The anatomy lesson of the SARS-CoV-2 pandemic: irreplaceable tradition (cadaver work) and new didactics of digital technology.

AIM: To compare the efficacy of different components of online and contact anatomy classes as perceived by medical students. METHODS: An anonymous course evaluation survey was conducted at the end of the academic year 2019/2020. The organization of classes due to the SARS-CoV-2 pandemic provided our students with a unique opportunity to compare online and contact classes. Students' responses were analyzed according to the type of obtained data (ratio, ordinal, and categorical). RESULTS: The response rate was 95.58%. Approximately 90% of students found anatomical dissection and practical work in general to be the most important aspect of teaching, which could not be replaced by online learning. During online classes, students missed the most the interaction with other students, followed by the interaction with student teaching assistants and teaching staff. Very few students found contact lectures useful, with most students reporting that they could be replaced with recorded video lectures. In contrast, recorded video lectures were perceived as extremely helpful for studying. Regular weekly quizzes were essential during online classes as they gave students adequate feedback and guided their learning process. Students greatly benefitted from additional course materials and interactive lessons, which were made easily available via e-learning platform. CONCLUSIONS: Anatomical dissection and interaction during contact classes remain the most important aspects of teaching anatomy. However, online teaching increases learning efficiency by allowing alternative learning strategies and by substituting certain components of contact classes, thus freeing up more time for practical work.

Fas receptor induces apoptosis of synovial bone and cartilage progenitor populations and promotes bone loss in antigen-induced arthritis.

Rheumatoid arthritis (RA) is an inflammatory joint disease that eventually leads to permanent bone and cartilage destruction. Fas has already been established as the regulator of inflammation in RA, but its role in bone formation under arthritic conditions is not completely defined. The aim of this study was to assess the effect of Fas inactivation on the bone damage during murine antigen-induced arthritis. Subchondral bone of wild-type (WT) and Fas-knockout (Fas-/-) mice was evaluated by histomorphometry and microcomputerized tomography. Proportions of synovial bone and cartilage progenitors were assessed by flow cytometry. Synovial bone and cartilage progenitors were purified by fluorescence-activated cell sorting and expression of Fas and Fas-induced apoptosis were analyzed in vitro. Results showed that Fas-/- mice developed attenuated arthritis characterized by preserved epiphyseal bone and cartilage. A proportion of the earliest CD200+ bone and cartilage progenitors was reduced in WT mice with arthritis and was unaltered in Fas-/- mice. During osteoblastic differentiation in vitro, CD200+ cells express the highest levels of Fas and are removed by Fas ligation. These results suggest that Fas-induced apoptosis of early CD200+ osteoprogenitor population represents potential mechanism underlying the impaired bone formation in arthritis, so their preservation may represent the bone-protective mechanism during arthritis.-Lazic Mosler, E., Lukac, N., Flegar, D., Fadljevic, M., Radanovic, I., Cvija, H., Kelava, T., Ivcevic, S., Sucur, A., Markotic, A., Katavic, V., Marusic, A., Grcevic, D., Kovacic, N. Fas receptor induces apoptosis of synovial bone and cartilage progenitor populations and promotes bone loss in antigen-induced arthritis.

Perceptions of Ethical Climate and Research Pressures in Different Faculties of a University: Cross-Sectional Study at the University of Split, Croatia.

We determined the prevailing ethical climate at three different schools of a single university, in order to explore possible differences in the ethical climate related to different research fields: the School of Electrical Engineering, Mechanical Engineering, and Naval Architecture; the School of Humanities and Social Sciences; and the School of Medicine. We used the Ethical Climate Questionnaire to survey the staff (teachers and administration) at the three schools, and used the research integrity and organizational climate (RIOC) survey for early-stage researchers at the three schools. The dominant ethical climate type perceived collectively at the three university schools (response rate 49%, n = 294) was Laws and professional codes, which is associated with the cosmopolitan level of analysis and the ethical construct of principle. Individually, the same climate predominated at the schools for engineering and humanities, but the School of Medicine had the Self-interest ethical climate, which is associated with the individual level of analysis and the egoism ethical construct. In the RIOC survey (response rate 85%; n = 70), early-stage researchers from the three university schools did not differ in their perceptions of the organizational research integrity climate, or in their perceived individual, group or organizational pressures. Our study is the first, to the best of our knowledge, to show differences in perceived ethical climate at a medical school compared to other schools at a university. Further studies are needed to explore the reasons for these differences and how they translate to organizational outcomes, such as job satisfaction, commitment to the institution and dysfunctional behaviour, including research misconduct.

