In comparison, KOH etching for 5 h resulted sparser but nonetheless disordered surface morphology with only 8 spikes per µm2. The NaOH surface was more effective at eliminating Gram-negative pathogens, while the KOH surface was more effective against the Gram-positive strains. These findings may guide further research and development of bactericidal titanium surfaces which are optimised for the predominant pathogens associated with the intended application.Inspired by observations that the natural topography observed on cicada and dragonfly wings may be lethal to bacteria, researchers have sought to reproduce these nanostructures on biomaterials with the goal of reducing implant-associated infections. Titanium and its alloys are widely employed biomaterials with excellent properties but are susceptible to bacterial colonisation. Hydrothermal etching is a simple, cost-effective procedure which fabricates nanoscale protrusions of various dimensions upon titanium, depending on the etching parameters used. We investigated the role of etching time and the choice of cation (sodium and potassium) in the alkaline heat treatment on the topographical, physical, and bactericidal properties of the resulting modified titanium surfaces. Optimal etching times were 4 h for sodium hydroxide (NaOH) and 5 h for potassium hydroxide (KOH). NaOH etching for 4 h produced dense, but somewhat ordered, surface nanofeatures with 75 nanospikes per µm Worldwide, there is a growing demand for implantable medical devices, in part due to an aging population made possible by advancements in medicine and technology . TitaniuTo tackle the burden of IAI, much research has focused on the modification of implanted biomaterials to grant them with anti-infective properties. For example, surfaces can be coated with antimicrobial peptides or drug-eluting compounds ,26,27,28Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), and Pseudomonas aeruginosa (P. aeruginosa). The Gram-positive coccus S. aureus was chosen due to its ubiquitous association with IAIs [E. coli and P. aeruginosa were chosen as representative Gram-negatives which frequently appear in implant infections [P. aeruginosa rods are typically longer than E. coli rods  [The purpose of this study is to examine the role of etchant type and processing time on the morphology of the resultant surfaces and their antibacterial capacity. Potassium hydroxide (KOH) and sodium hydroxide (NaOH) were used as etchants. Etching time was 1, 3, 4, and 5 h. The modified surface was challenged with three clinically relevant pathogens: ith IAIs , and welith IAIs  combinedith IAIs . E. colifections ,39. P. actively) ,41 which2) were received polished at a Ra of 0.5 µm  SDN BHD, Kedah, Malaysia). To create the nanostructured surface, the discs were hydrothermally etched at 150 °C, using either 1 M KOH or 1 M NaOH in a stainless-steel reactor . Next, the reactors were cooled, and the samples were cleaned in ultrapure water. Ti-6Al-4V discs were then dried, followed by heat treatment inside an oven and allowed to cool down overnight. The as-received titanium alloy discs were used as controls (AR-Ti) and hydrothermally etched discs, using 1 M NaOH and 1 M KOH aqueous etching solutions, were then cleaned and sterilised at 121 °C for 20 min prior to use. Samples were fabricated using both alkaline solutions, (1 M KOH and 1 M NaOH) with etching times of 1, 3, 4, and 5 h.Ti-6Al-4V discs , whereas the diameter was measured at mid-height in parallel orientation with the basal plane, and a correction factor of x/cos (45°) to amend dimensional distortion during linear measurements, where x equals the length of nanostructures. Spike height and diameter at mid height were expressed as mean ± standard deviation, and n = 5. The spacing between nanostructures was determined from four zero-degree tilted SEM images, considering the nanostructure tips in a 25 µm2 area and presented as mean ± standard deviation. Density was calculated by counting spike tip and expressing as nanostructures per μm2. The nanostructures dimensions were determined using ImageJ software version 1.53f51 .Scanning electron microscope (SEM) characterisation was performed on samples fabricated using both alkaline etchant solutions (1 M KOH and 1 M NaOH) with etching times of 1, 3, 4, and 5 h. The morphology and distribution of nanostructures upon the surface of the titanium substrate were analysed on a field emission gun scanning electron microscope Zeiss Merlin FEG-SEM , equipped with a secondary electron (SE) detector, at 2 KV with magnification from 5–50 K. The stage was tilted at 45 degrees for imaging of the nanostructures, and orthogonal when analysing density of individual nanostructures and imaging bacteria on the surfaces. The height of the nanostructures was determined by the linear distance between a basal plane and the highest point of each spike .Atomic force microscopy (AFM) was used to acquire 25 µmChemical composition of the upper 10 nm layer of AR-Ti, NaOH-4h, and KOH-5h were analysed using X-ray photoelectron spectroscopy (XPS). XPS survey spectra were collected using a Kratos AXIS Ultra DLD spectrometer  equipped with a magnetically confined charge compensation system, with monochromatic AlKα radiation (hν = 1486.7 eV). The sample area analysed was 300 μm × 700 μm at a pass energy of 160 eV. Data analysis was performed with CasaXPS software . All binding energies were referenced to the low energy, aliphatic C 1 s peak at 285.0 eV.Surface wettability was evaluated for AR-Ti, NaOH-4h, and KOH-5h using contact angle evaluated by the sessile drop method using a contact angle goniometer model RD-SDM02 . The contact angle from probe liquid, ultrapure water (4 μL) was measured by a tangent fitting method using plugin Contact_Angle.jar for Image J software version 1.53f51 .Escherichia coli ATCC 11303 (E. coli), Pseudomonas aeruginosa ATCC 15692 (P. aeruginosa), and Staphylococcus aureus ATCC 25923 (S. aureus) were cultured in tryptic soy broth  and incubated overnight at 37 °C. Prior to starting bacterial experiments, it was determined that an optical density of 1 measured at 600 nm (OD600) was approximately 1 × 109 CFU/mL6 CFU/mL, and 1 mL added to wells containing titanium discs  in triplicate, and incubated for 20 h at 37 °C in a humid chamber. Next, the samples were stained with LIVE/DEAD® BacLight™ Bacterial Viability Kit  as per the manufacturer’s instructions and analysed by an Olympus FV3000 laser confocal microscope . Samples were inverted onto a glass coverslip and four randomly selected regions were imaged. SYTO 9 and propidium iodide fluorescence excitation/emissions were monitored at 480/500 nm and 490/635 nm, respectively. The viability was determined using ImageJ software v1.53a .To compare the different etching times  and etchants (NaOH and KOH), overnight cultures of bacteria were adjusted to a concentration of 1 × 10E. coli, P. aeruginosa, and S. aureus incubated on AR-Ti, NaOH-, and KOH-etched samples were fixed for 1 h with 4% paraformaldehyde, 1.25% glutaraldehyde, and 4% sucrose in PBS. Following fixation, they were washed in PBS, followed by dehydration in an ascending ethanol order from 50% (v/v) to absolute ethanol and further dried using hexamethyldisilazane . After the dehydration process, the discs were mounted on aluminium stubs using double-sided carbon tape, sputter-coated with 2 nm platinum, and examined using a Zeiss Merlin FEG-SEM .Overnight cultures of v/v fetal bovine serum  and 1% (v/v) penicillin/streptomycin . Tissue culture plate (TCP) and AR-Ti discs were used as positive controls. HDF cells were seeded at a density of 2.5 × 104 cells/well. Short-term cytocompatibility was assessed using the resazurin assay . A stock solution of 100 µg/mL resazurin was prepared in phosphate buffered saline (PBS) and diluted to a final working concentration of 10 µg/mL. Briefly, the cells were incubated on TCP, AR-Ti, NaOH-4h, and KOH-5h for 48 h at 37 °C and 5% CO2, after which the culture medium was replaced with 250 μL media containing 10% resazurin of the stock solution and incubated for 1 h. Next, 100 µL of the supernatant was transferred into a 96 well plate and the fluorescent intensity was recorded using a plate reader. Resazurin is a fluorescent assay that detects cellular metabolic activity. The blue nonfluorescent resazurin reagent is reduced to highly fluorescent resorufin by dehydrogenase enzymes in metabolically active cells. The resorufin formed in the assay was quantified by measuring the fluorescent intensity using a plate reader . Percent of cell viability is calculated using the following formula, Primary human derived dermal fibroblast cells (HDF) were seeded on AR-Ti, NaOH-4h, and KOH-5h samples in a 48 well plate. Cultures were maintained in Dulbecco’s modified Eagle medium  supplemented with 10% hed work . Samplest-test followed by post hoc analysis using a Tukey’s multiple comparisons test. To compare nanostructure topographical dimensions, a Student’s t-test was performed. Statistical analysis was performed using GraphPad Prism version 8.3.0 . All experiments were undertaken in triplicate and reported as mean and standard deviation. In all experiments, significance was set at p < 0.05.Statistical analysis for surface parameters and viability was carried out using one-way ANOVA or Student’s A range of surface nanostructures were created on titanium alloy discs using hydrothermal etching with either NaOH or KOH aqueous solutions. Surfaces were etched for 1, 3, 4, or 5 h to investigate the changes in geometrical and morphological characteristics to investigate the nanostructure evolution with time. Surfaces were then annealed to improve corrosion resistance, alloy plasticity, abrasion