15) Systematic Evaluation of Chemically Distinct Tissue Optical Clearing Techniques in Murine Lymph Nodes.

Matryba P, Sosnowska A, Wolny A, Bozycki L, Greig A, Grzybowski J, Stefaniuk M, Nowis D, Gołąb J.
J Immunol. 2020,  204(5): 1395-1407

Abstract
Activation of adaptive immunity is a complex process coordinated at multiple levels in both time and the three-dimensional context of reactive lymph nodes (LNs). Although microscopy-based visualization of its spatiotemporal dynamics unravels complexities of developing immune response, such approach is highly limited by light-obstructing nature of tissue components. Recently, tissue optical clearing (TOC) techniques were established to bypass this obstacle and now allow to image and quantify the entire murine organs with cellular resolution. However, the spectrum of TOC is represented by wide variety of chemically distinct methods, each having certain advantages and disadvantages that were unsatisfactorily compared for suitability to LNs clearing. In this study, we have systematically tested 13 typical TOC techniques and assessed their impact on a number of critical factors such as LN transparency, imaging depth, change in size, compatibility with proteinaceous fluorophores, immunostaining, H&E staining, and light-sheet fluorescence microscopy. Based on the detailed data specific to TOC process of murine LNs, we provide a reliable reference for most suitable methods in an application-dependent manner.

14) Total Absence of Dystrophin Expression Exacerbates Ectopic Myofiber Calcification and Fibrosis and Alters Macrophage Infiltration Patterns.

Young CNJ, Gosselin MRF, Rumney R, Oksiejuk A, Chira N, Bozycki L, Matryba P, Łukasiewicz K, Kao AP, Dunlop J, Robson SC, Zabłocki K, Górecki DC.
Am J Pathol. 2020, 190(1):190-205

Abstract
Duchenne muscular dystrophy (DMD) causes severe disability and death of young men because of progressive muscle degeneration aggravated by sterile inflammation. DMD is also associated with cognitive and bone-function impairments. This complex phenotype results from the cumulative loss of a spectrum of dystrophin isoforms expressed from the largest human gene. Although there is evidence for the loss of shorter isoforms having impact in the central nervous system, their role in muscle is unclear. We found that at 8 weeks, the active phase of pathology in dystrophic mice, dystrophin-null mice (mdxβgeo) presented with a mildly exacerbated phenotype but without an earlier onset, increased serum creatine kinase levels, or decreased muscle strength. However, at 12 months, mdxβgeo diaphragm strength was lower, whereas fibrosis increased, compared with mdx. The most striking features of the dystrophin-null phenotype were increased ectopic myofiber calcification and altered macrophage infiltration patterns, particularly the close association of macrophages with calcified fibers. Ectopic calcification had the same temporal pattern of presentation and resolution in mdxβgeo and mdx muscles, despite significant intensity differences across muscle groups. Comparison of the rare dystrophin-null patients against those with mutations affecting full-length dystrophins may provide mechanistic insights for developing more effective treatments for DMD.

13) Src and ROCK Kinases Differentially Regulate Mineralization of Human Osteosarcoma Saos-2 Cells.

Strzelecka-Kiliszek A, Romiszewska M, Bozycki L, Mebarek S, Bandorowicz-Pikula J, Buchet R, Pikula S.
Int J Mol Sci. 2019,  20(12)

Abstract
Osteoblasts initiate bone mineralization by releasing matrix vesicles (MVs) into the extracellular matrix (ECM). MVs promote the nucleation process of apatite formation from Ca2+ and Pi in their lumen and bud from the microvilli of osteoblasts during bone development. Tissue non-specific alkaline phosphatase (TNAP) as well as annexins (among them, AnxA6) are abundant proteins in MVs that are engaged in mineralization. In addition, sarcoma proto-oncogene tyrosine-protein (Src) kinase and Rho-associated coiled-coil (ROCK) kinases, which are involved in vesicular transport, may also regulate the mineralization process. Upon stimulation in osteogenic medium containing 50 μg/mL of ascorbic acid (AA) and 7.5 mM of β-glycerophosphate (β-GP), human osteosarcoma Saos-2 cells initiated mineralization, as evidenced by Alizarin Red-S (AR-S) staining, TNAP activity, and the partial translocation of AnxA6 from cytoplasm to the plasma membrane. The addition of 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4-d] pyrimidine (PP2), which is an inhibitor of Src kinase, significantly inhibited the mineralization process when evaluated by the above criteria. In contrast, the addition of (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide hydrochloride (Y-27632), which is an inhibitor of ROCK kinase, did not affect significantly the mineralization induced in stimulated Saos-2 cells as denoted by AR-S and TNAP activity. In conclusion, mineralization by human osteosarcoma Saos-2 cells seems to be differently regulated by Src and ROCK kinases.  