Establishing Sensible and Practical Guidelines for Desk Rejections.

Publishing has become, in several respects, more challenging in recent years. Academics are faced with evolving ethics that appear to be more stringent in a bid to reduce scientific fraud, the emergence of science watchdogs that are now scrutinizing the published literature with critical eyes to hold academics, editors and publishers more accountable, and a barrage of checks and balances that are required between when a paper is submitted and eventually accepted, to ensure quality control. Scientists are often under increasing pressure to produce papers in an increasingly stringent publishing environment. In such a climate, timing is everything, as is the efficiency of the process. Academics appreciate that rejections are part of the fabric of attempting to get a paper published, but they expect the reason to be clear, based on careful evaluation of their work, and not on superficial or unsubstantiated excuses. A desk rejection occurs when a paper gets rejected even before it has entered the peer review process. This paper examines the features of some desk rejections and offers some guidelines that would make desk rejections valid, fair and ethical. Academics who publish are under constant pressure to do so quickly, but effectively. They are dependent on the editors' good judgment and the publisher's procedures. Unfair, unsubstantiated, or tardy desk rejections disadvantage academics, and editors and publishers must be held accountable for wasting their time, resources, and patience.

Genetic identification of a rare record of Ommastrephes Bartramii (Cephalopoda: Ommastrephidae) from the Eastern Adriatic Sea.

The neon flying squid Ommastrephes bartramii Lesueur, 1821 belongs to the Ommastrephidae, Cephalopoda family. The family Ommastrephidae (Mollusca: Cephalopoda) includes many commercially important species, dispersed around the world. The Ommastrephidae family is conventionally divided into three subfamilies (Illicinae, Todarodinae, and Ommastrephinae). We report a specimen of neon flying squid caught in the winter 2013 at Luka Sipanska, Island of Sipan, Croatia and identified at the genetic level using the standard mitochondrial COI barcode region. This record represents the first genetic identification of a neon flying squid from the Adriatic Sea.

Induction of osteoclast progenitors in inflammatory conditions: key to bone destruction in arthritis.

The inflammatory milieu favors recruitment and activation of osteoclasts, and leads to bone destruction as a serious complication associated with arthritis and with other inflammatory processes. The frequency and activity of osteoclast progenitors (OCPs) correspond to arthritis severity, and may be used to monitor disease progression and bone resorption, indicating the need for detailed characterization of the discrete OCP subpopulations. Collectively, current studies suggest that the most potent murine bone marrow OCP population can be identified among lymphoid negative population within the immature myeloid lineage cells, as B220(-)CD3(-)CD11b(-/lo)CD115(+)CD117(+)CX3CR1(+) and possibly also Ter119(-)CD11c(-)CD135(lo)Ly6C(+)RANK(-). In peripheral blood the OCP population bears the monocytoid phenotype B220(-)CD3(-)NK1.1(-)CD11b(+)Ly6C(hi)CD115(+)CX3CR1(+), presumably expressing RANK in committed OCPs. Much less is known about human OCPs and their regulation in arthritis, but the circulating OCP subset is, most probably, comprised among the lymphoid negative population (CD3(-)CD19(-)CD56(-)), within immature monocyte subset (CD11b(+)CD14(+)CD16(-)), expressing receptors for M-CSF and RANKL (CD115(+)RANK(+)). Our preliminary data confirmed positive association between the proportion of peripheral blood OCPs, defined as CD3(-)CD19(-)CD56(-)CD11b(+)CD14(+), and the disease activity score (DAS28) in the follow-up samples from patients with psoriatic arthritis receiving anti-TNF therapy. In addition, we reviewed cytokines and chemokines which, directly or indirectly, activate OCPs and enhance their differentiation potential, thus mediating osteoresorption. Control of the activity and migratory behaviour of OCPs as well as the identification of crucial bone/joint chemotactic mediators represent promising therapeutic targets in arthritis.