resistance, and potential for bone ongrowth, and reduce surface stress developed during fabrication ,44. The 2. The results are presented in p < 0.0001). The nanostructure diameter at mid-height, followed an upward trend for NaOH, with a significant increase between 1 h and 3 h etching times , and 1 h and 5 h etching times . However, the nanostructures’ diameter at mid-height remained almost constant from 1 to 5 h in the case of KOH-etching . The spacing between nanostructures remained unchanged for etching times on the NaOH-etched surfaces , and increased on KOH-etched surfaces . This indicates that there was no new nanostructure formation over time, only evolution of those that had already formed at the beginning of the hydrothermal process, the main outcome being the increase in nanostructure height. The density of the nanostructures was lower on the KOH-etched surface (8 ± 2 spikes/ µm2 at 5 h etching time) compared to NaOH-etched surface (32 ± 9 spikes/µm2 at 5 h etching time). The SEM micrographs were used to measure spike height, diameter at mid-height, spacing between, and density per µmE. coli, P. aeruginosa, and S. aureus on the surfaces generated by NaOH and KOH hydrothermal etching for 1, 3, 4, and 5 h are shown in S. aureus. This is somewhat expected as Gram-positive bacteria possess a thicker peptidoglycan layer which confers greater structural support compared to the relatively fragile Gram-negative species [S. aureus, a high proportion of damaged cells were observed on the KOH-5h surface . However, although the spike heights were not significantly different, the KOH-5h shows a much greater range in measured nanostructure height (175 nm) compared to the NaOH-4h (80 nm). This irregularity of spike heights can be clearly observed in both the SEM and AFM micrographs (p = 0.02) and greater spike density (p = 0.0001) when compared to the KOH-5h surface.To find a potential explanation for the observed bactericidal selectivity between NaOH-4h and KOH-5h, we first compared the spike dimensions of the fabricated nanostructures . We foun2) occurring during the hydrothermal etching process. Furthermore, the thicker oxide layer also resulted in a reduction in the surface concentration of vanadium and aluminium as XPS sampling depth is limited to the outermost 10 nm of the surface. This may prove to be advantageous for implantable biomaterials as wear particles and leaching of vanadium and aluminium from Ti-6Al-4V have raised concerns due to their potential toxicity and DNA damaging effects [Elemental composition detected on the AR-Ti, NaOH-4h, and KOH-5h surface using XPS is presented in  effects ,50,51. Ap < 0.0001, indicative of significantly increased hydrophilicity (p > 0.05). Such superhydrophilic behaviour is the result of the combination of surface roughness at the nanoscale and a hydrophilic TiO2 surface layer, consistent with the Wenzel and Cassie Baxter theory [To compare the hydrophilicity of samples before and after hydrothermal etching, water contact angles (WCA) were measured. The WCA for NaOH-4h surface (7.6° ± 1.2°) and KOH-5h (9.7° ± 1.1°) was significantly lower than that for the AR-Ti surface (76.0° ± 5.0°), hilicity B. There r theory ,53,54. Ar theory ,56.2 respectively, whereas for the NaOH-4h surface, these parameters were 88.4 nm, 71.4 nm, and 79.9 µm2, respectively. Compared to the NaOH-4h surface, the KOH-5h surface had a similar RMS value of 88.5 nm, and a decrease in both Ra and SA  incubated for 48 h are shown in p > 0.05 . These r2, compared to the sparser KOH-5h with a density of approximately 8 spikes per μm2 suggesting that a greater density of spikes supports bactericidal activity against Gram-negative rods, while a sparser nanostructures array supports killing of Gram-positive cocci. Short-term in vitro cytocompatibility study using HDF cells incubated on NaOH-4h and KOH-5h surfaces showed no unfavourable effects on morphology, viability, and proliferation. Further research is required to fully understand the mechanisms supporting the antibacterial properties of the nanostructured surface. However, this study highlights that with appropriate optimisation, substrates with selective antibacterial activity could be potentially achieved. We investigated the role of alkaline etchants and hydrothermal processing duration on the resultant surface nanotopography outcomes for titanium alloy discs. Nanostructured surfaces were challenged with three clinically relevant pathogens to determine their bactericidal potential. Our bacteriological analysis allowed us to narrow down the etching parameters to two favourable processing conditions involving either NaOH-etching for 4 h or KOH-etching for 5 h. NaOH-4h treatment exhibited spikes that are densely packed with a mean of 75 spikes per μm


SEVh characterization demonstrated increased protein abundance and diversity over normoxic SEV (SEVn), with enrichment in pro-invasive pathways. In normoxic cells, SEVh promotes invasive behavior through pro-migratory morphology, invadopodia development, ECM degradation, and matrix metalloprotease (MMP) secretion. The proteome profiling of 20% O2-cultured cells exposed to SEVh determined enrichment in metabolic processes and cell cycles, modulating cell health to escape apoptotic pathways. In hypoxia, SEVh was responsible for proteolytic and catabolic pathway inducement, interfering with integrin availability and gelatinase expression. Overall, our results demonstrate the importance of hypoxic signaling via SEV in tumors for the early establishment of metastasis.Hypoxia, a condition of low oxygenation frequently found in triple-negative breast tumors (TNBC), promotes extracellular vesicle (EV) secretion and favors cell invasion, a complex process in which cell morphology is altered, dynamic focal adhesion spots are created, and ECM is remodeled. Here, we investigated the invasive properties triggered by TNBC-derived hypoxic small EV (SEVh) in vitro in cells cultured under hypoxic (1% O Low oxygen tissue perfusion, or hypoxia, is established in most solid tumors as a consequence of cell proliferation, increased tumor mass, and abnormal tumor vascularization . Oxygen Tumor cells have increased extracellular vesicle (EV) secretion, which enables intercellular communication by stably transferring biomolecules between different compartments ,9. In trIn many cancer types, EV secretion is favored by hypoxic stress  and theiCell invasion involves the migration of tumor cells through adjacent tissues in combination with extracellular matrix (ECM) remodeling, dynamic focal adhesion spots, and F-actin cytoskeleton polarization. It is considered an early step of metastasis that favors intravasation, extravasation, and the colonization of a new metastatic niche ,20. Simi2) or hypoxia (1% O2). In this paper, we address (1) the phenotypic alterations elicited by SEVh; (2) the ECM remodeling by gelatinases ; (3) the direct interference of SEVh in in vitro invasion; and (4) the proteomic profiling of cells after SEVh exposure. We used oxygen levels of 20% to establish the condition of normoxia, which are those routinely employed in cell culture. However, it is necessary to consider that the physiological values of oxygen found in the tissues can be much lower, usually about 5% [In this study, we investigated the effects of TNBC-derived, hypoxic small extracellular vesicles (SEVh) in tumor cells in vitro, comparing the responses in the human TNBC model cell line MDA-MB-231 exposed to normoxia . In comparison, a wider difference was observed in MEV samples . NTA analysis provided a histogram displaying MEV . The top 144 proteins (p < 0.01) were considered for enrichment analysis (−6), a ubiquitin-like protein previously linked to integrin-mediated cell invasion [−4) and its regulator, RPTOR (1.05 × 10−3), critical for controlling cancer metabolism [−3; 1.78 × 10−3; 7.3 × 10−3, respectively). Two protein families were abundant in SEVh: the SMADs (1.12 × 10−3), related to the activation of TGF-β pathway [−3), predicted to act on the nuclear pore and to aid GTPase activity [Univariate analysis identified 552 statistically relevant proteins between SEVh and SEVn , five of the TGF-β pathway , and eleven of the VEGFA/VEGFR2 pathway . We identified that pathways related to vesicle-mediated transport (6.7E-03), RNA metabolism (1.5 × 10−2), DNA replication (3.01 × 10−2), and cell cycle (0.21) were statistically upregulated in SEVh  a transwell invasion assay with Matrigel coating a, and ib. MatrigSEVh signaling in normoxia was a driving force for gelatin invasion after 48 h, as was hypoxia (FBS- group), with non-statistical tendencies appearing after 24 h . The sizSince gelatinase activity was detected, we investigated the effects of SEVh and/or hypoxia in MMP-2 and MMP-9. Our results suggest a significant increase in the activity of soluble MMP-2 e and MMPUpon visual interpretation, cell morphology was modified slightly by SEVh treatment in all conditions, particularly on the Matrigel coating a. UntreaOverall, cell circularity decreased after exposure to collagen-based matrices for 24 and 48 h c–e. In uSince there is an inherent phenotypical diversity of MDA-MB-231 cells, which morphs according to matrix stiffness and function [SEVh treatment did not alter the gene and protein expression of integrin subunits β1 and β3, which regulate migration and invasion . On the The same promoting effect of hypoxia towards the integrin gene and protein expression was found for the β3 subunit; however, there was no downregulation of surface-available β3 integrin i–k. Alth2 atmosphere increased the late apoptosis percentage when compared to the untreated normoxic control . Its protein levels followed the same pattern from the