a two-way mixed model analysis of variance. Results indicate that while microcracks between individuals differ significantly, differences between the left and right corresponding pairs within individuals and the pleural and cutaneous cortices within each rib did not. These results suggest that systemic influences, such as differential metabolic activity, affect the accumulation of linear microcracks. Furthermore, variation in remodeling rates between individuals may be a major factor contributing to differential fracture risk in the elderly. Future work should expand to include a wider age range to examine differences in in vivo microdamage accumulation across the lifespan, as well as considering the influence of bisphosphonates on microdamage accumulation in the context of compromised remodeling rates in the elderly.

The molecular epidemiology of HIV-1 in the Comunidad Valenciana (Spain): analysis of transmission clusters.

HIV infections are still a very serious concern for public heath worldwide. We have applied molecular evolution methods to study the HIV-1 epidemics in the Comunidad Valenciana (CV, Spain) from a public health surveillance perspective. For this, we analysed 1804 HIV-1 sequences comprising protease and reverse transcriptase (PR/RT) coding regions, sampled between 2004 and 2014. These sequences were subtyped and subjected to phylogenetic analyses in order to detect transmission clusters. In addition, univariate and multinomial comparisons were performed to detect epidemiological differences between HIV-1 subtypes, and risk groups. The HIV epidemic in the CV is dominated by subtype B infections among local men who have sex with men (MSM). 270 transmission clusters were identified (>57% of the dataset), 12 of which included ≥10 patients; 11 of subtype B (9 affecting MSMs) and one (n = 21) of CRF14, affecting predominately intravenous drug users (IDUs). Dated phylogenies revealed these large clusters to have originated from the mid-80s to the early 00 s. Subtype B is more likely to form transmission clusters than non-B variants and MSMs to cluster than other risk groups. Multinomial analyses revealed an association between non-B variants, which are not established in the local population yet, and different foreign groups.

The future of metabolomics in ELIXIR.

Metabolomics, the youngest of the major omics technologies, is supported by an active community of researchers and infrastructure developers across Europe. To coordinate and focus efforts around infrastructure building for metabolomics within Europe, a workshop on the "Future of metabolomics in ELIXIR" was organised at Frankfurt Airport in Germany. This one-day strategic workshop involved representatives of ELIXIR Nodes, members of the PhenoMeNal consortium developing an e-infrastructure that supports workflow-based metabolomics analysis pipelines, and experts from the international metabolomics community. The workshop established metabolite identification as the critical area, where a maximal impact of computational metabolomics and data management on other fields could be achieved. In particular, the existing four ELIXIR Use Cases, where the metabolomics community - both industry and academia - would benefit most, and which could be exhaustively mapped onto the current five ELIXIR Platforms were discussed. This opinion article is a call for support for a new ELIXIR metabolomics Use Case, which aligns with and complements the existing and planned ELIXIR Platforms and Use Cases.

Examination of Factors Potentially Influencing Osteon Size in the Human Rib.

Previous research demonstrates that the size of secondary osteons varies considerably between individuals, though what factors act in the delineation of osteon size remain uncertain. This study explores the influence of age, sex, percent cortical area (%Ct.Ar), percent cortical porosity (%Po.Ar), and loading environment on osteon area (On.Ar) in human ribs. The sample consisted of midshaft 6(th) ribs from 80 individuals, 6-94 years of age. T-tests demonstrated no significant differences in On.Ar between the sexes (P=0.383). Age showed a significant correlation with both %Ct.Ar and %Po.Ar, so a hierarchical regression model was used to control for the effects of age on the other variables. Results indicate that age is the most significant factor of those tested in this study (P=0.004), with %Ct.Ar playing a much smaller but still significant role (P=0.014), while %Po.Ar had no significant influence on On.Ar (P=0.443). Age demonstrates an inverse relationship with On.Ar, while %Ct.Ar has a direct relationship with On.Ar. Significant differences in On.Ar between the pleural and cutaneous cortices are attributed to variation in %Ct.Ar of each cortex. Therefore, age and %Ct.Ar account for the majority of osteon size variability in this study, although it is likely genetics play an important role as well. Understanding the biological mechanisms that act in remodeling and determine osteon size is essential for accurately addressing and interpreting histological findings, work that is invaluable in its implications for bone biology.

Low platelet counts alone do not cause bleeding in an experimental immune thrombocytopenic purpura in mice.

BACKGROUND AND OBJECTIVES: The physiopathogenesis of hemorrhagic phenomena in patients with autoimmune thrombocytopenic purpura is associated with low platelet levels. In the present work the relation between thrombocytopenia and bleeding was examined. The possible participation of endothelial cells in bleeding was also investigated. DESIGN AND METHODS: Immune thrombocytopenia and bleeding were studied in mice injected with anti-mouse and anti-human platelet polyclonal rabbit IgG. Platelet levels were measured at different times and bleeding signs were systematically recorded. ANOVA tests were used to compare platelet levels. Binding of anti-platelet antibodies to vascular endothelial cells was analyzed by immunohistochemistry. RESULTS: Three different doses of anti-platelet IgG caused the same low platelet levels, but bleeding occurred only with high doses of anti-platelet IgG irrespective of the platelet levels. No inflammation around blood vessels was observed in paraffin-embedded tissue sections of organs. Immunohistochemistry demonstrated anti-platelet antibodies bound to vascular endothelium. INTERPRETATION AND CONCLUSIONS: We showed lack of correlation between platelet counts and bleeding in a murine model. The binding of anti-platelet IgG to endothelial cells of small vessels is an indication that antibody-mediated endothelial activation, in addition to thrombocytopenia, could be participating in bleeding.