12) Quantitative atomic force microscopy provides new insight into matrix vesicle mineralization.

Plaut JS, Strzelecka-Kiliszek A, Bozycki L, Pikula S, Buchet R, Mebarek S, Chadli M, Bolean M, Simao AMS, Ciancaglini P, Magrini A, Rosato N, Magne D, Girard-Egrot A, Farquharson C, Esener SC, Millan JL, Bottini M.
Arch Biochem Biophys. 2019, 667:14-21

Abstract 
Matrix vesicles (MVs) are a class of extracellular vesicles that initiate mineralization in cartilage, bone, and other vertebrate tissues by accumulating calcium ions (Ca2+) and inorganic phosphate (Pi) within their lumen and forming a nucleation core (NC). After further sequestration of Ca2+ and Pi, the NC transforms into crystalline complexes. Direct evidence of the existence of the NC and its maturation have been provided solely by analyses of dried samples. We isolated MVs from chicken embryo cartilage and used atomic force microscopy peak force quantitative nanomechanical property mapping (AFM-PFQNM) to measure the nanomechanical and morphological properties of individual MVs under both mineralizing (+Ca2+) and non-mineralizing (–Ca2+) fluid conditions. The elastic modulus of MVs significantly increased by 4-fold after incubation in mineralization buffer. From AFM mapping data, we inferred the morphological changes of MVs as mineralization progresses: prior to mineralization, a punctate feature, the NC, is present within MVs and this feature grows and stiffens during mineralization until it occupies most of the MV lumen. Dynamic light scattering showed a significant increase in hydrodynamic diameter and no change in the zeta potential of hydrated MVs after incubation with Ca2+. This validates that crystalline complexes, which are strongly negative relative to MVs, were forming within the lumen of MVs. These data were substantiated by transmission electron microscopy energy dispersive X-ray and Fourier transform infrared spectroscopic analyses of dried MVs, which provide evidence that the complexes increased in size, crystallinity, and Ca/P ratio within MVs during the mineralization process.

11) Vesicles of the intracellular and extracellular transport - key structures in the process of tissue differentiation towards bone and cartilage.

Strzelecka-Kiliszek A, Bożycki Ł, Komiażyk M, Dudek J, Gasik J, Pikuła S. 
Postepy Biochem. 2018,  64(3): 253-260

Abstract 
Differentiation of cells of the skeletal tissue, such as osteoblasts and chondrocytes, into mineralization-competent cells is a necessary step of the physiological process of bone and cartilage mineralization. Vascular cell calcification accompanies a pathological process of atherosclerotic plaque formation, which occurs due to trans-differentiation of vascular smooth muscle cells into cells resembling bone mineralization-competent cells. The activity of tissue-nonspecific alkaline phosphatase (TNAP), an enzyme necessary for physiological mineralization, is also induced in vascular cells in response to inflammation. TNAP acquires its mineralizing function when anchored to the plasma membrane (PM) of mineralizing cells and to the surface of vesicles derived from these cells. Numerous important reports indicate that various types of vesicles play a crucial role in initiating cell differentiation. In this review, we would like to highlight various functions of different types of vesicular structures of the cellular transport machinery such as intracellular vesicles (IVs), extracellular vesicles (EVs) or matrix vesicles (MVs) at distinct stages of both physiological and pathological processes of tissue differentiation.

10) Whole-body clearing, staining and screening of calcium deposits in the mdx mouse model of Duchenne muscular dystrophy.

Bozycki L, Łukasiewicz K, Matryba P, Pikula S. 
Skelet Muscle. 2018, 8(1):21

Abstract 
Duchenne muscular dystrophy (DMD) is a fatal, X-linked genetic disorder. Although DMD is the most common form of muscular dystrophy, only two FDA-approved drugs were developed to delay its progression. In order to assess therapies for treating DMD, several murine models have recently been introduced. As the wide variety of murine models enlighten mechanisms underlying DMD pathology, the question on how to monitor the progression of the disease within the entire musculoskeletal system still remains to be answered. One considerable approach to monitor such progression is histological evaluation of calcium deposits within muscle biopsies. Although accurate, histology is limited to small tissue area and cannot be utilized to evaluate systemic progression of DMD. Therefore, we aimed to develop a methodology suitable for rapid and high-resolution screening of calcium deposits within the entire murine organism. We present new methodology to monitor calcium deposits in situ in the mouse model of Duchenne muscular dystrophy. Sample imaging with the presented setup is feasible and applicable for whole-organ/body imaging. Accompanied by the development of custom-made LSFM apparatus, it allows targeted and precise characterization of calcium deposits in cleared muscles. Hence, presented approach might be broadly utilized to monitor degree to which muscles of the entire organism are affected by the necrosis and how is it altered by the treatment or physical activity of the animal. We believe that this would be a valuable tool for studying organs alternations in a wide group of animal models of muscle dystrophy and bone-oriented diseases.

9) Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis.

Bozycki L, Komiazyk M, Mebarek S, Buchet R, Pikula S, Strzelecka-Kiliszek A.
J Vis Exp. 2018, (136)

Abstract 
This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. These cell lines, after treatment with ascorbic acid (AA) and β-glycerophosphate (β-GP), undergo complete osteogenic transdifferentiation from proliferation to mineralization and produce matrix vesicles (MVs) that trigger apatite nucleation in the extracellular matrix (ECM). Based on Alizarin Red-S (AR-S) staining and analysis of the composition of minerals in cell lysates using ultraviolet (UV) light or in vesicles using TEM imaging followed by EDX quantitation and ion mapping, we can infer that osteosarcoma Saos-2 and osteoblastic hFOB 1.19 cells reveal distinct mineralization profiles. Saos-2 cells mineralize more efficiently than hFOB 1.19 cells and produce larger mineral deposits that are not visible under UV light but are similar to hydroxyapatite (HA) in that they have more Ca and F substitutions. The results obtained using these techniques allow us to conclude that the process of mineralization differs depending on the cell type. We propose that, at the cellular level, the origin and properties of vesicles predetermine the type of minerals.

8) Optimized perfusion-based CUBIC protocol for the efficient whole-body clearing and imaging of rat organs.

Matryba P, Bozycki L, Pawłowska M, Kaczmarek L, Stefaniuk M. 
J Biophotonics. 2018 , 11(5)

Abstract 
Whole-organ and whole-body optical tissue clearing methods allowing imaging in 3 dimensions are an area of profound research interest. Originally developed to study nervous tissue, they have been successfully applied to all murine organs, yet clearing and imaging of rat peripheral organs is less advanced. Here, a modification of CUBIC clearing protocol is presented. It provides a rapid and simple approach to clear the entire adult rat organism and thus all organs within as little as 4 days. Upgraded perfusion-based rat CUBIC protocol preserves both anatomical structure of organs and signal from proteinaceous fluorophores, and furthermore is compatible with antibody staining. Finally, it enables also volumetric cells analyses and is tailored for staining of calcium deposits within unsectioned soft tissues.

7) Matrix vesicles from chondrocytes and osteoblasts: Their biogenesis, properties, functions and biomimetic models.

Bottini M, Mebarek S, Anderson KL, Strzelecka-Kiliszek A, Bozycki L, Simão AMS, Bolean M, Ciancaglini P, Pikula JB, Pikula S, Magne D, Volkmann N, Hanein D, Millán JL, Buchet R. 
Biochim Biophys Acta Gen Subj. 2018, 1862(3):532-546

Abstract 
Matrix vesicles (MVs) are released from hypertrophic chondrocytes and from mature osteoblasts, the cells responsible for endochondral and membranous ossification. Under pathological conditions, they can also be released from cells of non-skeletal tissues such as vascular smooth muscle cells. MVs are extracellular vesicles of approximately 100-300nm diameter harboring the biochemical machinery needed to induce mineralization. The review comprehensively delineates our current knowledge of MV biology and highlights open questions aiming to stimulate further research. The review is constructed as a series of questions addressing issues of MVs ranging from their biogenesis and functions, to biomimetic models. It critically evaluates experimental data including their isolation and characterization methods, like lipidomics, proteomics, transmission electron microscopy, atomic force microscopy and proteoliposome models mimicking MVs. MVs have a relatively well-defined function as initiators of mineralization. They bind to collagen and their composition reflects the composition of lipid rafts. We call attention to the as yet unclear mechanisms leading to the biogenesis of MVs, and how minerals form and when they are formed. We discuss the prospects of employing upcoming experimental models to deepen our understanding of MV-mediated mineralization and mineralization disorders such as the use of reconstituted lipid vesicles, proteoliposomes and, native sample preparations and high-resolution technologies. MVs have been extensively investigated owing to their roles in skeletal and ectopic mineralization. MVs serve as a model system for lipid raft structures, and for the mechanisms of genesis and release of extracellular vesicles.