The Zagreb Skull Collection-The unique identified collection of human skulls from fetuses to centenarians.

Skull anatomy and development have been extensively studied due to their significance in evolutionary biology, forensic anthropology, and clinical medicine. Bone collections are an indispensable resource for conducting such anthropological and anatomical studies. However, worldwide there are only few skull collections containing specimens covering the entire fetal and postnatal period. Herein we describe the Zagreb Skull Collection, an identified collection comprising more than 1100 skulls and skull bone sets from the early fetal period to centenarians. The Zagreb Skull Collection consists of two main parts: the unique Collection of Skull Bones containing 386 sets of separated skull bones from the early fetal period to adulthood and the Collection of Skulls containing 742 skulls (age range 4-101 years). The collection was the core source for numerous anatomical studies on the development, postnatal changes, and anatomical variations of the skull. However, the Zagreb Skull Collection is still an underexploited resource for anthropological, forensic, and anatomical studies with translatability to contemporary clinical practice.

In the eye of the beholder - how course delivery affects anatomy education.

BACKGROUND: The COVID-19 pandemic caused major shifts in students' learning strategies as well as teaching environments that profoundly affected the delivery of anatomy courses in medical schools. The Department of Anatomy at the University of Zagreb School of Medicine had a unique experience where the anatomy course in 2019/2020 was first taught in-person before transferring to an online course delivery, while the inverse happened in 2020/2021. The core curriculum, course material and examination criteria were the same in both academic years. The aim of the study was to determine whether course delivery affected students' perceptions of the course and whether it impacted students' engagement and success. METHODS: The students' perceptions of the course were assessed via an anonymous course survey (student evaluation of teaching, SET). The questions in the SET assessed the usefulness of teaching modalities rather than students' satisfaction. Most questions were in the form of statements to which students responded with their level of agreement on a five-point Likert scale. Differences between responses in 2019/2020 and 2020/2021 were analyzed using the Mann-Whitney test. Effect size was estimated using Cliff's delta and association between responses was assessed using Spearman's r coefficient. RESULTS: Students' perceptions were significantly affected by changes in course delivery. Students' success and engagement were higher in 2019/2020 when in-person teaching preceded online teaching. Furthermore, students' views on course organization and the usefulness of continuous assessment were more positive in 2019/2020. Finally, students' perceptions of the usefulness of online materials and activities were more positive in 2019/2020. All comparisons between the two academic years were statistically significant (P ≤ 0.0001 for all comparisons, Mann-Whitney test). CONCLUSIONS: Students' perceptions of the anatomy course were dependent on the teaching environment they were exposed to at the beginning of the course. A transfer from in-person to online course delivery was more successful than vice-versa. This has important implications for structuring hybrid courses in medical education in the future.

Preventive CCL2/CCR2 Axis Blockade Suppresses Osteoclast Activity in a Mouse Model of Rheumatoid Arthritis by Reducing Homing of CCR2hi Osteoclast Progenitors to the Affected Bone.