gene expression analysis, but with a significant increase in HIF-1α in SEVh-treated hypoxic cells  controls the balance between pro- and anti-apoptotic signals . HIF-1 iic cells d. The paThe label-free proteomic analysis of cells exposed to SEVh, under normoxia and hypoxia conditions, showed a distinct proteome profile for each group. Overall, a total of 2615 proteins were detected, with 1441 shared among all groups. We found 11 proteins exclusive to normoxic groups and 124 in hypoxia, with 196 unique proteins in untreated cells in normoxia (PBS-N), 18 in SEVh-treated cells in normoxia (SEVh-N), 108 in untreated cells in hypoxia (PBS-H), and 120 in SEVh-treated cells in hypoxia (SEVh-H). Shared proteins between groups are described in the Venn diagram in p < 0.05), with the 62 most divergent proteins , hence the increasing microtubule nucleation (FDR: 2.6 × 10−4) and protein polymerization (FDR: 3.3 × 10−4). Other additional pathways induced by SEVh signaling include mitochondrial biogenesis, the citric acid cycle, and the synthesis of ubiquitin E1 and E2 enzymes (FDR: 1.71 × 10−1). All together, these results indicate that SEVh signaling in normoxia promotes substantial alterations in cell metabolism, favoring the cell cycle and survival of tumoral cells.SEVh directly interferes with the proteome of cells in normoxia, especially with the upregulation of γ-tubulin complex binding by TUBGCP6, TUBG1, TUBGCP3, and TUBGCP2 (FDR: 9.7 × 10−2) and other metabolic processes (FDR: 2.39 × 10−2). Particularly, it led to an increase in mRNA biogenesis and splicing , DNA repair , and the degradation of cysteine and homocysteine (FDR: 8.04 × 10−2). Overall, these results reveal an oxygen-dependent modulation for SEVh signaling in tumoral cells, generating distinct survival mechanisms.In hypoxia, SEVh treatment led to distinct responses, favoring response to stress  and untreated cells (6.02 × 10−3) and the response to stress of the control groups (3.34 × 10−1). Although programmed cell death, a staple of hypoxia signaling, was slightly favored by our proteomic analysis, our results also show an increase in autophagy . Akin to the literature, several proteins from hypoxic cells are related to DNA damage and repair, including USP7, BABAM1, RBX1, and POLR2I (3.34 × 10−1). On the other hand, SEVh signaling induces major mitochondrial and proteolytic pathways in hypoxia when compared to normoxia, such as the transport and synthesis of PAPS (8.99 × 10−2) and metalloprotease deubiquitinating enzymes (DUBs) (2.05 × 10−1). Details of enriched biological pathways may be found in Hypoxia alone also interferes with the cell metabolism, increasing particularly the catabolic activity of both SEVh-treated  inhibit prolyl-hydroxylase domain enzymes (PHDs) from cleaving this residue, thus enabling the binding of HIF subunits to form a transcriptional regulator that interferes with metabolism, redox homeostasis, angiogenesis, tumorigenesis, and inflammation . Our resInterestingly, the protein expression of HIF-1α was also decreased by SEVh treatment in normoxia, indicating the importance of oxygen in regulating HIF-mediated cell responses. Although HIF-1 activation promotes pro-tumoral responses such as ECM remodeling—either by inducing ECM protein secretion  or contrECM remodeling interferes directly with the expression pattern of integrins, cellular adhesion receptors that mediate outside-in and inside-out responses . IntegriA consequence of integrin differential expression and availability is an impact in cellular morphology due to the link between the cytoplasmic tail of the β subunit to F-actin via talin-1. Therefore, when the extracellular segment binds to ECM components, molecular clutches are formed to propel the cell onwards, thus creating a leading edge and promoting a migratory phenotype ,32,62. T2) and harsh (0.2% O2) hypoxia was recently published [We unveiled the baseline pathways and proteins favored by SEVh signaling and/or hypoxia in MDA-MB-231 cells plated in uncoated settings, which may differ from investigations in which cells are adhered to ECM components. We found that (1) each group presented a distinct protein and pathway enrichment and (2) the proteome of SEVh-treated groups diverged from the raw proteome of SEVh, which indicates a likely internalization of these particles to elicit cellular responses. In particular, the activation of survival pathways, the modulation of the cell cycle by SEVh in normoxia, and the increase in proteolysis and DNA damage in hypoxia confirm how oxygenation is important for determining cell fate whenever SEVh signaling is available. While the proteomes of both TNBC cell line MDA-MB-231  and its ublished , here weNevertheless, our study has limitations that should be addressed. We based our study on a single TNBC cell line; therefore, further investigation in different systems is necessary to unveil a thorough mechanism for SEVh-mediated invasion. The investigation focused on SEVh isolated by a differential ultracentrifugation method that, although characterized at length, has disregarded other EV subtypes and preparations with additional methods for increased EV purity. We also did not investigate oxidative stress in our cellular experiments, nor EV tracking to determine how they interact with cells. Lastly, we did not cultivate cells at a physiological oxygen atmosphere, therefore rendering our normoxic groups closer to hyperoxia and hindering our ability to translate basic research to clinical practice. Since oxygen levels in normal tissues can be as low as 5%, further studies are necessary to determine if the same results would be obtained using tumor cells cultured in this condition. Furthermore, due to the low diffusion rate of oxygen in culture medium, the equilibrium oxygen concentration in culture medium is usually much lower than in the incubator atmosphere . Therefo2. A hypoxic atmosphere  was set in an H35 Hypoxystation , and cells were incubated immediately after plating and for no longer than 4 days. EV-depleted FBS (UC-FBS) was collected after ultracentrifugation  and filtration through a 0.22 µm syringe filter.Triple-negative breast cancer cells (MDA-MB-231) were purchased from ATCC and maintained in DMEM supplemented with glucose (4.6 g/L), sodium pyruvate (10 mg/L), and fetal bovine serum (10% FBS) at 37 °C, 5% CO4 cells/cm2) were plated in DMEM 10% UC-FBS and incubated in hypoxia for 24 h. Culture media were exchanged to Opti-MEM reduced serum media (Gibco) for a further 48-h incubation. Conditioned media were collected and submitted to a 4-step differential centrifugation process in order to sediment live cells , large-EV , medium-EV , and small-EV . EV pellets were collected, washed, and resuspended in filtered PBS. This procedure was replicated under normal culture conditions  for comparison.MDA-MB-231  with NTA software . EV samples were diluted in ultrapure water (1:1000 to 1:4000) and analyzed by five 60-s captures, with the following parameters: screen gain: 2.0, camera level: 14, blur: auto, max jump distance: 14.6, min track length: auto. For processing, we used a screen gain of 10.0 and a detection threshold of 4.0.2 F20 HRTEM microscope on 40,000× magnification.EVs were diluted (1:2) in PBS and deposited in copper grids covered with formvar and carbon  for 20 min, RT. Samples were fixed in 2% paraformaldehyde (PFA) in 0.2 M PBS, pH 7.4 for 20 min, RT, followed by extensive washes in deionized water. Grids were exposed to 4% Uranyl-acetate (pH 4) and 2% methylcellulose solution for 10 min on ice and in the dark . After d2 <0.99.Isolated EV samples, cell lysates, and conditioned media were quantified using either a microBCA kit  or a standard BCA assay kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. Assays were considered successful if the BSA standard curve reached R6 cells/well) were plated in DMEM 10% UC-FBS and incubated for adhesion in hypoxia (1% O2) or normoxia (~20% O2) for 24 h. Cell media were replaced under sterile conditions to contain SEVh (5 µg/mL), and cells were once again incubated in hypoxia or normoxia for 24 h prior to collection. Untreated controls  were kept in parallel.In a 6-well plate, MDA-MB-231 cells , and resuspended in lysis buffer  for 2 h on ice. Samples were centrifuged  to collect the supernatant.Samples (10 µg) were mixed with Laemmli sample buffer, boiled , and applied to SDS-PAGE gels with Precision Plus Dual Color  as the loading control. Protein samples were separated by electrophoresis, transferred to nitrocellulose membranes , and stained with Ponceau S dye for 2 min for quality control. Non-specific bindings were blocked with 3% BSA in Tween-TBS buffer  for 1 h prior to probing with primary antibodies for: Alix , Calnexin , CD63 , Flotillin-1 , HIF-1α , integrin β1 , integrin β3 , MMP-2 , and MMP-9 . Appropriate secondary antibodies were applied for 1 h: IgG Goat anti-Mouse HRP  and IgG Goat anti-Rabbit HRP . Membranes were revealed with ECL substrate  for 3 min, scanned on a ChemiDoc imaging system (Bio-Rad), and analyzed using FIJI [®Ultra 0.5 column , centrifuged , concentrated , and re-quantified (BCA). Proteins were solubilized with RapiGest SF  at 80 °C for 15 min, followed by the addition of dithiothreitol  for 30 min, 60 °C and iodoacetamine  for 30 min, RT. After overnight digestion in trypsin solution in 50 mM NH4HCO3 (0.05 µg/µL) at 37 °C, samples were transferred to MS vials and lyophilized.Approximately 200 µg of protein was applied to the Amicon−1 in an oven compartment heated to 50 °C. To analyze samples from whole-proteome digests, we used a gradient starting with a linear increase from 2% B to 17% B over 60 min, followed by further linear increases to 25% B in 30 min and to 37% B in 