Remodeling Cycle Research Articles

RNAmetasome network for macromolecule biogenesis in human cells

RNA plays a central role in macromolecule biogenesis for various pathways, such as gene expression, ribosome biogenesis, and chromatin remodeling. However, RNA must be converted from its nascent to functional forms for that role. Here, we describe a large RNA metabolic network (RNAmetasome network) for macromolecule biogenesis in human cells. In HEK293T, the network consists of proteins responsible for gene expression, splicing, ribosome biogenesis, chromatin remodeling, and cell cycle. Reciprocal immunoprecipitations show that MKI67, GNL2, MDN1, and ELMSAN1 are core proteins of the network, and knockdown of either MKI67 or GNL2 affects the state of the other protein, MDN1, and some other network members. Furthermore, GNL2 knockdown retards cell proliferation. Several proteins of the RNAmetasome network are diminished in Hela.cl1, and this diminishment is associated with low expression of MDN1 and elevated MKI67 degradation. These results together suggest that the RNAmetasome network is present in human cells and associated with proliferation, and that MKI67, GNL2, and MDN1 play an important role in organizing the RNAmetasome network.

Practical Considerations for the Clinical Application of Bone Turnover Markers in Osteoporosis.

Bone turnover markers (BTMs) are released during the bone remodelling cycle and are measurable in blood or urine, reflecting bone remodelling rate. They have been useful in elucidating the pharmacodynamics and effectiveness of osteoporosis medication in clinical trials and are increasingly used in routine clinical management of osteoporosis, especially for monitoring therapy, in addition to their use in other metabolic bone disease such as Paget's disease of bone and osteomalacia. Serum β isomerised C-terminal telopeptide of type I collagen and pro-collagen I N-terminal propeptide have been designated as reference BTMs for use in osteoporosis. In addition, bone-specific isoenzymeof alkaline phosphatase (B-ALP) secreted by osteoblasts and tartrate-resistant acid phosphatase 5b (TRACP-5b) secreted by osteoclasts are also found to be specific markers of bone formation and resorption, respectively. The concentrations of the latter enzymes in blood measured by immunoassay provide reliable measures of bone turnover even in the presence of renal failure. B-ALP is recommended for use in the assessment of renal bone disease of chronic kidney disease, and TRACP-5b shows promise as a marker of bone resorption in that condition. BTMs in blood do not suffer from biological variation to the same extent as the older BTMs that were measured in urine. Appropriate patient preparation and sample handling are important in obtaining accurate measures of BTMs for clinical use. Reference change values and treatment targets have been determined for the reference BTMs for their use in monitoring osteoporosis treatment. Further ongoing studies will enhance their clinical applications.

Lipid Interactions Between Flaviviruses and Mosquito Vectors.

Mosquito-borne flaviviruses, such as dengue (DENV), Zika (ZIKV), yellow fever (YFV), West Nile (WNV), and Japanese encephalitis (JEV) viruses, threaten a large part of the human populations. In absence of therapeutics and effective vaccines against each flaviviruses, targeting viral metabolic requirements in mosquitoes may hold the key to new intervention strategies. Development of metabolomics in the last decade opened a new field of research: mosquito metabolomics. It is now clear that flaviviruses rely on mosquito lipids, especially phospholipids, for their cellular cycle and propagation. Here, we review the biosyntheses of, biochemical properties of and flaviviral interactions with mosquito phospholipids. Phospholipids are structural lipids with a polar headgroup and apolar acyl chains, enabling the formation of lipid bilayer that form plasma- and endomembranes. Phospholipids are mostly synthesized through the de novo pathway and remodeling cycle. Variations in headgroup and acyl chains influence phospholipid physicochemical properties and consequently the membrane behavior. Flaviviruses interact with cellular membranes at every step of their cellular cycle. Recent evidence demonstrates that flaviviruses reconfigure the phospholipidome in mosquitoes by regulating phospholipid syntheses to increase virus multiplication. Identifying the phospholipids involved and understanding how flaviviruses regulate these in mosquitoes is required to design new interventions.

Antigen receptor therapy in bone metastasis via optimal control for different human life stages

In this work we propose a bone metastasis model using power law growth functions in order to describe the biochemical interactions between bone cells and cancer cells. Experimental studies indicate that bone remodeling cycles are different for human life stages: childhood, young adulthood, and adulthood. In order to include such differences in our study, we estimate the model parameter values for each human life stage via bifurcation analysis. Results reveal an intrinsic relationship between the active period of remodeling cycles and the proliferation of cancer cells. Subsequently, using optimal control theory we analyze a possible antigen receptor therapy as a new treatment for bone metastasis. Theoretical results such as existence of optimal solutions are proved. Numerical simulations for late stages of bone metastasis are presented and a discussion of our results is carried out.