Detailed characterization of medullary and extramedullary reservoirs of osteoclast progenitors (OCPs) is required to understand the pathophysiology of increased periarticular and systemic bone resorption in arthritis. In this study, we focused on identifying the OCP population specifically induced by arthritis and the role of circulatory OCPs in inflammatory bone loss. In addition, we determined the relevant chemokine axis responsible for their migration, and targeted the attraction signal to reduce bone resorption in murine collagen-induced arthritis (CIA). OCPs were expanded in periarticular as well as circulatory compartment of arthritic mice, particularly the CCR2hi subset. This subset demonstrated enhanced osteoclastogenic activity in arthritis, whereas its migratory potential was susceptible to CCR2 blockade in vitro. Intravascular compartment of the periarticular area contained increased frequency of OCPs with the ability to home to the arthritic bone, as demonstrated in vivo by intravascular staining and adoptive transfer of splenic LysMcre/Ai9 tdTomato-expressing cells. Simultaneously, CCL2 levels were increased locally and systemically in arthritic mice. Mouse cohorts were treated with the small-molecule inhibitor (SMI) of CCR2 alone or in combination with methotrexate (MTX). Preventive CCR2/CCL2 axis blockade in vivo reduced bone resorption and OCP frequency, whereas combining with MTX treatment also decreased disease clinical score, number of active osteoclasts, and OCP differentiation potential. In conclusion, our study characterized the functional properties of two distinct OCP subsets in CIA, based on their CCR2 expression levels, implying that the CCR2hi circulatory-like subset is specifically induced by arthritis. Signaling through the CCL2/CCR2 axis contributes to OCP homing in the inflamed joints and to their increased osteoclastogenic potential. Therefore, addition of CCL2/CCR2 blockade early in the course of arthritis is a promising approach to reduce bone pathology.

Fas receptor is required for estrogen deficiency-induced bone loss in mice.

Bone mass is determined by bone cell differentiation, activity, and death, which mainly occur through apoptosis. Apoptosis can be triggered by death receptor Fas (CD95), expressed on osteoblasts and osteoclasts and may be regulated by estrogen. We have previously shown that signaling through Fas inhibits osteoblast differentiation. In this study we analyzed Fas as a possible mediator of bone loss induced by estrogen withdrawal. At 4 weeks after ovariectomy (OVX), Fas gene expression was greater in osteoblasts and lower in osteoclasts in ovariectomized C57BL/6J (wild type (wt)) mice compared with sham-operated animals. OVX was unable to induce bone loss in mice with a gene knockout for Fas (Fas -/- mice). The number of osteoclasts increased in wt mice after OVX, whereas it remained unchanged in Fas -/- mice. OVX induced greater stimulation of osteoblastogenesis in Fas -/- than in wt mice, with higher expression of osteoblast-specific genes. Direct effects on bone cell differentiation and apoptosis in vivo were confirmed in vitro, in which addition of estradiol decreased Fas expression and partially abrogated the apoptotic and differentiation-inhibitory effect of Fas in osteoblast lineage cells, while having no effect on Fas-induced apoptosis in osteoclast lineage cells. In conclusion, the Fas receptor has an important role in the pathogenesis of postmenopausal osteoporosis by mediating apoptosis and inhibiting differentiation of osteoblast lineage cells. Modulation of Fas effects on bone cells may be used as a therapeutic target in the treatment of osteoresorptive disorders.

RNA sequencing data from osteochondroprogenitor populations in synovial joints of mice during murine model of rheumatoid arthritis.

The aim of this study was to analyze the transcriptome of TER119-CD31-CD45-CD51+CD200+CD105- population (further, CD200+), potential early osteocondroprogenitors, whose frequency is reduced in the joints of mice with antigen-induced arthritis (AIA) [1]. A population defined by similar surface markers has been previously identified as murine skeletal stem cells in bone [2]. In order to confirm their identity this population was compared to TER119-CD31-CD45-CD51+CD200-CD105+ (further, CD105+) cells, which possibly represent committed progenitors, or other non-progenitor population such as synovial fibroblasts. In order to asses changes in CD200+ population in inflammatory setting, it was also compared to the same population from healthy mice. AIA was induced by immunization of mice with methylated bovine serum albumin (mBSA) and subsequent intra-articular injection of mBSA, while non-immunized mice were injected with phosphate-buffered saline at all timepoints. Ten days after intra-articular injection, knee joints were harvested and synovial cells were released by collagenase digestion. Using fluorescence-activated cell sorting, 200-500 cells from selected populations were sorted directly into cell lysis buffer, RNA was reversely transcribed, and first strand cDNA product was amplified. cDNA amplicons were used for library preparation. Bioinformatics analysis was performed using cutadapt [3], HISAT2 [4], Samtools [5] and StringTie [6] tools, and egdeR [7], limma [8], and ClusterProfiler [9] Bioconductor packages. In addition to access to raw data at the NCBI Gene Expression Omnibus repository, this article also provides sample similarity analysis, tables of differentially expressed genes, graphic visualisations of differential expression and gene set enrichment analysis performed on publicly available GO terms. Interpretation of osteochondroprogenitor phenotype of CD200+ population based on analysis of presented data is provided in the article "What do we know about bone morphogenetic proteins and osteochondroprogenitors in inflammatory conditions?" [10]. Reuse of this data may help researchers elucidate alterations of synovial stromal and osteochondroprogenitor populations in inflammatory settings and define their role in structural damage in rheumatoid arthritis.