10 min, and finally to 95% B in 10 min, which was held constant for 10 min. The column was equilibrated using 4 volumes of solvent A. The mass spectrometer was operated in data-dependent PASEF [0 = 1.6 to 0.6 vs. cm−2 using equal ion accumulation and ramp time in the dual TIMS analyzer of 100 ms each. Suitable precursor ions for MS/MS analysis were isolated in a window of 2 Th for m/z < 700 and 3 Th for m/z > 700 by rapidly switching the quadrupole position in sync with the elution of precursors from the TIMS device. The collision energy was lowered stepwise as a function of increasing ion mobility, starting at 20 eV for 1/K0 = 0.6 vs. cm−2 and 59 eV for 1/K0 = 1.6 vs. cm−2. We made use of the m/z and ion mobility information to exclude singly charged precursor ions with a polygon filter mask and further used “dynamic exclusion” to avoid the re-sequencing of precursors that reached a “target value” of 20,000 a.u. Ion mobility dimension was calibrated linearly using three ions from the Agilent ESI LC/MS tuning mix .LC–MS was performed on a NanoElute system  coupled online to a hybrid TIMS-quadrupole TOF mass spectrometer  , transformed by −log10, and scaled following Paretto’s parameter. Principal component analysis (PCA), hierarchical clustering, and univariate statistical analysis were performed in MetaboAnalyst (v. 5.0) [p < 0.05). False discovery rates (FDR) were considered as confidence requirements for enrichment analysis. Mass spectrometry proteomics data were deposited to the ProteomeXchange Consortium  via the PRIDE partner repository [MS scans were cross-referenced with UniProt Annotated Complete Human Proteome  using Ma(v. 5.0) . Data en(v. 5.0)  and Reac(v. 5.0)   for 20 h at 37 °C. Gels were dyed using Coomassie Brilliant Blue overnight and de-stained for band revelation [n = 3).Samples (10 µg) were prepared with 50% non-reducing sample buffer and loaded into 10% gelatin (100 µg/mL) SDS-PAGE gels for electrophoresis at 4 °C. Gels were washed with 2.5% Triton X-100 for 40 min, RT, and submersed in refolding buffer . A total of 15,000 events were captured in each replicate (n = 3). Doublets were removed using the standard gating technique , and resuspended in PBS for binding with primary antibodies: anti-integrin β1  and anti-integrin β3, . After 1 h incubation at 4 °C, tubes were washed twice with PBS  before secondary antibody probing . Samples were washed twice with PBS  and resuspended for flow cytometry analysis on BD Accuriechnique , and sin5/well) were treated with SEVh (5 µg/mL) in FBS-free media on ice and applied to the top chamber. Inserts were assembled atop wells containing DMEM 10% FBS (negative control: FBS-free DMEM) and incubated for 6 and 16 h in normoxia or hypoxia (1% O2). Membranes were fixed with 4% PFA for 10 min, washed, and cleaned with a cotton swab. Cell nuclei were stained with DAPI (0.7 ng/µL) for 10 min. Membranes were detached and assembled in slides for epifluorescence imaging  at 10× magnification, followed by an automated nuclei count. The cell invasion index was calculated as the ratio between the total nuclei count from groups and the total nuclei count from untreated, normoxic cells.An invasion chamber was assembled using 8.0 µm-pore inserts  coated with Matrigel (Corning) for 2 h at 37 °C, installed in a 24-well plate (662160-01 Greiner). Cells (10TM mounting media (Thermo Fisher Scientific). Epifluorescence images were acquired for field analysis  under 20× magnification, and the representative imaging was performed under 60× magnification in a confocal microscope (Olympus FV10i). We quantified the total degraded area over a 72 × 72 µm frame and then normalized the values by cell count.Round coverslips were coated with poly-L-lysine (0.1 mg/mL) for 20 min, RT, cross-linked with 0.5% glutaraldehyde for 15 min, and covered with a thin layer of fluorescent gelatin  overnight at 4 °C. MDA-MB-231  were treated with SEVh (5 µg/mL) and plated in FBS-free DMEM (positive control: DMEM 10% FBS). Cells were incubated for 24 and 48 h in normoxia or hypoxia, protected from light. Cells were fixed with 4% PFA for 10 min, permeabilized for 5 min with 0.1% Triton X-100, and blocked for 1 h with 1% BSA-PBS, RT. Tubulin was immunoprobed  overnight at 4 °C, followed by a 1-h incubation with the secondary antibody AlexaFluor 647 . DAPI (0.7 ng/µL) was used for nuclei staining. Slides were assembled using Prolongv/v). After incubation in normoxia or hypoxia for 24 h or 48 h, samples were fixated with 4% PFA for 10 min, RT, washed, and permeabilized with 0.1% Triton X-100 for 5 min, RT. Cell nuclei and F-actin were stained with DAPI (0.7 ng/µL) + 2.5% Phalloidin-FITC for 20 min. Image acquisition was performed at 20× magnification . The parameters of cell circularity, aspect ratio (AR), and perimeter/area ratio (n = 300).Cells were plated on round uncoated plates or coverslips coated with gelatin (0.2 mg/mL) or Matrigel , washed on iced PBS , and scrapped in binding buffer for staining with Annexin V-PE and 7AAD (7-aminoactinomycin) using the BD PharmingenTM PE Annexin V Apoptosis Detection Kit I . Cells were centrifuged  and resuspended in binding buffer for flow cytometry analysis on BD AccuriTM C6 (BD Biosciences), where 15,000 events were analyzed. Experimental controls included unstained and untreated cells in both incubation settings and single-stained and double-stained controls , the upper translucent fraction was collected and mixed with isopropanol for 10 min, RT. Samples were centrifuged at 12,000 rpm for 10 min at 4 °C and washed twice with 75% ethanol . After dried, pellets were resuspended in ultrapure water and quantified . RNA samples were treated with DNase I , and reverse transcription was performed using the High-capacity cDNA Reverse Transcription kit .Cells were scraped in ice-cold TRIzol (Invitrogen) and mixed with chloroform at RT for 15 min. After centrifugation . DNA sequences were checked using Blast . The primer sequences for targeted genes were: HIF1A , ITGB1 , ITGB3 , MMP2 , and MMP9 . Housekeeping genes GAPDH  and HPRT  were used as normalizers. Reactions were set to start at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s; 59–68 °C for 30 s; and 72 °C for 30 s. The melt curve was produced by increasing the temperature from 65 °C to 95 °C in 0.5 °C increments every 30 s. PCR products were determined based on Ct values (threshold cycle), in which each gene expression variation was equal to 2−∆∆Ct [Primers for the analysis of HIF-1α (NM_001243084.2), MMP-2 (NM_004530.4), MMP-9 (NM_004994.2), integrin β1 (NM_133376.3), and integrin β3 (NM_000212.3) expression by qPCR were designed using the online tool . In case of lognormality, datasets were transformed into log10. Parametric data were analyzed using ANOVA one-way with Tukey’s multiple comparison post-hoc. Non-parametric data were tested using the Kruskal-Wallis analysis of variance, with Dunn’s multiple comparison post-test. Values of p < 0.05 were considered statistically relevant. Results are presented as mean ± standard deviation (SD), standard error of mean (SEM) if parametric, or median ± interquartile range if non-parametric. Data analysis and graph design were made on GraphPad Prism (v. 9.3).All assays were performed in technical triplicates and at least three independent experiments. Data were submitted to ROUT’s outlier detection test and normality tests  and integrin  expression and availability. These modifications interfere with the F-actin cytoskeleton to create a migratory phenotype coupled with invadopodia development. Protein profiling of SEVh-treated, 20% O


Magnetospirillum gryphiswaldense by fusing biotin carboxyl carrier protein (BCCP) with membrane protein MamF of BMPs. The engineered strain (MSR−∆F−BF) grew well and synthesized small-sized (20 ± 4.5 nm) BMPs and were cultured in a 42 L fermenter; the yield (dry weight) of cells and BMPs reached 8.14 g/L and 134.44 mg/L, respectively, approximately three-fold more than previously reported engineered strains and BMPs. The genetically engineered BMPs (BMP−∆F−BF) were successfully linked with streptavidin or streptavidin-labelled horseradish peroxidase and displayed better storage stability compared with chemically constructed biotinylated BMPs. This study systematically demonstrated the biosynthesis of engineered magnetic nanoparticles, including its construction, characterization, and production and detection based on MTB. Our findings provide insights into biomanufacturing multiple functional magnetic nanomaterials.Biosynthesis provides a critical way to deal with global sustainability issues and has recently drawn increased attention. However, modifying biosynthesized magnetic nanoparticles by extraction is challenging, limiting its applications. Magnetotactic bacteria (MTB) synthesize single-domain magnetite nanocrystals in their organelles, magnetosomes (BMPs), which are excellent biomaterials that can be biologically modified by genetic engineering. Therefore, this study successfully constructed in vivo biotinylated BMPs in the MTB  The heightened awareness of actively mitigating climate change, reducing pollution, and improving food security has driven the need for biomanufacturing. Biosynthesis provides a key platform for bioproduction , includi3O4 nanoparticles (NPs) is performed by precipitating Fe2+/Fe3+ [Among various materials, magnetic nanomaterials display a higher specific area and increased magnetic, optical, and electrical stability and are widely applied in imaging ,11, cance2+/Fe3+ ,18. Orgae2+/Fe3+ ,20,21. Te2+/Fe3+ ,23. Howe3O4) nanocrystals in their organelles, magnetosomes (BMPs) [MTBs are environmental microorganisms that can synthesize single-domain magnetite (Fes (BMPs) ,25,26. Bs (BMPs) . Thus, Bs (BMPs) . BMPs cas (BMPs) ,30. BMPss (BMPs) ,32,33,34s (BMPs) ,36. Mores (BMPs) ,38,39.BMPs can be modified chemically and biologically. Chemical modification is performed by using a chemical crosslinking agent, coupling functional molecules to the BMP surface. However, most crosslinking agents are toxic and cannot ensure the activity of the molecules modified on the BMPs. Biological methods are the most efficient mode for BMPs modification. Recombinant MTB strains can be constructed using the BMP surface display technique to produce genetically engineered BMPs ,41,42,43Magnetospirillum gryphiswaldense, the model strain for analyzing BMPs synthesis and bioproduction [Biotin binds specifically with streptavidin/avidin, forming a biotin–avidin system (BAS). BAS is one of the most available tools for biological research, used in protein purification, protein localization analysis, and immobilization ,47,48,49oduction . MoreoveIn this study, we successfully constructed a recombinant strain (MSR−∆F−BF) for biotinylated BMP (BMP−∆F−BF) production, evaluated the cell yield, and engineered BMPs in a 42 L fermenter. BMP−∆F−BF extraction conditions were developed and optimized. BMP−∆F−BF coupled with streptavidin was invested and compared with commercially and chemically constructed biotin-labeled beads.E. coli S17-1 BF harboring plasmid pBBR−bccp−mamF was used as a donor strain for biparental conjugation; it was cultured in Luria–Bertani (LB) medium containing kanamycin (50 μg/mL). The mamF mutant strain (MSR−∆F) was used as the recipient strain and cultured at 30 °C for 24 h in sodium lactate/ammonium chloride/yeast extract (LAY) medium containing gentamycin (5 μg/mL) and nalidixic acid (5 μg/mL), as described previously [The plasmids, mutants, and bacterial strains used in this study are listed in eviously . MSR-1 t565) and magnetic response (Cmag) were measured every 2 h using a UV–visible spectrophotometer in the bacteria culture process . MSR−∆F−BF cell growth and Cmag curves were constructed to detect the status of strain growth and magnetosome synthesis.The cell growth and magnetic response of MSR−∆F−BF were measured, and wild-type MSR-1 and MSR−∆F were used as controls. The cell density 3 between fusion genes. bccp-mamF was cloned into a broad host-vector pBBR1MCS-2 to construct the recombinant plasmids pBBR−bccp−mamF. The pBBR−bccp−mamF was transformed into MSR−∆F by biparental conjugation to construct a recombinant strain, MSR−∆F−BF. Selecting MSR−∆F as the host strain can increase the amount of biotin displayed on engineered BMPs. The authigenic biotin ligase link biotin to a lysine residue of biotin carboxyl carrier protein (BCCP) displayed on BMP provided a recombinant magnetosome, BMP−∆F−BF. The MSR−∆F native mamF gene in the genome was replaced by the gentamycin-resistance gene. To detect the expression of fusion genes bccp−mamF, the gfp−marked expression plasmids pBBR−bccp−mamF-gfp were constructed. Western blotting was performed to evaluate the green fluorescent protein (GFP) expression level.The recombinant plasmids were constructed as shown in 2O. Cultured strains (1 mL) were suspended in 50 μL ddH2O. Then, 10 μL of the suspension was dropped onto a copper mesh for 10 min, dried for 30 min using infrared light, and observed under a TEM .A TEM was used to observe the morphology of cell and magnetosomes of MSR−∆F−BF, MSR−WT, and MSR−∆F. The strains were cultured in LAY media for 24 h at 30 °C and collected by centrifugation at 12,000 rpm for 2 min at 4 °C. Pellets were rinsed three times with ddH–10 Am2) from the Paleomagnetism and Geochronology Laboratory.Structural details of the magnetosomes in MSR−∆F−BF were determined using a high-resolution transmission electron microscope (HRTEM) at ESPCI  on a JEOL 2010F microscope at 200 kV, equipped with a Gatan cooled CCD camera (1024 × 1024 pixels). For each particle, the structural parameters of the crystals were determined from the two-dimensional fast Fourier transform of the HRTEM image. The magnetic properties of BMP−∆F−BF were determined using a vibrating sample magnetometer . Cell and magnetosome yields were calculated, as described previously [v/v) inoculum was transferred to the 42 L fermenter for submerged culture.The recombinant strain eviously . The ino4Cl, 6.0 g MgSO4.7H2O, 15.0 g K2HPO4.3H2O, 15.0 g yeast extract, and 17.5 mL Wolfe mineral solution. The feeding medium contained 500 g lactic acid, 90 mL NH3.H2O, 6.0 g MgSO4.7H2O, 15.0 g yeast extract, 15.0 g K2HPO4.3H2O, 10.0 g FeCl3.6H2O, and 17.5 mL Wolfe mineral solution was dissolved in 700 mL ddH2O. The culture and feeding medium were sterilized for 30 min at 121 °C. Gentamycin (5 μg/mL), kanamycin (5 μg/mL), and nalidixic acid (5 μg/mL) were added before inoculation. The pH was maintained at 6.8 by automated supplementation of the feeding medium supplemented with high concentration of lactate, and the temperature was maintained at 30 °C. The initial airflow was 0.5 L/min, and the stirring rate was 100 rpm. Once the dissolved oxygen (dO2) decreased to 15%, we increased the airflow to 1 L/min and maintained dO2 between 0% and 1% by regulating agitation (10 rpm every 2 h). When the dO2 decreased to 5%, 7.5 mL 1M isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to induce the bccp-mamF gene expression. The cell density and magnetic response were measured every 4 h during the fermentation process.The 42 L fermenter was filled with a 30 L culture medium containing 20 g (70–80%) sodium lactate, 5.0 g NHFermenter−cultured MSR−∆F−BF was collected by centrifugation at 10,000 rpm at 4 °C for 15 min. The collected cells were resuspended in 20 mM phosphate−buffered saline (PBS) with a mass volume ratio of 1:10 (pH 7.4) and disrupted using an ultrasonic homogenizer  for 20 min at 3 s/time with a time interval of 5 s at 400 W to lyse the cells. BMPs were collected from the disrupted cell suspension using a columnar neodymium−boron magnet. The supernatant was discarded, and the separated magnetosomes were resuspended in 10 mM PBS (pH 7.4), sonicated for 15 min at 3 s/time with a time interval of 5 s at 150 W, and washed twice. Then, we reduced the power to 100 W and washed the sample several times until the supernatant protein concentration was lower than 0.1 mg/mL. The purified BMP−∆F−BF was resuspended in 25% glycerol and stored at 4 °C.Purified BMP−∆F−BF and wild-type BMP were observed under a TEM. For this purpose, a small number of BMPs was suspended in 1 mL of deionized water and thoroughly dispersed by ultrasonication for 10 min. Then, 10 μL of the suspension was dropped onto a copper mesh for 10 min, air-dried, and observed under a TEM. Hydrated radii and zeta potential of BMP−∆F−BF were detected at Nanjing Dongna Biological Technology Co., Ltd., Nanjing, China.BMPs are membrane-enclosed magnetite nanocrystals. The primary amines of the BMP membrane were biotinylated by NHS−Biotin to achieve biotin-labeled BMPs (BMP−biotin). Then, 2 mg wild-type BMP was suspended in 1 mL 10 mM PBS (pH 7.4), and 13.5 μL 20 mM NHS−Biotin was added. The reaction mixture was ultrasonicated (70 W) for 1 min, incubated with shaking (200 rpm) for 30 min at 25 °C, and placed over a magnet to isolate BMP−biotin. BMP−biotin was suspended in a 25% glycerol and stored at 4 °C.2SO4 was added to stop the reaction. The supernatant was transferred to a new 96−well microtiter. Absorbance at 450 nm was measured on a microplate reader . The OD450 value was calculated to compare the BMPs and MB linkage rates.The linkage rate of BMP−∆F−BF and BMP−biotin with HRP−streptavidin was compared with commercial biotin-labeled magnetic beads . BMP−∆F−BF, BMP−biotin, and MB−biotin were incubated with 1% bovine serum albumin (BSA) and stored at 4 °C. One−step ELISA was used to detect the linkage rate. A 96−well microtiter plate coated with 300 μL 2% BSA was incubated at 4 °C overnight and washed thrice with 250 μL 10 mM PBST (pH 7.4). Then, 2 mg BMP−∆F−BF, BMP−biotin, and MB−biotin each were added  and captured using a magnetic rack. The sample was then washed thrice with PBST, and 100 μL 1:2000 diluted HRP-streptavidin  was added to each well, with coupling for 1 h at 600 rpm at room temperature, captured by a magnetic rack and washed five times with PBST. The color was developed using 100 μL tetramethylbenzidine (TMB) for each well, reacted for 3 min at room temperature, and then 50 μL 2 M Hbccp and mamF genes were fused into the bccp-mamF gene by fusion PCR then cloned into a broad host-vector pBBR1MSC-2 to construct fusion expression vector pBBR−bccp−mamF. Then, pBBR−bccp−mamF was transformed into MSR−∆F by biparental conjugation to construct an engineered strain, MSR−∆F−BF. The authigenic biotin ligase can link biotin to a lysine residue of BCCP protein, obtained biotin-labeled engineered BMPs (BMP−∆F−BF). MSR−∆F native mamF gene in the genome was replaced by the gentamicin-resistance gene. Choosing MSR−∆F as the host strain can increase the amount of biotin displayed on engineered BMPs.Previously, we adopted the “BMP surface display technique” to exhibit the functional molecule (protein A) on BMPs  used forgfp-labeled three-gene fusion expression vector was constructed. The expression of fusion protein BCCP-MamF-GFP on engineered BMPs was confirmed by Western blotting  at 22 h, approaching MSR−WT (~1.01) at 20 h  and compared with that of MSR−WT. Hysteresis loops for MSR−WT displayed a bigger gap between two lines than that for MSR−∆F−BF B. The coMagnetospirillum gryphiswaldense MSR-1, Magnetospirillum magnetotacticum MS-1, Magnetospirillum magneticum AMB-1, Magnetovibrio blakemorei MV-1, and Desulfovibrio magneticus RS-1 can only be cultivated on the laboratory scale. The BMP production level is low. The order