Are Osteoclasts Mechanosensitive Cells?

Skeleton metabolism is a process in which osteoclasts constantly remove old bone and osteoblasts form new osteoid and induce mineralization; disruption of this balance may cause diseases. Osteoclasts play a key role in bone metabolism, as osteoclastogenesis marks the beginning of each bone remodeling cycle. As the only cell capable of bone resorption, osteoclasts are derived from the monocyte/macrophage hematopoietic precursors that terminally adhere to mineralized extracellular matrix, and they subsequently break down the extracellular compartment. Bone is generally considered the load-burdening tissue, bone homeostasis is critically affected by mechanical conductions, and the bone cells are mechanosensitive. The functions of various bone cells under mechanical forces such as chondrocytes and osteoblasts have been reported; however, the unique bone-resorbing osteoclasts are less studied. The oversuppression of osteoclasts in mechanical studies may be because of its complicated differentiation progress and flexible structure, which increases difficulty in targeting mechanical structures. This paper will focus on recent findings regarding osteoclasts and attempt to uncover proposed candidate mechanosensing structures in osteoclasts including podosome-associated complexes, gap junctions and transient receptor potential family (ion channels). We will additionally describe possible mechanotransduction signaling pathways including GTPase ras homologue family member A (RhoA), Yes-associated protein/transcriptional co-activator with PDZ-binding motif (TAZ), Ca2+ signaling and non-canonical Wnt signaling. According to numerous studies, evaluating the possible influence of various physical environments on osteoclastogenesis is conducive to the study of bone homeostasis.

Systems-Level Proteomics Evaluation of Microglia Response to Tumor-Supportive Anti-Inflammatory Cytokines.

BackgroundMicroglia safeguard the CNS against injuries and pathogens, and in the presence of certain harmful stimuli are capable of inducing a disease-dependent inflammatory response. When exposed to anti-inflammatory cytokines, however, these cells possess the ability to switch from an inflammatory to an immunosuppressive phenotype. Cancer cells exploit this property to evade the immune system, and elicit an anti-inflammatory microenvironment that facilitates tumor attachment and growth.ObjectiveThe tumor-supportive biological processes that are activated in microglia cells in response to anti-inflammatory cytokines released from cancer cells were explored with mass spectrometry and proteomic technologies.MethodsSerum-depleted and non-depleted human microglia cells (HMC3) were treated with a cocktail of IL-4, IL-13, IL-10, TGFβ, and CCL2. The cellular protein extracts were analyzed by LC-MS/MS. Using functional annotation clustering tools, statistically significant proteins that displayed a change in abundance between cytokine-treated and non-treated cells were mapped to their biological networks and pathways.ResultsThe proteomic analysis of HMC3 cells enabled the identification of ~10,000 proteins. Stimulation with anti-inflammatory cytokines resulted in the activation of distinct, yet integrated clusters of proteins that trigger downstream a number of tumor-promoting biological processes. The observed changes could be classified into four major categories, i.e., mitochondrial gene expression, ECM remodeling, immune response, and impaired cell cycle progression. Intracellular immune activation was mediated mainly by the transducers of MAPK, STAT, TGFβ, NFKB, and integrin signaling pathways. Abundant collagen formation along with the expression of additional receptors, matrix components, growth factors, proteases and protease inhibitors, was indicative of ECM remodeling processes supportive of cell-cell and cell-matrix adhesion. Overexpression of integrins and their modulators was reflective of signaling processes that link ECM reorganization with cytoskeletal re-arrangements supportive of cell migration. Antigen processing/presentation was represented by HLA class I histocompatibility antigens, and correlated with upregulated proteasomal subunits, vesicular/viral transport, and secretory processes. Immunosuppressive and proangiogenic chemokines, as well as anti-angiogenic factors, were detectable in low abundance. Pronounced pro-inflammatory, chemotactic or phagocytic trends were not observed, however, the expression of certain receptors, signaling and ECM proteins indicated the presence of such capabilities.ConclusionsComprehensive proteomic profiling of HMC3 cells stimulated with anti-inflammatory cytokines revealed a spectrum of microglia phenotypes supportive of cancer development in the brain via microenvironment-dependent biological mechanisms.