Effector diversification within compartments of the Leptosphaeria maculans genome affected by Repeat-Induced Point mutations.

Fungi are of primary ecological, biotechnological and economic importance. Many fundamental biological processes that are shared by animals and fungi are studied in fungi due to their experimental tractability. Many fungi are pathogens or mutualists and are model systems to analyse effector genes and their mechanisms of diversification. In this study, we report the genome sequence of the phytopathogenic ascomycete Leptosphaeria maculans and characterize its repertoire of protein effectors. The L. maculans genome has an unusual bipartite structure with alternating distinct guanine and cytosine-equilibrated and adenine and thymine (AT)-rich blocks of homogenous nucleotide composition. The AT-rich blocks comprise one-third of the genome and contain effector genes and families of transposable elements, both of which are affected by repeat-induced point mutation, a fungal-specific genome defence mechanism. This genomic environment for effectors promotes rapid sequence diversification and underpins the evolutionary potential of the fungus to adapt rapidly to novel host-derived constraints.

[Meningitis outbreak caused by Echovirus serotype 30 in the Valencian Community]. / Brote de meningitis por echovirus serotipo 30 en la Comunidad Valenciana.

BACKGROUND AND OBJECTIVE: Aseptic meningitis can be caused by several agents, and in many cases the etiology remains unknown. The aim of this study to analyze the clinical and epidemiological characteristics of a meningitis outbreak detected in Health Department 11 of the Valencian Community (Spain). MATERIAL AND METHODS: The study was performed in children hospitalized between November and December 2006 with meningitis symptoms, CSF pleocytosis, and negative CSF bacteriological culture. An epidemiological survey was conducted among cases and family members. Virus detection and phylogenetic analysis were performed with molecular biology techniques. RESULTS: The outbreak affected at least 44 children, with a mean age (standard deviation) of 5.5 years (2.9). The average hospital stay was 3.1 days and outcome was favorable in all cases. In 24 patients the CSF specimen sufficed for viral detection by PCR; enteroviruses ultimately serotyped as echovirus 30 were detected in 12 of them (50%). This serotype has been recently found in other parts of our country. CONCLUSIONS: Detection of echovirus 30 in CSF and the epidemiological presentation of cases enabled determination of the etiology of the outbreak. This finding coincided in time with other outbreaks of echovirus 30 in Spain, a fact that may explain the epidemic situation in the Valencian Community during 2006. Establishment of a national surveillance network for monitoring systemic enterovirus infection would provide data on the circulation patterns and identify new emerging serotypes.

Use of penetrating peptides interacting with PP1/PP2A proteins as a general approach for a drug phosphatase technology.

Protein phosphatase types 1 (PP1) and 2A (PP2A) represent two major families of serine/threonine protein phosphatases that have been implicated in the regulation of many cellular processes, including cell growth and apoptosis in mammalian cells. PP1 and PP2A proteins are composed of oligomeric complexes comprising a catalytic structure (PP1c or PP2AC) containing the enzymatic activity and at least one more interacting subunit. The binding of different subunits to a catalytic structure generates a broad variety of holoenzymes. We showed here that casein kinase 2alpha (Ck2alpha) and simian virus 40 small t antigen share a putative common beta-strand structure required for PP2A1 trimeric holoenzyme binding. We have also characterized DPT-sh1, a short basic peptide from Ck2alpha that interacted only in vitro with the PP2A-A subunit and behaves as a nontoxic penetrating shuttle in several cultivated human cell lines and chick embryos. In addition, DPT-sh1 specifically accumulated in human red cells infected with Plasmodium falciparum malaria parasites. We therefore designed bipartite peptides containing DPT-sh1 and PP1- or PP2A-interacting sequences. We found that DPT-5, a DPT-sh1-derived peptide containing a short sequence identified in CD28 antigen, interacts with PP2A-Balpha, and DPT-7, another DPT-sh1-derived peptide containing a short sequence identified in Bad as a PP1 catalytic consensus docking motif, induce apoptosis in cultivated cell lines. These results clearly indicate that the rational design of PP1/PP2A interacting peptides is a pertinent strategy to deregulate intracellular survival pathways.

In vitro activity of linezolid, clarithromycin and moxifloxacin against clinical isolates of Mycobacterium kansasii.

OBJECTIVES: To compare the activity of linezolid with a range of drugs used in the treatment of Mycobacterium kansasii infections. RESULTS: The percentages of resistant isolates against isoniazid, rifampicin and ethambutol were 2.9%, 1.9% and 2.9%, respectively. All isolates were susceptible to clarithromycin and moxifloxacin both with MIC(90) values of 0.125 mg/L. Linezolid was active against all isolates with MIC(50) and MIC(90) values of 0.5 and 1 mg/L, respectively, both below the susceptibility breakpoint established for mycobacteria. CONCLUSION: Linezolid, clarithromycin or moxifloxacin, could be used as alternative drugs for treatment of infections due to rifampicin-resistant isolates as well as short-course or intermittent therapy of M. kansasii lung disease.