of the BMP yield is MSR-1 > AMB-1 > MS-1 [M. gryphiswaldense ∆F-FA) on a large scale and achieved the highest cell and BMP dry weight of 2.26 g/L and 62.29 mg/L, respectively [Large-scale cultivation of the engineered strains is the basis for engineered BMP application. However, MTB large-scale cultivation is challenging, especially the cultivation of engineered strains. Similarly, MTB such as 1 > MS-1 . Previou1 > MS-1 . Later, ectively .M. gryphiswaldense MSR−∆F−BF in a 42 L fermenter and detected the cell and magnetosome yield. BMP synthesis began when dO2 decreased to ~1% . The average particle size was 20 ± 4.5 nm, smaller than that of MSR−WT (35 ± 7.8 nm). BMP−∆F−BF and BMP−WT displayed a narrow diameter distribution D. Then, The linkage rate of BMP−∆F−BF to streptavidin was detected and compared with BMP−biotin, constructed through chemical modification and commercial MB−biotin. First, the conditions for the linkage between BMP−∆F−BF and streptavidin were optimized, including the coupling time, buffer, and BMP−∆F−BF and streptavidin weight rate A. The bo2 group of the BMP membrane and modify biotin onto BMP, forming biotin-labeled BMP. BMP−∆F−BF linkage with the HRP-labeled streptavidin (Streptavidin−HRP) rate was compared with those of BMP−biotin and commercial MB−biotin (BMP−∆F−BF linkage with the streptavidin rate was compared with chemically modified BMP−biotin. The schematic diagram of BMP−biotin construction is shown in B−biotin C. BMP−∆FInterest in nanomaterials and especially nanoparticles has exploded in the past decades primarily due to their novel or enhanced physical and chemical properties compared to the bulk material. These extraordinary properties have created a multitude of innovative applications in the fields of medicine and pharma, electronics, agriculture, chemical catalysis, the food industry, and many others. More recently, nanoparticles are also being synthesized “biologically” through the use of plant− or microorganism−mediated processes, as an ecofriendly alternative to the expensive, energy−intensive, and potentially toxic physical and chemical synthesis methods . Among tMagnetospirillum—M. gryphiswaldense MSR−1 and M. magneticum AMB-1—could be cultivated in bulk. Unfortunately, the magnetosome productions of these strains were much lower than the chemical synthesized magnetic nanoparticles [Magnetosomes are excellent biomaterials, but only a few MTBs could achieve tractability and straightforward cultivation because of their restricted living conditions. Only two strains in the genus articles , and thearticles . In thisarticles . In addiarticles , which iThe process of magnetosome biotinylation in this work could also be considered an effective biotin surface display technique. The surface display is a recombinant technology that expresses target proteins on the surface of biomembranes, biostructures, or even biomolecules, and can be applied to almost all types of biological entities from viruses to mammalian cells. Phage display, mRNA display, ribosome display, bacteria display, and yeast display are among the most commonly used methods. These techniques have been used for various biotechnical and biomedical applications such as therapy of disease, drug screening, biocatalysts, library screening, quantitative assays, and biosensors ,57,58. HUp until now, biotin has been the only nonprotein biomolecule employed in the magnetosome surface display system. Biotin is widely distributed in animal and plant tissues and has a molecular weight of 244.31 Da with two ring structures: imidazolone ring, the main site noncovalently binding with avidin/streptavidin or special receptors with high specificity and affinity ; and thiParticularly, bioengineered biotinylation of magnetosomes brings an important new function to the display system. It is known that biotin receptors are overexpressed in different tumor cells, including breast, ovarian, and lung cancer ,65. ThusIn brief, we systematically introduced the bioengineering and biofabrication process of biotinylated magnetosomes. Both the strategy and resulting engineered magnetosome could be promising examples for preparing multiple functional biogenic magnetic nanomaterials.


Progesterone treatment is commonly employed to promote and support pregnancy. While maternal tissues are the main progesterone targets in humans and mice, its receptor (PGR) is expressed in the murine embryo, questioning its function during embryonic development. Progesterone has been previously associated with murine blastocyst development. Whether it contributes to lineage specification is largely unknown. Gastrulation initiates lineage specification and generation of the progenitors contributing to all organs. Cells passing through the primitive streak (PS) will give rise to the mesoderm and endoderm. Cells emerging posteriorly will form the extraembryonic mesodermal tissues supporting embryonic growth. Cells arising anteriorly will contribute to the embryonic heart in two sets of distinct progenitors, first (FHF) and second heart field (SHF). We found that PGR is expressed in a posterior–anterior gradient in the PS of gastrulating embryos. We established in vitro differentiation systems inducing posterior (extraembryonic) and anterior (cardiac) mesoderm to unravel PGR function. We discovered that PGR specifically modulates extraembryonic and cardiac mesoderm. Overexpression experiments revealed that PGR safeguards cardiac differentiation, blocking premature SHF progenitor specification and sustaining the FHF progenitor pool. This role of PGR in heart development indicates that progesterone administration should be closely monitored in potential early-pregnancy patients undergoing infertility treatment. One of the leading causes of recurring early pregnancy loss is embryo implantation failure and consequent embryonic death . HormonaFurthermore, progesterone is also associated with early murine embryonic development, as low progesterone concentrations lead to the developmental delay of blastocysts in vitro . FollowiWhether progesterone could have a dual role in supporting maternal tissues and embryonic development at gastrulation is unknown. We found that PGR is expressed in a posterior–anterior gradient in the PS of gastrulating murine embryos, resembling the BMP signaling gradient. We established in vitro cell culture systems to model gastrulation and assessed the role of PGR in post-implantation cells. We employed mouse epiblast stem cells (EpiSCs) that correspond to the undifferentiated epiblast of post-implantation embryos and are primed to differentiate in vitro into different germ layer lineages ,11. To sWe found that PGR is expressed in EpiSCs and demonstrated that it is involved in early mesoderm specification, modulating extraembryonic and cardiac mesoderm differentiation. Furthermore, we show that PGR has a specific effect on the maturation of the PS-derived progenitors, promoting the differentiation of the BMP-responding cell populations and thus favoring the posterior mesoderm progenitors such as extraembryonic mesoderm and first heart field. We also report that during differentiation, progesterone signaling differentially modulates the expression of actin cytoskeleton remodeling, focal adhesion, and cell motility proteins implicated in the process of mesoderm formation. In conclusion, we demonstrate that PGR plays a role in the early specification of extraembryonic mesoderm and cardiac progenitors at gastrulation. Given progesterone is used as a treatment for recurring early miscarriages and improved in vitro fertilization (IVF) success rate, the offspring of patients undergoing infertility treatment should be monitored with special emphasis on cardiovascular conditions.Evx1, Bmp4, Hand1, Tbx3, Foxf1, and Pgr, demonstrating that a high level of BMP restricts the fate of PPS towards posterior and extraembryonic mesoderm, while the anterior markers Gsc, Sox17, Mesp1, Tbx6, Eomes, and Foxa2 are more abundant in the APS cells, suggesting that this population is enriched in progenitor cells with endoderm, cardiac, and paraxial mesoderm signatures.Primed pluripotency is the state that immediately precedes germ layer specification, including PS induction and mesoderm differentiation ,14. We s  . The expd Bcl11b E, suggesantation ,18,19,20 in vivo F. These Pgr function during gastrulation, we assess PGR protein levels and localization in embryonic day (E) 6.0–6.5 mouse embryos. PS formation represents the initiation of gastrulation, the process in which ectoderm, mesoderm, and endoderm cell fates are established [To further characterize ablished ,8. The tablished . These dPgr expression among different lineages, we confirm that Pgr is highly expressed in PPS compared with definitive endoderm and neural lineages  are screened by PCR genotyping, and the lack of the receptor protein is confirmed by Western blot  binding, cell adhesion, cytoskeleton remodeling, and cell motility are mechanistic factors strongly influencing PS induction and mesoderm formation . PGR actmotility A. RNA-seresponse . RNAseq mesoderm B. While Furthermore, in cardiac mesoderm, Ncam1 , the cadThe importance of progesterone signaling in preparing and supporting pregnancy as well as maintaining ovulation is well-documented ,20,47,48T-Bra, supporting a potential role in mesoderm differentiation.We reported that, in vivo, PGR is expressed in the PS of gastrulating mouse embryos in a posterior-to-anterior gradient, and enriched in primed pluripotent stem cells, EpiSCs, which represent the cellular state preceding gastrulation. In vitro, during EpiSCs differentiation towards mesoderm, both the transcript and protein peak in a 24 h window, which coincides with the high expression