Nanomaterial-induced mesenchymal stem cell differentiation into osteoblast for counteracting bone resorption in the osteoporotic rats

The current approach was designed to unearth the therapeutic potential of osteoblasts infusion, yielded from cultivating rat mesenchymal stem cells of bone marrow source in osteogenic differentiation media supplied with either hydroxyapatite nanoparticles (HA-NPs), chitosan/hydroxyapatite nanomaterials (C/HA-NPs), or chitosan nanoparticles, in the osteoporotic rats. The successful migration of the osteoblasts to the diseased bones of rats in C/HA-NPs and HA-NPs groups was evidenced by PCR screening of the Y-linked sex-determining gene (SRY) in the femoral bone tissue. Serum bone biomarker levels and gene expression patterns of cathepsin K, receptor activator of nuclear factor kappa B ligand (RANKL), and osteoprotegerin (OPG) were assessed. Additionally, histological examination of the femoral bone tissues of rats was performed. The current outcomes revealed that osteoblast implantation, resulted from C/HA-NPs or HA-NPs group, significantly lessened bone sialoprotein level. In Addition, it yielded a significant decline in the gene expression patterns of cathepsin K, RANKL, and RANKL/OPG proportion as well as up-regulation in BMP-2 and Runx-2 gene expression levels as opposed to the untreated ovariectomized animals. Moreover, it could restrain bone resorption and refine bone histoarchitecture. Conclusively, this study sheds light on the therapeutic significance of osteoblasts transplantation in alleviating the intensity of the bone remodeling cycle, consequently representing a hopeful therapeutic approach for primary osteoporosis.

Экспрессия PCNA как маркера пролиферации в доброкачественных и высокодифференцированных злокачественных опухолях щитовидной железы человека (обзор литературы и собственные данные)

Определение пролиферативного потенциала клеток опухоли — важная задача для диагностики рака щитовидной железы (ЩЖ). Поэтому необходим поиск маркеров, характеризующих состояние пролиферативных процессов. Ядерный антиген пролиферирующих клеток (PCNA) — белок, необходимый для организации компонентов, участвующих в процессах репликации и репарации ДНК, является наряду с Ки-67 одним из основных маркеров пролиферативных процессов. Последние исследования показали его способность взаимодействовать со многими факторами, участвующими в нескольких метаболических путях, включая репарацию ДНК, транслезионный синтез ДНК, метилирование ДНК, ремоделирование хроматина и регуляцию клеточного цикла. В обзоре приведены данные о его структуре, функции, регуляции и участии в злокачественной трансформации клеток. Для определения биомаркеров, полезных для диагностики рака ЩЖ и установки панели маркеров по раннему выявлению метастатических карцином ЩЖ, был проведен ряд исследований, показавших, что в метастатических опухолях уровень PCNA выше почти в 2 раза, чем в неметастатических. Отмечена повышенная экспрессия PCNA в карциномах щитовидной железы по сравнению с аденомами. Самый высокий уровень экспрессии PCNA наблюдался в наиболее агрессивных типах рака ЩЖ — анапластической и медуллярной карциномах. В дифференцированных опухолях количество антигена было несколько меньше, но заметно возрастало в инвазивных вариантах этих опухолей. Экспрессия PCNA была усилена в B-RAF V600E-позитивных папиллярных карциномах по сравнению с B-RAF V600E-отрицательными. Анализ выживаемости больных, которую связывали с уровнями маркеров PCNA и Ki-67, показал лучший прогноз у пациентов с низкими показателями PCNA. Худшие результаты наблюдались у пациентов со средними значениями PCNA, а наихудший прогноз характерен для пациентов с высокими уровнями антигена. Обсуждаются возможности подавления PCNA специфическими ингибиторами для терапии некоторых видов рака.

Abstract 2082: Deciphering the nucleosome acidic patch binding at amino acid resolution