What do we know about bone morphogenetic proteins and osteochondroprogenitors in inflammatory conditions?

Osteochondroprogenitors are crucial for embryonic bone development and postnatal processes such as bone repair in response to fracture injury, and their dysfunction may contribute to insufficient repair of structural damage in inflammatory arthritides. In the fracture healing, the early inflammatory phase is crucial for normal callus development and new bone formation. This process involves a complex interplay of many molecules and cell types, responsible for recruitment, expansion and differentiation of osteochondroprogenitor populations. In inflammatory arthritides, inflammation induces bone resorption and causes insufficient bone formation, which leads to local and systemic bone loss. While bone loss is a predominant feature in rheumatoid arthritis, inflammation also induces pathologic bone formation at enthesial sites in seronegative spondyloarthropathies. Bone morphogenetic proteins (BMP) are involved in cell proliferation, differentiation and apoptosis, and have fundamental roles in maintenance of postnatal bone homeostasis. They are crucial regulators of the osteochondroprogenitor pool and drive their proliferation, differentiation, and lifespan during bone regeneration. In this review, we summarize the effects of inflammation on osteochondroprogenitor populations during fracture repair and in inflammatory arthritides, with special focus on inflammation-mediated modulation of BMP signaling. We also present data in which we describe a population of murine synovial osteochondroprogenitor cells, which are reduced in arthritis, and characterize their expression of genes involved in regulation of bone homeostasis, emphasizing the up-regulation of BMP pathways in early progenitor subset. Based on the presented data, it may be concluded that during an inflammatory response, innate immune cells induce osteochondroprogenitors by providing signals for their recruitment, by producing BMPs and other osteogenic factors for paracrine effects, and by secreting inflammatory cytokines that may positively regulate osteogenic pathways. On the other hand, inflammatory cells may secrete cytokines that interfere with osteogenic pathways, proapoptotic factors that reduce the pool of osteochondroprogenitor cells, as well as BMP and Wnt antagonists. The net effect is strongly context-dependent and influenced by the local milieu of cells, cytokines, and growth factors. Further elucidation of the interplay between inflammatory signals and BMP-mediated bone formation may provide valuable tools for therapeutic targeting.

The Long Pentraxin 3 Plays a Role in Bone Turnover and Repair.

Pentraxin 3 (PTX3) is an inflammatory mediator acting as a fluid-phase pattern recognition molecule and playing an essential role in innate immunity and matrix remodeling. Inflammatory mediators also contribute to skeletal homeostasis, operating at multiple levels in physiological and pathological conditions. This study was designed to investigate the role of PTX3 in physiological skeletal remodeling and bone healing. Micro-computed tomography (µCT) and bone histomorphometry of distal femur showed that PTX3 gene-targeted female and male mice (ptx3-/- ) had lower trabecular bone volume than their wild-type (ptx3+/+ ) littermates (BV/TV by µCT: 3.50 ± 1.31 vs 6.09 ± 1.17 for females, p < 0.0001; BV/TV 9.06 ± 1.89 vs 10.47 ± 1.97 for males, p = 0.0435). In addition, µCT revealed lower trabecular bone volume in second lumbar vertebra of ptx3-/- mice. PTX3 was increasingly expressed during osteoblast maturation in vitro and was able to reverse the negative effect of fibroblast growth factor 2 (FGF2) on osteoblast differentiation. This effect was specific for the N-terminal domain of PTX3 that contains the FGF2-binding site. By using the closed transversal tibial fracture model, we found that ptx3-/- female mice formed significantly less mineralized callus during the anabolic phase following fracture injury compared to ptx3+/+ mice (BV/TV 17.05 ± 4.59 vs 20.47 ± 3.32, p = 0.0195). Non-hematopoietic periosteal cells highly upregulated PTX3 expression during the initial phase of fracture healing, particularly CD51+ and αSma+ osteoprogenitor subsets, and callus tissue exhibited concomitant expression of PTX3 and FGF2 around the fracture site. Thus, PTX3 supports maintenance of the bone mass possibly by inhibiting FGF2 and its negative impact on bone formation. Moreover, PTX3 enables timely occurring sequence of callus mineralization after bone fracture injury. These results indicate that PTX3 plays an important role in bone homeostasis and in proper matrix mineralization during fracture repair, a reflection of the function of this molecule in tissue homeostasis and repair.