of the PS marker Here, we report that PGR is involved in regulating extraembryonic and cardiac mesoderm differentiation. We show that PGR has a lineage-specific effect on the maturation of extraembryonic mesodermal progenitors, promoting hemangioblast differentiation over allantois fate.Hand1 and Foxf1, as well as Hcn4 and Tbx3, which label the heart conductive progenitors, suggesting that it could support the formation of atrial and ventricular cardiomyocytes with FHF origin. Interestingly, PGR has an opposite effect on SHF cardiac genes, inhibiting the expression of Hand2, Mef2c, Myl4, Myl7, Tnnt2, and Tnni1. These results suggest that PGR promotes the differentiation of FHF derivatives and represses SHF fate. While Pgr mutant embryos do not have evident cardiac defects, it is possible that an alternative hormonal receptor compensatory mechanism exists in vivo. Another possibility is that SHF-derived cells could rescue the FHF deficiency. In zebrafish embryos, ablation experiments showed that SHF could compensate for the absence of FHF [Additionally, during cardiac differentiation, PGR promotes the expression of the FHF markers e of FHF .Pgr-KO cells exposed to differentiation conditions towards extraembryonic and cardiac mesoderm that rely on BMP signaling suggests that PGR modulates BMP-driven mesoderm specification. The posterior–anterior gradient of PGR in the embryos also resembles BMP levels at gastrulation, supporting a possible BMP–PGR interaction. It has previously been reported that BMP signaling upregulates PGR expression in the uterus. Similarly, BMP could induce PGR in the posterior PS [Gata4 and Nkx2-5 expression at E8.0, but not for their maintenance, and that the BMP receptor is crucial for the specification of the FHF. SHF progenitor differentiation instead strongly relies on canonical WNT signaling [Id1, which is critical for the cardiac induction of FHF genes [The observation that the in vitro phenotype is more severe in erior PS . Therefoignaling . MoreoveHF genes . On the HF genes . Pgr mutant mice do not have strong cardiac defects. The dual role of PGR as cardiac progenitor modulator is clearer in the overexpression experiments. While overexpression of PGR has a moderate effect on FHF markers, it has, on the contrary, a strong effect on SHF, suggesting that PGR blocks SHF progenitors if present at higher levels. We can speculate the existence of a BMP-dependent safe-lock mechanism blocking premature SHF differentiation or transdifferentiation and sustaining concomitantly the FHF progenitor pool. To work as a safeguard of premature differentiation could be the major role of PGR, and it could explain why the 57. Again postnatally, progesterone supplementation promotes neonatal cardiomyocytes proliferation through the upregulation of YAP signaling [FHF cells mostly become cardiomyocytes, while SHF-derived cells are multipotent and give rise to various cardiac cell types, such as cardiomyocytes, smooth muscle, endothelial, and fibroblast cells. In adult mice, it was reported that progesterone has an anti-atherosclerotic effect and inhibits proliferation and migration of aortic smooth muscle cells ,55. Thusignaling . Thus, PItga3 and Itga4 are specifically expressed in extraembryonic and cardiac mesoderm, respectively, indicating that specific cell–matrix interactions could differentially affect their migratory and differentiation properties. Integrins can signal across the plasma membrane in both directions, transducing extracellular and intracellular signals affecting cell fate [Pgr mutant cells, the upregulation of Itgb3 in ExM suggest that a PGR-ITGB3-dependent mechanism could mediate the establishment of a specific microenvironment promoting extraembryonic mesoderm differentiation. ITGB3 is historically associated with hemangioblast differentiation, and its upregulation coincides with increased levels of Tal1 and Gata2, thereby indicating a shift of Pgr-KO ExM cells towards a hemangioblast fate [Mesoderm delamination from the epiblast requires basal membrane disruption, apical constriction, loss of apicobasal polarity, changes in cytoskeletal properties, and acquisition of cell motility. Thus, after exiting pluripotent epiblast state, the different mesodermal populations acquire a specific set of cytoskeletal components necessary for cell motility and adhesion, which can underlie the differences in their migratory properties. A recent study showed that during mouse gastrulation, extraembryonic and embryonic mesoderm showed differences in cell migratory properties . ExM andell fate . ITGB3 mell fate . In Pgr ast fate . These dAltogether, the observed phenotypes could be attributed to the previously described PGR–cSRC interaction . The perIn other contexts, PGR is known to directly affect cytoskeleton actin dynamics and focal adhesion formation in breast cancer, ovarian, neural, and human endothelial cells. Furthermore, previous reports showed that progesterone is involved in cytoskeletal remodeling and cell migration during gastrulation in zebrafish embryos . While tThe PGR protein exists in at least two isoforms—a longer PGR-B and an N-terminally truncated PGR-A ,42. The Progesterone and its synthetic analogues are used as contraceptive agents  and a trThe potential impact of this study, however, extends beyond the consequences for embryonic development. Stem-cell-based therapies are considered to be one of the possible groundbreaking solutions for degenerative diseases such as type 1 diabetes, Parkinson’s, and heart failure, as they can be differentiated into virtually any somatic cell type ,65,66. AUnless stated otherwise, all chemical reagents were purchased from Sigma Aldrich, all cell culture reagents from Thermo Fisher Scientific , and all the cytokines from Qkine .A total of 129 EpiSCs were cultured on fibronectin-coated plates , FC01015) in N2B27 supplemented with 20 ng/mL Activin A Plus, 10 ng/mL bFGF and 1 µM XAV939 (EpiSC medium). N2B27 was prepared in the following way: 200 mL DMEM:F12, 200 mL Neurobasal Medium, 4 mL L-Glutamine, 400 µL β-mercaptoethanol, 2 mL N2 supplement, and 4 mL B27 supplement. N2 reagent was prepared by adding 10 mL 7.5% BSA to 68.75 mL DMEM:F12, mixing well, and adding the following components drop-wise: 250 mg insulin re-suspended in 10 mL 0.01 HCl, 1 g apo-transferrin dissolved in 10 mL MilliQ water, 330 μL progesterone (stock solution 0.6 mg/mL), 1 mL putrescine (stock solution 160 ng/mL), and 100 μL Na selenite (stock solution 3 mM). Mouse embryonic stem cells (mESCs) were cultured on gelatin-coated plates in N2B27 medium supplemented with LIF, 1 μM PD-0325901, and 3 µM CHIR99021. As part of standard cell culture procedure, all cell lines were mycoplasma-tested and confirmed to have normal karyotype.3 cells/cm2 in chemically defined EpiSC medium 24 h prior to induction of differentiation. PPS cells were induced by supplementing N2B27 with 30 ng/mL Activin A, 40 ng/mL BMP4, 6 µM CHIR-99021, 20 ng/mL bFGF, and 100 nM PIK-90, while APS cells induction medium consisted of N2B27 with 30 ng/mL Activin A, 4 µM CHIR-99021, 20 ng/mL bFGF, and 100 nM PIK-90. In both cases, cells were cultured in the appropriate media for 24 h. PPS cells were further differentiated to extraembryonic mesoderm (ExM) for another 24 h by supplementing N2B27 with 1 μM A-83-01, 30 ng/mL BMP4, and 1 μM C59. Lateral mesoderm (LM) was obtained by applying the ExM medium on APS cells for 24 h, and finally paraxial mesoderm (PM) was induced from APS with N2B27 supplemented with 1 µM A-83-01, 3 µM CHIR-99021, 250 nM LDN193189, and 20 ng/mL bFGF for 24 h.Cells were differentiated in a monolayer and seeded on fibronectin-coated wells at 10.5 × 10Tbp and Sdha) were used for normalization, and all samples were run in duplicates. The list of primer sequences can be found in Cells were washed once with PBS and detached from the culture dish using Accutase. The cells were collected, and RNA was extracted using QIAGEN RNeasy Mini Kit  according to the manufacturer’s instructions. A total of 1 μg of RNA was reverse transcribed using SuperScript III enzyme  and random hexamers , according to the producer’s protocol. All qPCR experiments were run on Roche LightCycler480 Real-Time PCR System using SYBR Green master mix . A mix of concentrated cDNA was used as a standard curve, two housekeeping genes (w/v) bromophenol blue). We ran the samples in NuPAGE Novex 4–12% Bis-Tris Protein Gels  using 1× NuPAGE MES SDS running buffer  at 150 V for 70 min. We transferred the protein to Amersham Protran Nitrocellulose Blotting membrane 0.45 µm  in 1× NuPAGE transfer buffer  at 100 V for 1.5 h in a cold room. Membranes were shortly rinsed in PBS and blocked using 5% skim milk in PBS with 0.1% Tween-20 for 1 h at room temperature (RT) and next incubated with primary antibodies at dilution 1:1000 overnight at 4 °C. For blotting PGR, we used Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb . The following day, membranes were washed 3 × 10 min with 0.1% Tween-20 in PBS and incubated with HRP-conjugated secondary antibodies for 2 h at RT ). Finally, the membranes were washed as previously, and the bands were visualized using Amersham ECL Prime Western Blotting Detection reagent  and ChemiDoc MP (Bio-Rad).Cells were washed once with cold PBS on ice and scraped in Laemmli buffer. Cells were sonicated at 20% for 10 s to release the protein content using Branson Digital Sonifier Model S-450D , centrifuged at maximum speed for 10 min at 4 °C, and stored at −80 °C until the gel could be run. The protein concentration was measured on NanoDrop (Thermo Fisher Scientific) A280, and 25–30 μg of total protein was loaded in each lane, diluted in Laemmli for the final volume of 30 μL with 2 μL 0.1 M DTT