Abstract In all eukaryotic cells, nucleosomes provide basic organization of genomic DNA and serve as signaling hubs for gene expression, DNA replication and DNA damage repair. These genome-templated processes are tuned by chromatin enzymes that introduce chemical modifications to nucleosomes and control their positioning and occupancy. Mutations in genes encoding chromatin enzymes are highly correlated with numerous cancer types. A thorough exploration of how these chromatin enzymes act on their physiologic target, the nucleosome, is critical to understanding abnormal DNA methylation, histone posttranslational modifications, and chromatin composition, leading to cancer. In our recent nucleosome interactome proteomic screen, we demonstrated that the vast majority of nuclear proteins require a group of acidic residues on the nucleosome disk surface to interact with nucleosomes. This acidic patch and an adjacent region were recognized by over 50% and 18% of nuclear proteins, respectively, while almost half of the nucleosome disk surface contributed only minimally to nucleosome recognition. Overall, we paired about 300 proteins representing diverse nuclear functions including DNA methylation (DNMT1 and 3, Tet1), histone modification (HAT complexes, kinases), nucleosome remodeling (SWI/SNF), DNA repair (MRN complex), DNA replication (ORC complex), RNA processing, and cell cycle progression (APC/C) with specific nucleosome surface requirements. We have now repeated our screen at amino acid resolution using a library of fifteen nucleosomes containing single point mutations in and around the acid patch, including potential histone oncomutations identified to be overrepresented in cancers. Using our nucleosome library, we preformed pulldowns from human embryonic kidney 293 cell nuclear lysates to comprehensively evaluate the role of single histone residues in nucleosome recognition. Overall, this study dissects the primary nucleosome binding hot-spot at high resolution and provide a robust resource for further exploration of the nucleosome interactome in normal and cancer cell biology. Citation Format: Aleksandra Skrajna, Holly Simmons, Charlotte Smith, Jeanne-Marie E. Mcpherson, Dennis Goldfarb, Robert K. McGinty. Deciphering the nucleosome acidic patch binding at amino acid resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2082.

HER2 in uterine serous carcinoma: Testing platforms and implications for targeted therapy.

5580 Background: HER2 is an emerging prognostic and therapeutic target in uterine serous carcinoma (USC). Testing algorithms and platforms in breast and gastric cancers are well studied and validated, but optimal HER2 testing in uterine cancer is not yet established. We aimed to assess the concordance of chromogenic in situ hybridization (CISH), immunohistochemistry (IHC), and next generation sequencing (NGS) platforms to aid in the development of USC specific testing guidelines. We also evaluated the rate of downstream mutations that may affect response to HER2 directed therapy. Methods: A total of 2,192 USC tumors were analyzed using NGS (NextSeq, 592 Genes and WES, NovaSEQ), a subset of 1,423 tumors were also tested by IHC and CISH (Caris Life Sciences, Phoenix, AZ). HER2 positivity through IHC (4B5, Ventana) and CISH (INFORM DUAL HER2 ISH Assay, Ventana) was determined based on 2007 and 2018 ASCO/CAP HER2 breast cancer guidelines. PD-L1 expression was tested by IHC using SP142 (Spring Biosciences) (positive cut-off >1%). Microsatellite instability (MSI) was tested by fragment analysis (FA), IHC and NGS. Tumor mutational burden (TMB) was measured by totaling somatic mutations per tumor (TMB-high cut-off > 10 mutations per Mb). Statistical significance was determined using chi-square. Results: Rates of HER2 positivity were comparable using the 2018 and 2007 breast cancer guidelines (19.5% vs 17.5%; p=0.25). Based on 2018 guidelines, the concordance between IHC and CISH was 98.9%. Specifically, 229/1423 patients (16%) were IHC+/CISH+, 5 patients (0.4%) were IHC+/CISH- and 11 patients (0.8%) were IHC-/CISH+ (Table). Common pathway alterations in HER2+ USC include TP53, RTK RAS, PI3K, NOTCH, chromatin remodeling and cell cycle genes. Single gene alterations in HER2+ tumors that may implicate HER2 therapy resistance (based on pathway analyses in other tumor types) included PI3K (36%), KRAS (2.6%), and PTEN (2.1%). HER2+ tumors had low immunotherapy biomarker profiles (0.3% MSI-H, 0.8% TMB, 17.1% PD-L1). Conclusions: High concordance rates were observed between CISH and IHC. Ultimately these testing platforms need to be validated by response to HER2 targeted therapies in order to develop USC specific HER2 testing guidelines.[Table: see text]

Comparative characteristics of bone marrow cell composition, stroma, and trabecular bone in allogenic hematopoietic stem cell transplantation in patients with primary myelofibrosis