Role of editors and journals in detecting and preventing scientific misconduct: strengths, weaknesses, opportunities, and threats.

Scientific journals have a central place in protecting research integrity because published articles are the most visible documentation of research. We used SWOT analysis to audit (S)trengths and (W)eaknesses as internal and (O)pportunities and (T)hreats as external factors affecting journals' responsibility in addressing research integrity issues. Strengths include editorial independence, authority and expertise, power to formulate editorial policies, and responsibility for the integrity of published records. Weaknesses stem from having no mandate for legal action, reluctance to get involved, and lack of training. Opportunities for editors are new technologies for detecting misconduct, policies by editorial organization or national institutions, and greater transparency of published research. Editors face threats from the lack of legal regulation and culture of research integrity in academic communities, lack of support from stakeholders in scientific publishing, and different pressures. Journal editors cannot be the policing force of the scientific community but they should actively ensure the integrity of the scientific record.

Chemokine signals are crucial for enhanced homing and differentiation of circulating osteoclast progenitor cells.

BACKGROUND: The peripheral blood (PB) monocyte pool contains osteoclast progenitors (OCPs), which contribute to osteoresorption in inflammatory arthritides and are influenced by the cytokine and chemokine milieu. We aimed to define the importance of chemokine signals for migration and activation of OCPs in rheumatoid arthritis (RA) and psoriatic arthritis (PsA). METHODS: PB and, when applicable, synovial fluid (SF) samples were collected from 129 patients with RA, 53 patients with PsA, and 110 control patients in parallel to clinical parameters of disease activity, autoantibody levels, and applied therapy. Receptors for osteoclastogenic factors (CD115 and receptor activator of nuclear factor-κB [RANK]) and selected chemokines (CC chemokine receptor 1 [CCR1], CCR2, CCR4, CXC chemokine receptor 3 [CXCR3], CXCR4) were determined in an OCP-rich subpopulation (CD3-CD19-CD56-CD11b+CD14+) by flow cytometry. In parallel, levels of CC chemokine ligand 2 (CCL2), CCL3, CCL4, CCL5, CXC chemokine ligand 9 (CXCL9), CXCL10, and CXCL12 were measured using cytometric bead array or enzyme-linked immunosorbent assay. Sorted OCPs were stimulated in culture by macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand, and they were differentiated into mature osteoclasts that resorb bone. Selected chemokines (CCL2, CCL5, CXCL10, and CXCL12) were tested for their osteoclastogenic and chemotactic effects on circulatory OCPs in vitro. RESULTS: The OCP population was moderately enlarged among PB cells in RA and correlated with levels of tumor necrosis factor-α (TNF-α), rheumatoid factor, CCL2, and CCL5. Compared with PB, the RANK+ subpopulation was expanded in SF and correlated with the number of tender joints. Patients with PsA could be distinguished by increased RANK expression rather than total OCP population. OCPs from patients with arthritis had higher expression of CCR1, CCR2, CCR4, CXCR3, and CXCR4. In parallel, patients with RA had increased levels of CCL2, CCL3, CCL4, CCL5, CXCL9, and CXCL10, with significant elevation in SF vs PB for CXCL10. The subset expressing CXCR4 positively correlated with TNF-α, bone resorption marker, and rheumatoid factor, and it was reduced in patients treated with disease-modifying antirheumatic drugs. The CCR4+ subset showed a significant negative trend during anti-TNF treatment. CCL2, CCL5, and CXCL10 had similar osteoclastogenic effects, with CCL5 showing the greatest chemotactic action on OCPs. CONCLUSIONS: In our study, we identified distinct effects of selected chemokines on stimulation of OCP mobilization, tissue homing, and maturation. Novel insights into migratory behaviors and functional properties of circulatory OCPs in response to chemotactic signals could open ways to new therapeutic targets in RA.