6) Possibility of inclusion of the sobibór forests into importand birds areas and special protection areas networks

Kanclerska K, Karpińska O, Woźniak B, Grzȩbkowski M,  Aftyka S, Bełcik M, Bozycki L, Celej M, Chodkiewicz T, Kuszner M,  Litwiniak K, Pawelec M, Pietrasz K, Rózycki AŁ, Sikora D, Smith L, Stasiak K, Szewczuk W, Śliwiński S, Keller M
Sylwan 2018, 162(2):146-154

Abstract
One of the greatest challenges of nature conservation is to identify and locate networks of pro tected areas, which will preserve the biodiversity. The paper aims to show the ornithological value of the Sobibór Forests (E Poland) and to emphasize the fact that the network of Important Bird Areas (IBA) and Special Protection Areas within Natura 2000 (SPA) may require additions. The study was conducted in the period of 2008−2016, mainly on forested areas occupying 256 km². The Sobibór Forests are characterized by a large diversity of habitats, but are mainly covered by Scots pine Pinus sylvestris. High diversity of wetlands and marshlands make the Sobibór Forest unique within the country. Three methodologies were used to estimate population size of chosen species: sampling, census and random observations. 31 species from Annex I of the Birds Directive plus 25 species of SPEC or non−SPECE category were observed which allows to consider this area as one of the most valuable for avifauna in Poland. Six breeding species classify the Sobibór Forests as the IBA and SPA. These species include Black Stork Ciconia nigra (8−13 breeding pairs), Common Crane Grus grus (80−120 breeding pairs), Grey Great Owl Strix nebulosa (0−7 breeding pairs i.e. 50−100% of Polish breeding population), Eagle Owl Bubo bubo (6 breeding pairs), White−backed Woodpecker Dendrocopos leucotos (40−70 breeding pairs) and Grey−headed Woodpecker Picus canus (20−40 breeding pairs). Six other species show potential for qualification, but not enough data has been collected yet. The list of this specie includes: Short−toed Snake Eagle Circaetus gallicus, Common Snipe Gallinago gallinago, Stock Dove Columba oenas, European Nightjar Caprimulgus europaeus, Collared Flycatcher Ficedula albicollis and Bluethroat Luscinia svecica. The Sobibór Forests occupy a small area compared to other IBA forests. However, the number of breeding species classifies the surveyed area as 7th among other IBAs. Moreover all eligible species may be considered as a bioindicators of the natural state of the forest. This is proof of the high natural value of this area.

5) Characteristics of minerals in vesicles produced by human osteoblasts hFOB 1.19 and osteosarcoma Saos-2 cells stimulated for mineralization.

Strzelecka-Kiliszek A, Bozycki L, Mebarek S, Buchet R, Pikula S. 
J Inorg Biochem. 2017, 171:100-107

Abstract 
Bone cells control initial steps of mineralization by producing extracellular matrix (ECM) proteins and releasing vesicles that trigger apatite nucleation. Using transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) we compared the quality of minerals in vesicles produced by two distinct human cell lines: fetal osteoblastic hFOB 1.19 and osteosarcoma Saos-2. Both cell lines, subjected to osteogenic medium with ascorbic acid (AA) and β-glycerophosphate (β-GP), undergo the entire osteoblastic differentiation program from proliferation to mineralization, produce the ECM and spontaneously release vesicles. We observed that Saos-2 cells mineralized better than hFOB 1.19, as probed by Alizarin Red-S (AR-S) staining, tissue nonspecific alkaline phosphatase (TNAP) activity and by analyzing the composition of minerals in vesicles. Vesicles released from Saos-2 cells contained and were surrounded by more minerals than vesicles released from hFOB 1.19. In addition, there were more F and Cl substituted apatites in vesicles from hFOB 1.19 than in those from Saos-2 cells as determined by ion ratios. Saos-2 and h-FOB 1.19 cells revealed distinct mineralization profiles, indicating that the process of mineralization may proceed differently in various types of cells. Our findings suggest that TNAP activity is correlated with the relative proportions of mineral-filled vesicles and mineral-surrounded vesicles. The origin of vesicles and their properties predetermine the onset of mineralization at the cellular level.

4) Cross-talk between Src kinases and Rho small GTPases regulates biomineralization and simplify imaging of the mineralization process.