Introduction. Primary myelofibrosis (PMF) is a clonal disease violating the cell composition, histological topography and stroma in bone marrow (BM). Allogeneic haematopoietic stem cell transplantation (allo-HSCT) is a curative therapy in PMF.Aim — description of change in the haematopoietic tissue cell composition and stroma, as well as in trabecular bone in allo-HSCT patients with fibrotic PMF.Materials and methods. We studies 24 trephine biopsy samples from nine PMF patients with allo-HSCT at the intervals: I — 1 month prior to, II — past 1–3 months and III — past 4–6 months from allo-HSCT. BM trephine biopsy slides were prepared in a standard histological assay with haematoxylin—eosin and additional staining with Gomori’s silver and Masson’s trichrome. Morphological change was evaluated in reticulin and collagen stroma, bone trabeculae, cellularity and topography of haematopoietic tissue.Results. The BM trephine biopsies of interval I were morphologically distinguished in three types by haematopoietic cellularity, stromal and trabecular sclerotic change. Post-transplant intervals II and III (3–6 months after allo-HSCT) did not reveal these types but showed an evident myelofibrosis and osteosclerosis reduction and signs of a restoring bone remodelling cycle. Myelopoietic lineages recovered in stages: the erythroid germ restored in three, granulocytic — in six months, and megakaryocytic cellularity did not fully recover in six months. Myelopoietic cellularity recovery outpaced blood recovery, which may be due to induced myelodysplasia or disruption of stromal niches.Conclusion. Allo-HSCT leads to the disappearance of PMF-pathognomonic BM morphology reflecting a histological remission. The reduction of myelofibrosis and osteosclerosis and normalisation of the trabecular bone remodelling cycle in post-transplant periods indicates an impact of cell microenvironment on PMF pathogenesis and warrants research into the composition and histological topography of cell microenvironment in PMF.

Disturbance of osteonal bone remodeling and high tensile stresses on the lateral cortex in atypical femoral fracture after long-term treatment with Risedronate and Alfacalcidol for osteoporosis

An 83 year-old Japanese woman complained of left lateral thigh pain following a low-energy fall 4 months prior to admission. She had been treated for osteoporosis with Risedronate and Alfacalcidol for the previous five years. She was diagnosed with an atypical femoral fracture (AFF) according to the American Society for Bone and Mineral Research (ASBMR) Task Force revised criteria. Radiographs revealed cortical thickening and a transverse radiolucent fracture line in the lateral cortex of the shaft. MRI showed a high intensity signal on the T2WI image 1 cm long in the lateral cortex. The patient had normal levels of bone resorption and formation biomarkers except for low 25(OH) Vitamin D. Double fluorescent labeling was done preoperatively.Due to significant bowing, a corrective osteotomy and intramedullary nailing were performed, and the resected bone wedge was analyzed by bone histomorphometry. Three ground sections of the lateral cortex at the fracture site showed many and large pores, with or without tetracycline labeling. Histomorphometric assessment was done on intracortical pores, classified by a novel criteria, only to assess size of the pores to know prolonged osteoclastic activity and its characteristics of inner surfaces to assess whether bone formation has been occurring or not in labeling period in remodeling cycle, and coalition of multi-pores. Increased size with widespread variation of pores suggested prolonged osteoclastic activity in the reversal/resorptive phase. Bone labeling showed lamellar bone on the endocortical surface.We hypothesize that the case had developed from a regional disturbance of osteonal remodeling in the lateral cortex, in which accumulated microcracks might have initiated a resorption process resulting in resorption cavities, i.e., pores, which became larger due to prolonged activity of secondary osteoclasts. Various sized pores could form lamellar bone, still forming at the time of biopsy, some had formed lamellar bone, but stopped to form before labeling and not to start to form at all, probably due to incomplete coupling. Endocortical lamellar bone might had started to resorbed to smooth off endocortical surface, followed by formation of lamellar bone. The endocortical bone formation was assessed and its formation period is about 2.7 years.A finite element analysis using preoperative CT data revealed high tensile stresses on the lateral aspect of the femur. Histomorphometric results suggest that there might be more pores in the tensile area than the compressive area. These findings may subsequently connect accumulation of microcracks, an increase of size and number of pores and coalition and subsequent fracture in the lateral cortex.

Distinct signaling by insulin and IGF-1 receptors and their extra- and intracellular domains

Insulin and insulin-like growth factor 1 (IGF-1) receptors share many downstream signaling pathways but have unique biological effects. To define the molecular signals contributing to these distinct activities, we performed global phosphoproteomics on cells expressing either insulin receptor (IR), IGF-1 receptor (IGF1R), or chimeric IR-IGF1R receptors. We show that IR preferentially stimulates phosphorylations associated with mammalian target of rapamycin complex 1 (mTORC1) and Akt pathways, whereas IGF1R preferentially stimulates phosphorylations on proteins associated with the Ras homolog family of guanosine triphosphate hydrolases (Rho GTPases), and cell cycle progression. There were also major differences in the phosphoproteome between cells expressing IR versus IGF1R in the unstimulated state, including phosphorylation of proteins involved in membrane trafficking, chromatin remodeling, and cell cycle. In cells expressing chimeric IR-IGF1R receptors, these differences in signaling could be mapped to contributions of both the extra- and intracellular domains of these receptors. Thus, despite their high homology, IR and IGF1R preferentially regulate distinct networks of phosphorylation in both the basal and stimulated states, allowing for the unique effects of these hormones on organismal function.