Bone morphogenetic protein 2 induces cyclo-oxygenase 2 in osteoblasts via a Cbfal binding site: role in effects of bone morphogenetic protein 2 in vitro and in vivo.

We tested the hypothesis that induction of cyclo-oxygenase (COX) 2 mediates some effects of bone morphogenetic protein (BMP) 2 on bone. BMP-2 induced COX-2 mRNA and prostaglandin (PG) production in cultured osteoblasts. BMP-2 increased luciferase activity in calvarial osteoblasts from mice transgenic for a COX-2 promoter-luciferase reporter construct (Pluc) and in MC3T3-E1 cells transfected with Pluc. Deletion analysis identified the -300/-213-bp region of the COX-2 promoter as necessary for BMP-2 stimulation of luciferase activity. Mutation of core-binding factor activity 1 (muCbfal) consensus sequence (5'-AACCACA3') at -267/-261 bp decreased BMP-2 stimulation of luciferase activity by 82%. Binding of nuclear proteins to an oligonucleotide spanning the Cbfal site was inhibited or supershifted by specific antibodies to Cbfal. In cultured osteoblasts from calvariae of COX-2 knockout (-/-) and wild-type (+/+) mice, the absence of COX-2 expression reduced the BMP-2 stimulation of both ALP activity and osteocalcin mRNA expression. In cultured marrow cells flushed from long bones, BMP-2 induced osteoclast formation in cells from COX-2(+/+) mice but not in cells from COX-2(-/-) mice. In vivo, BMP-2 (10 microg/pellet) induced mineralization in pellets of lyophilized collagen implanted in the flanks of mice. Mineralization of pellets, measured by microcomputed tomography (microCT), was decreased by 78% in COX-2(-/-) mice compared with COX-2(+/+) mice. We conclude that BMP-2 transcriptionally induces COX-2 in osteoblasts via a Cbfal binding site and that the BMP-2 induction of COX-2 can contribute to effects of BMP-2 on osteoblastic differentiation and osteoclast formation in vitro and to the BMP-2 stimulation of ectopic bone formation in vivo.

The surface antigen CD45R identifies a population of estrogen-regulated murine marrow cells that contain osteoclast precursors.

We examined the osteoclastogenic potential of murine bone marrow cells that were fractionated according to their expression of the surface antigen CD45R. Osteoclast-like cells (OCL) with many authentic osteoclast characteristics readily formed in purified CD45R(+) murine bone marrow cell cultures after treatment with receptor activator of nuclear factor kappaB ligand (RANKL) and M-CSF. Ovariectomy (Ovx) caused a 1.5- to 2-fold increase in OCL number in unfractionated and CD45R(+) murine bone marrow cell cultures without affecting OCL formation in CD45R(-) marrow cells. Limiting dilution assays confirmed that Ovx caused an increase in osteoclast precursor cell number in CD45R(+) but not CD45R(-) cells. Mice deficient in the type 1 IL-1 receptor (IL-1R1 KO) do not lose bone mass after Ovx. We found that unfractionated, CD45R(+), and CD45R(-) bone marrow cells from IL-1R1 KO mice showed no increase in OCL formation in vitro after Ovx. In both the wild-type (WT) and the IL-1R1 KO mice Ovx was associated with a 2-fold increase in pre-B-lymphocytes. About 1.3-3.5% of murine marrow cells expressed surface RANK (the receptor for RANKL) while about 11.9-15% of murine bone marrow cells expressed c-Fms (the receptor for M-CSF). There was little effect of Ovx on cells expressing either RANK or c-Fms. These results demonstrate that CD45R expression identifies a subset of murine bone marrow cells whose ability to form OCL in vivo is regulated by estrogen in WT but not IL-1R1 KO cells. The effects of estrogen on bone mass may be related to these responses.