Strzelecka-Kiliszek A, Bożycki Ł. 
Postepy Biochem. 2017, 63(2):93-109

Abstract 
The process of physiological mineralization that occurs during bone ossification is a tightly regulated cascade of molecular events leading to formation of bony skeleton. Its deregulation associated with aging leads to pathological mineralization not only in osseous but also in soft tissues. Recent discoveries in the field of bone biology indicate the participation of the Src family of tyrosine kinases as well as the Rho family of small GTPases in mineral formation. Cross-talk between these two signaling pathways is activated during an early step of the mineralization process, and leads to reorganization of the cytoskeleton of mineralization-competent cells and to matrix vesicles release. The understanding of mechanisms of the cross-talk between Src kinases and Rho small GTPases that regulate the mineralization process is crucial for the development of novel simply imaging techniques and therapeutic strategies in relation to pathological mineralization.

3) Simultaneous Two-photon in Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex.

Łukasiewicz K, Robacha M, Bożycki Ł, Radwanska K, Czajkowski R.
J Vis Exp. 2016,  (109)

Abstract 
This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo. The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging. The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAV(mCherry)) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

2) On the heat stability of amyloid-based biological activity: insights from thermal degradation of insulin fibrils.

Surmacz-Chwedoruk W, Malka I, Bożycki Ł, Nieznańska H, Dzwolak W.
PLoS One. 2014, 9(1)

Abstract
Formation of amyloid fibrils in vivo has been linked to disorders such as Alzheimer's disease and prion-associated transmissible spongiform encephalopathies. One of the characteristic features of amyloid fibrils is the high thermodynamic stability relative both to native and disordered states which is also thought to underlie the perplexingly remarkable heat resistance of prion infectivity. Here, we are comparing high-temperature degradation of native and fibrillar forms of human insulin. Decomposition of insulin amyloid has been studied under helium atmosphere and in the temperature range from ambient conditions to 750°C using thermogravimetry and differential scanning calorimetry coupled to mass spectrometry. While converting native insulin into amyloid does upshift onset of thermal decomposition by ca. 75°C, fibrils remain vulnerable to covalent degradation at temperatures below 300°C, as reflected by mass spectra of gases released upon heating of amyloid samples, as well as morphology and infrared spectra of fibrils subjected to incubation at 250°C. Mass spectra profiles of released gases indicate that degradation of fibrils is much more cooperative than degradation of native insulin. The data show no evidence of water of crystallization trapped within insulin fibrils. We have also compared untreated and heated amyloid samples in terms of capacity to seed daughter fibrils. Kinetic traces of seed-induced insulin fibrillation have shown that the seeding potency of amyloid samples decreases significantly already after exposure to 200°C, even though corresponding electron micrographs indicated persisting fibrillar morphology. Our results suggest that amyloid-based biological activity may not survive extremely high temperature treatments, at least in the absence of other stabilizing factors.

1) An FT-IR study on packing defects in mixed β-aggregates of poly(L-glutamic acid) and poly(D-glutamic acid): a high-pressure rescue from a kinetic trap.

Yamaoki Y, Imamura H, Fulara A, Wójcik S, Bożycki L, Kato M, Keiderling TA, Dzwolak W.
J Phys Chem B. 2012, 116(17):5172-8

Abstract
Under favorable conditions of pH and temperature, poly(L-glutamic acid) (PLGA) adopts different types of secondary and quaternary structures, which include spiral assemblies of amyloid-like fibrils. Heating of acidified solutions of PLGA (or PDGA) triggers formation of β(2)-type aggregates with morphological and tinctorial properties typical for amyloid fibrils. In contrast to regular antiparallel β-sheet (β(1)), the amide I' vibrational band of β(2)-fibrils is unusually red-shifted below 1600 cm(-1), which has been attributed to bifurcated hydrogen bonds coupling C═O and N-D groups of the main chains to glutamic acid side chains. However, unlike for pure PLGA, the amide I' band of aggregates precipitating from racemic mixtures of PLGA and PDGA (β(1)) is dominated by components at 1613 and 1685 cm(-1)-typically associated with intermolecular antiparallel β-sheets. The coaggregation of PLGA and PDGA chains is slower and biphasic and leads to less-structured assemblies of fibrils, which is reflected in scanning electron microscopy images, sedimentation properties, and fluorescence intensity after staining with thioflavin T. The β(1)-type aggregates are metastable, and they slowly convert to fibrils with the infrared characteristics of β(2)-type fibrils. The process is dramatically accelerated under high pressure. This implies the presence of void volumes within structural defects in racemic aggregates, preventing the precise alignment of main and side chains necessary to zip up ladders of bifurcated hydrogen bonds. As thermodynamic costs associated with maintaining void volumes within the racemic aggregate increase under high pressure, a hyperbaric treatment of misaligned chains leads to rectifying the packing defects and formation of the more compact form of fibrils.

Using Format