Abstract IA007: The mutational landscape of normal human endometrial epithelium

Abstract Human endometrium is a highly dynamic tissue that undergoes numerous cycles of breakdown, rapid repair and remodelling in response to the oscillating levels of oestrogen and progesterone. The marked regenerative capacity of the tissue’s epithelial compartment is maintained by the intra-glandular adult stem cells (ASCs) that are retained during menstruation. Although the endometrial epithelial ASCs were first described over a decade ago, they remain poorly characterised in comparison to their counterparts in other tissues, such as the small and arge intestine. In particular, the size of the stem cell pool within individual glands, clonal dynamics and genomic landscape are largely unknown. In this study, we laser-capture microdissected 257 morphologically normal endometrial glands from pre- and post-menopausal women (aged 19 to 81). In selected donors, we also obtained samples of normal endocervical and Fallopian tubular epithelium. Isolated cellular material was subjected to our modified library preparation protocol and subsequent whole genome sequencing (WGS). Analysis of the WGS data identified that the majority of the glands were clonal cell populations sharing a recent common ancestor. Somatic mutations accumulated at a rate of 29 substitutions per year. Total mutation burden in the normal endometrial epithelium is higher than in other normal cells and manyfold lower than in endometrial cancers. Overall, despite the heterogeneity in age, reproductive history and BMI, we found relatively homogenous mutational processes to be operative in this tissue with only occasional outliers. Remarkably, we not only identify recurrent acquisitions of certain cancer-associated mutations, particularly those that are advantageous to cell growth, proliferation and migration, but also show that such events occur early in life, potentially even before adolescence. Using a generalised linear mixed effect model, we found that age has a positive association with the accumulation of driver mutations, while parity has a negative association. Over time, the mutant ASCs serve as a reservoir for the acquisition of further driver mutations to the extent that in some cases, the entire sampled endometrium becomes ‘neoplastic’ on the genomic level while still retaining the apparently normal phenotype. Our data identify a distinct mutational landscape in normal endometrium that is in keeping with the presence of early positive selection in this highly regenerative tissue. Citation Format: Luiza Moore. The mutational landscape of normal human endometrial epithelium [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr IA007.

Trabecular bone organoid model for studying the regulation of localized bone remodeling.

Trabecular bone maintains physiological homeostasis and consistent structure and mass through repeated cycles of bone remodeling by means of tightly localized regulation. The molecular and cellular processes that regulate localized bone remodeling are poorly understood because of a lack of relevant experimental models. A tissue-engineered model is described here that reproduces bone tissue complexity and bone remodeling processes with high fidelity and control. An osteoid-inspired biomaterial-demineralized bone paper-directs osteoblasts to deposit structural mineralized bone tissue and subsequently acquire the resting-state bone lining cell phenotype. These cells activate and shift their secretory profile to induce osteoclastogenesis in response to chemical stimulation. Quantitative spatial mapping of cellular activities in resting and activated bone surface coculture showed that the resting-state bone lining cell network actively directs localized bone remodeling by means of paracrine signaling and cell-to-cell contact. This model may facilitate further investigation of trabecular bone niche biology.

GDNF secreted by pre-osteoclasts induces migration of bone marrow mesenchymal stem cells and stimulates osteogenesis

Bone resorption is linked to bone formation via temporal and spatial coupling within the remodeling cycle. Several lines of evidence point to the critical role of coupling factors derived from pre-osteoclasts (POCs) during the regulation of bone marrow-derived mesenchymal stem cells (BMMSCs). However, the role of glial cell-derived neurotrophic factor (GDNF) in BMMSCs is not completely understood. Herein, we demonstrate the role of POC-derived GDNF in regulating the migration and osteogenic differentiation of BMMSCs. RNA sequencing revealed GDNF upregulation in POCs compared with monocytes/macrophages. Specifically, BMMSC migration was inhibited by a neutralizing antibody against GDNF in pre-osteoclast-conditioned medium (POC-CM), whereas treatment with a recombinant GDNF enhanced migration and osteogenic differentiation. In addition, POC-CM derived from GDNF knockdowned bone marrow macrophages suppressed BMMSC migration and osteogenic differentiation. SPP86, a small molecule inhibitor, inhibits BMMSC migration and osteogenic differentiation by targeting the receptor tyrosine kinase RET, which is recruited by GDNF into the GFRα1 complex. Overall, this study highlights the role of POC-derived GDNF in BMMSC migration and osteogenic differentiation, suggesting that GDNF regulates bone meta-bolism.