Non-functional Fas ligand increases the formation of cartilage early in the endochondral bone induction by rhBMP-2.

It has previously been shown that mice with a defect in Fas ligand-mediated apoptosis have an enhancement of ectopic bone formation. We investigated the expression of bone-related markers--alkaline phosphatase, collagen, bone sialoprotein, osteocalcin, osteopontin, and bone morphogenetic proteins (BMP) -2, -4, and -7; and cytokines interleukin-1alpha (IL-1), IL-1beta, and tumor necrosis factor-alpha (TNF-alpha) in ectopic new bone induced by recombinant human (rh) BMP-2 in mice without functional Fas-ligand (gld mice). At day 6 after rhBMP-2 implantation, gld mice formed more cartilage and mesenchyme compared with their wild type littermates. At later stages, gld mice did not differ from the control mice in the volume of newly formed tissue, expressing higher level of BMP genes and lower levels of genes involved in osteoblast maturation--bone sialoprotein and osteopontin. Differences in the levels of expression of IL-1alpha and TNF-alpha were observed only at day 12 after rhBMP-2 implantation. These results suggest that gld mice have an increased recruitment of cells of mesenchymal origin and an abnormal pattern of differentiation and maturation of the newly formed mesenchymal tissues.

Retractions of scientific publications: responsibility and accountability.

This evidence-based opinion piece gives a short overview of the increase in retractions of publications in scientific journals and discusses various reasons for that increase. Also discussed are some of the recent prominent cases of scientific misconduct, the number of authors with multiple retractions, and problems with reproducibility of published research. Finally, some of the effects of faulty research on science and society, as well as possible solutions are discussed.

Acute hematopoietic stress in mice is followed by enhanced osteoclast maturation in the bone marrow microenvironment.

Osteoclasts are components of hematopoietic stem cell (HSC) niches, but their role as contributors to the HSC homeostasis and release are still controversial. We aimed to investigate whether an acute blood loss of 10% of total blood content, along with the consequent intense hematopoiesis, would affect osteoclast differentiation and activity. Isolated peripheral blood, spleen, and bone marrow (BM) cells from bones of hind limbs were investigated for the presence of specific subpopulations of osteoclast precursors: B220(-)CD3(-)NK1.1(-)CD11b(-/low)CD115(+)CD117(+) cells in BM, and B220(-)CD3(-)NK1.1(-)Gr-1(-)CD11b(+)CD115(+) cells in peripheral blood and spleen as well as the receptor activator of nuclear factor κ-B(+) cycle-arrested quiescent osteoclast precursors. Expression of osteoclastogenesis-related genes CD115, receptor activator of nuclear factor κ-B, and cathepsin K, the potential of BM cells to form osteoclast-like cells in vitro, and osteoclast activity in vivo were also evaluated. We observed an increase in spleen cellularity and myelopoiesis during week 1 following blood loss, without any significant effects on BM cellularity or BM myeloid precursors, including cells with high osteoclastogenic potential. However, at 1 week postbleeding, hematopoiesis significantly promoted the expression of cathepsin K, interleukin-34, and bone morphogenetic protein-6. Quiescent osteoclast precursors increased significantly in spleen 2 days following bleeding, whereas osteoclast activity remained unchanged up to 2 weeks postbleeding. Osteoclast-dependent B-cell differentiation was affected at the pre-B stage of maturation in BM, whereas the Lin(-)Sca-1(+)c-kit(+) population expanded in BM and spleen after 2 days postbleeding. Our data demonstrate that an acute blood loss promotes differentiation and maturation of osteoclasts at 1 week but does not enhance osteoresorption at 2 weeks postbleeding. Our data also identify osteoclast differentiation as a consequent and important event in establishing HSC homeostasis following hematopoietic stress.