Response of Gli1+ Suture Stem Cells to Mechanical Force Upon Suture Expansion.

Normal development of craniofacial sutures is crucial for cranial and facial growth in all three dimensions. These sutures provide a unique niche for suture stem cells (SuSCs), which are indispensable for homeostasis, damage repair, as well as stress balance. Expansion appliances are now routinely used to treat underdevelopment of the skull and maxilla, stimulating the craniofacial sutures through distraction osteogenesis. However, various treatment challenges exist due to a lack of full understanding of the mechanism through which mechanical forces stimulate suture and bone remodeling. To address this issue, we first identified crucial steps in the cycle of suture and bone remodeling based on the established standard suture expansion model. Observed spatiotemporal morphological changes revealed that the remodeling cycle is approximately 3to4 weeks, with collagen restoration proceeding more rapidly. Next, we traced the fate of the Gli1+ SuSCs lineage upon application of tensile force in three dimensions. SuSCs were rapidly activated and greatly contributed to bone remodeling within 1 month. Furthermore, we confirmed the presence of Wnt activity within Gli1+ SuSCs based on the high co-expression ratio of Gli1+ cells and Axin2+ cells, which also indicated the homogeneity and heterogeneity of two cell groups. Because Wnt signaling in the sutures is highly upregulated upon tensile force loading, conditional knockout of β-catenin largely restricted the activation of Gli1+ SuSCs and suppressed bone remodeling under physiological and expansion conditions. Thus, we concluded that Gli1+ SuSCs play essential roles in suture and bone remodeling stimulated by mechanical force and that Wnt signaling is crucial to this process. © 2022 American Society for Bone and Mineral Research (ASBMR).

The Dramatic Role of IFN Family in Aberrant Inflammatory Osteolysis.

Skeletal system has been considered a highly dynamic system, in which bone-forming osteoblasts and bone-resorbing osteoclasts go through a continuous remodeling cycle to maintain homeostasis of bone matrix. It has been well acknowledged that interferons (IFNs), acting as a subgroup of cytokines, not only have crucial effects on regulating immunology but also could modulate the dynamic balance of bone matrix. In the light of different isoforms, IFNs have been divided into three major categories in terms of amino acid sequences, recognition of specific receptors and biological activities. Currently, type I IFNs consist of a multi-gene family with several subtypes, of which IFN-α exerts pro-osteoblastogenic effects to activate osteoblast differentiation and inhibits osteoclast fusion to maintain bone matrix integrity. Meanwhile, IFN-β suppresses osteoblast-mediated bone remodeling as well as exhibits inhibitory effects on osteoclast differentiation to attenuate bone resorption. Type II IFN constitutes the only type, IFN-γ, which exerts regulatory effects on osteoclastic bone resorption and osteoblastic bone formation by biphasic ways. Interestingly, type III IFNs are regarded as new members of IFN family composed of four members, including IFN-λ1 (IL-29), IFN-λ2 (IL-28A), IFN-λ3 (IL-28B) and IFN-λ4, which have been certified to participate in bone destruction. However, the direct regulatory mechanisms underlying how type III IFNs modulate the metabolic balance of bone matrix, remains poorly elucidated. In this review, we have summarized functions of IFN family during physiological and pathological conditions and described the mechanisms by which IFNs maintain bone matrix homeostasis via affecting the osteoclast-osteoblast crosstalk. In addition, the potential therapeutic effects of IFNs on inflammatory bone destruction diseases such as rheumatoid arthritis (RA), osteoarthritis (OA) and infectious bone diseases are also well displayed, which are based on the predominant role of IFNs in modulating the dynamic equilibrium of bone matrix.

Finite element analysis of human skull bone adaptation to mechanical loading

Bones self-optimize their mechanical behavior in response to mechanical stimulus. The objective of this research was to develop an integrated bone remodeling and stress binning algorithms into a finite element environment to elucidate the evolution of the bone properties as a function of loading. The bone remodeling algorithm was used to calculate the change in the density and elastic modulus based on the strain energy stimulus. The stress-binning procedure seeks to assign the properties to each element based on the levels of stress from the previous cycle, eliminating pseudo-lazy-zoning and stress dilation effects. The developed algorithms were used to analyze the response skull to loading associated with orthodontic devices. Specifically, a load was applied between the roots of the canine teeth and the first premolars while constraining the foramen magnum. Full-field contours of the displacement, strain, and strain energy were extracted after each remodeling cycle at nine commonly cephalometric landmarks. The results indicate that the overall mechanical response and the associated properties reached a steady-state behavior after nearly 50 cycles of applying the algorithm, where different zones within the skull exhibited unique evolution based on the locations from the loading and boundary sites. When approaching this steady-state condition, it was found that the upper incisor displacement is reduced by 72%, and the density is reduced by almost 7.5%. The finite element approach can be used in defining the treatment process by dynamically changing the loads. Future research will focus on integrating the time-dependent behavior of the bone.

Unraveling Actionable Target Mutations in Formalin Fixed Tissue in Mantle Cell Lymphoma

Introduction: Mantle Cell Lymphoma (MCL) is a B-cell neoplasm with poor prognosis and high relapse frequency. It follows an heterogenous clinical course and, despite clear improvements in survival among young and fit patients, most patients eventually relapse. Recurrent mutations have been previously described, pinpointing alterations in DNA damage pathways, cell survival and cell-cycle regulation. Nevertheless, the current genomic knowledge of the disease cannot fully explain its high clinical variability. The present study aimed at revealing the mutation profile for the development of personalized treatment approaches in a population-based cohort of patients diagnosed with MCL. Material & Methods: We performed sequencing across ~230 lymphomagenesis-related genes on 75 formalin-fixed paraffin-embedded (FFPE) samples from symptomatic patients diagnosed with MCL in Southern Sweden. The panel covered the exonic parts of the chosen genes with capture probes provided by TWIST Bioscience. Sequence alignment was done against the human genome version 37 and variant calling was performed resorting to Sention TNscope. IGV was used for manual validation of each identified mutation. Samples were pre-selected according to DNA quality. Data on patient characteristics, prognostic factors and treatment was extracted from the Swedish Lymphoma Register. Results: MCL tumors were highly heterogenous with respect to the mutational status of the analyzed genes, ranging between 0 and 45 mutated genes in this cohort. ATM was the most commonly mutated gene (49%), followed by KMT2D, NOTCH2, TP53, FAT4, BIRC3, CCND1, UBR5, MEF2B, NOTCH1,SMARCA4, DNAH5,SAMHD1 and SP140 (Figure 1). Our results are in accordance with the previously published analyses, although the frequency of samples with mutated NOTCH2 was higher (18.7%, compared to 3-12.5% of affected tumors in previous studies), and with less samples with mutated NOTCH3 (6.7% of the studied tumors were mutated, vs 12.5% reported by Yang et al., (2018)). We found mutations in DNAH5, PCLO, FAT3 and BRCA1 genes, which have not been previously reported in MCL. Additionally, FAT4 (14.7%) showed recurrent mutations in our cohort, but has only been referred as commonly mutated in MCL before by Zhang et al. (2014). Main affected pathways were NOTCH-related and TP53-related pathways, followed by chromatin remodeling and cell cycle pathways. Due to low numbers of patients with non-classic morphology, no mutations were significantly different comparing classical and non-classical histology, although there was an enrichment of TP53 and NOTCH1 mutations in non-classic cases. It seems that cases with non-classic morphology have stronger driver mutations, which may reflect their aggressiveness. Cases with high proliferation (Ki67 >30%) showed a higher frequency of SMARCA4 (30%, vs 7.7% for low proliferation tumors) and TP53 mutations (36% in highly proliferative tumors vs. 17% in low proliferative tumors). No differences in mutation patterns were found in cases with low vs. high SOX11 expression. Unsurprisingly, TP53 mutations were the strongest independent factor for prognosis among the studied genes. Conclusions: Using the results from a population-based cohort of MCL, we report the feasibility of applying a targeted panel and sequencing technologies on DNA from formalin fixed tissue, as a method applicable in clinical routine in tailoring treatment approaches for MCL. Figure 1. Top 15 genes mutated in the cohort studied. Frequency of mutations on each gene is presented on the right panel and in the upper panel the proportion of affected genes by sample can be seen. Darker blue identifies a missense mutation, light blue corresponds to a frameshift deletion, pink color matches frameshift insertions, orange represent in frame deletions, whereas red in frame insertions. Light green identifies nonsense mutations and dark green stands for different types of mutation affecting that gene in that sample. Figure Disclosures Jerkeman: Roche: Research Funding; Abbvie: Research Funding; Celgene: Research Funding; Gilead: Research Funding; Janssen: Research Funding.