and 3 μL loading dye  at a density of 10–20 k cells/well, and fixed 24–48 h afterwards using 4% PFA for 15 min at RT. Next, we permeabilized the cells using 0.5% Triton in PBS for 10 min at RT and blocked the non-specific binding sites using 3% BSA in 0.1% Triton/PBS for 20 min at RT. We incubated cells with primary antibody solution (1% BSA in 0.1% Triton/PBS with antibodies) containing mouse anti-PGR  overnight at 4 °C. The following day, we washed cells 3 × 5 min using washing solution (1% BSA in 0.1% Triton/PBS) and incubated with fluorophore-conjugated secondary antibodies and nuclear dye DAPI in darkness for 2 h at RT. Finally, we washed the cells 3 × 5 min in washing solution and 1 × 5 min in PBS. The cells were either immediately imaged or stored at 4 °C and imaged within 1 month.v/v) Triton X-100 for 10 min at RT, shaking. Embryos were blocked overnight at 4 °C in CAS-Block . Embryos were incubated with the 1:100 diluted primary antibodies for 48 h at 4 °C, and the following antibodies were used: mouse anti-PGR , goat anti-SOX2 , and rabbit anti-BRACHYURY . Embryos were washed 3 x 10 min with CAS-Block and then overnight at 4 °C. Next, they were incubated with secondary antibodies for 24 h in 4 °C. Embryos were washed 3 × 10 min in CAS-Block and then overnight at 4 °C. Afterwards, they were incubated with 1 μg/mL DAPI for 1 h at RT. Finally, embryos were washed 3 × 10 min at RT and dehydrated in ascending concentrations of methanol, eventually reaching 100%. Embryos were optically cleared in 1:2 solution of benzyl alcohol:benzyl benzoate (BABB). Cells and embryos were imaged using Leica TCS SP8 confocal microscope, and the images were analyzed using ImarisTM software .Mouse embryos were dissected and fixed in 4% PFA at RT for 30 min at RT. They were permeabilized using PBS/PVP + 1% (6 cells per condition were washed with ice-cold FACS buffer (10% (v/v) FBS in PBS) and resuspended in 1 mL ice-cold FACS buffer containing 1 µg/mL DAPI. Single cells were sorted into Eppendorf Polypropylene U-shaped 384-well Twin Tec PCR Microplates  containing 2 μL of lysis solution (0.2% (v/v) Triton X-100) supplemented with 0.4 U/μL RNasin Ribonuclease Inhibitor  and 400 nM indexed RT primer from group 1 (1–96 barcodes) or group 2 (97–192 barcodes). Wild-type (WT) EpiSCs were sorted into each plate, as spike-in control for batch-effect correction. Capture plates were prepared on the Bravo automated liquid handling robot station  using 384-filtered tips . Index sorting was performed using a FACS Aria III cell sorter  at the DanStem Flow Cytometry Platform , sorting singlets and live cells only. Directly after sorting, plates were briefly centrifuged, snap-frozen on dry ice, and stored at −80 °C for further procedures. Semi-automated library preparation was carried out using 10–12 total cycles of PCR amplification and AMPure XP beads .Massively Parallel Single-Cell RNA-Sequencing technology (MARS-Seq) was performed for scRNA-Seq. EpiSCs were seeded at a density of 60 k cells per well of a six-well cell culture plate pre-coated with 15 µg/mL human Fibronectin and differentiated to PPS and ExM according to differentiation protocols described above. At 24 h and 48 h time-points, cells were dissociated using Accutase for 3 min at 37 °C and counted. A total of 0.5 × 10DNA concentration was measured with a Qubit Fluorometer  and fragment size was determined with a Fragment analyzer (Agilent). Libraries were paired-end sequenced on a Next-Seq 500 Sequencer  at the DanStem Genomics Platform . Between 1146 and 1528 cells were sequenced per lane.Data for PPS can be retrieved under the following accession number E-MTAB-12106, and the data for ExM under E-MTAB-12107.EpiSCs were seeded at 60 k cells/well in six-well cell culture plates pre-coated with 15 µg/mL human Fibronectin and differentiated to PPS, APS, ExM, LM, and PM as described above. At chosen time points (either 24 h or 48 h), cells were lysed, and total RNA was extracted using the QIAGEN RNeasy Mini Kit according to the manufacturer’s instructions . RNA concentration was quantified by NanoDrop, and quality was verified with a Fragment analyzer. Library preparation was carried out using 0.5 μg of RNA with NEBNext Ultra II RNA library Prep Kit  according to the manufacturer’s instructions. Libraries were amplified for 4 total PCR cycles and purified with AMPure XP beads. DNA concentration was measured with a Qubit Fluorometer, and fragment size was determined with a Fragment analyzer. All samples were sequenced in biological triplicates on a Next-Seq 500 Sequencer (Illumina) at the DanStem Genomics Platform .Bulk RNA-seq data can be accessed on ArrayExpress under E-MTAB-12104 accession number.Pgr sequences corresponding to isoforms A and B using the following primers:RNA was extracted from WT EpiSCs using QIAGEN RNeasy Mini Kit , and 1 μg was reverse transcribed using SuperScript III enzyme  and random hexamers  according to the manufacturer’s protocol. We PCR amplified PGR-B forward: 5′ CACC-ATGACTGAGCTGCAGGCAAAG 3′PGR-B reverse: 5′ TCACTTTTTGTGAAAGAGGAGCGG 3′PGR-A forward: 5′ CACC-ATGAGTCGGCCAGAGATCAAG 3′PGR-A reverse: 5′ TCACTTTTTGTGAAAGAGGAGCG 3′.E. coli, which was part of the kit, according to the producer’s protocol. We then used Gateway™ LR Clonase™ Enzyme mix  and, following the manufacturer’s protocol, cloned the sequences encoding the two isoforms into destination vector pLEX307 (kindly provided by the Helin laboratory). The resulting constructs carrying the sequences encoding either PGR-A or PGR-B were verified by sequencing.We ran the PCR product on 1.5% agarose gel and extracted the DNA from gel using QIAquick Gel Extraction Kit . Purified PCR product was cloned into pENTR™/D-TOPO™  vector using TOP10™ chemically competent 6 cells/dish. The following day cells were transfected using the following mix for each 10 cm dish: 15 μg pLEX307-PgrA or pLEX-PgrB, 10 μg packaging plasmid psPAX2, and 5 μg envelope-encoding pCMV-VSV-G with 90 μL 10 mg/mL PEI  in 500 μL Opti-MEM™ reduced serum medium . Transfection was performed in the late afternoon and medium was changed the following morning. Medium was collected 24 h and 48 h later for virus concentration. The virus was concentrated using Amicon® Ultra 15 mL Centrifugal Filters . Briefly, collected medium was centrifuged for 15 min at maximum speed to remove cellular debris. Supernatant was loaded on the Amicon® Ultra 15 mL Centrifugal Filters and centrifuged at maximum speed for 15–20 min. The loading of the media and centrifugation was repeated until all the virus-containing medium was passed through the filter and concentrated to 500–1000 μL total volume. The concentrated virus was stored in −80 °C for up to one year.To prepare lentiviral vectors, HEK293T cells were seeded on 10 cm plastic cell culture dishes at a density of 6 × 10Cells were seeded on fibronectin-coated 6-well plates at a density of 100 k cells/well in the appropriate culture media. The following day cells were transduced at MOI > 0.7, and the following day medium was changed to EpiSC medium containing 1 μg/mL puromycin . The selection pressure was maintained for the following two days, when the cells were passaged and seeded for differentiation experiment on 6-well plates at a density 100 k cells/well. The following day medium was changed to differentiation medium. Cell lysates were collected 24 h and 48 h after induction of mesoderm differentiation for the analysis.® Green Master Mix . At least 10 ng of gDNA was used for each PCR reaction. The product of the reaction was run on 2% agarose gel, and the resulting bands were imaged using ChemiDoc MP (Bio-Rad). For karyotyping cells were sent to Cell Guidance Systems, Cambridge, UK.Cell lines were genotyped using GoTaqAll animals used in this study were maintained in laboratory animal housing facilities at macroenvironmental temperature and humidity ranges of 20–24 °C and 45–65%, respectively, with a 12/12 h light/dark cycle. All animal work was carried out in accordance with European legislation. All work was authorized by and carried out under the Project License 2017-15-0201-01255 issued by the Danish Regulatory Authority. Mouse embryos were dissected at stages E6.5-7.5, with the stage described as E6.75 dissected in the evening.


Pediatric non-Wilms renal tumors (NWRTs), which comprise a small proportion of renal tumors, are a heterogeneous group of neoplasms with variable malignant potential, mortality, and response to treatment. We performed this study to determine the clinical characteristics, management and prognosis of children with Pediatric NWRTs.Medical records of all patients (n = 139) treated for NWRTs over a 12-year period (2008.01–2019.10) at a single center were reviewed retrospectively.The histopathological groups of NWRTs included malignant rhabdoid tumor of the kidney (MRTK) , renal cell cancer (RCC) , clear cell sarcoma of the kidney (CCSK) , congenital mesoblastic nephroma (CMN) , cystic nephroma (CN) , metanephric tumors , renal angiomyoliporma (RAML) , renal primitive neuroectodermal tumor , renal hemangioma , inflammatory myofibroblastic tumor , ossifying renal tumor of infancy (ORTI) . The distribution of all malignant NWRTs, including MRTK, CCSK, RCC and PNET, according to stage was as follows: stages I (n = 26), II (n = 16), III (n = 29), and IV (n = 11). The summary table shows the treatment offered to children with NWRTs. A total of 123 children were followed up for an average of 42 months. Sixteen children were lost to follow-up. Tumor-free survival was observed in 94 children. One patient who suffered from RCC is currently receiving targeted therapy and survives with the tumor. Twenty-eight children (22.8%) died.Pediatric NWRTs comprise 19.1% of all renal tumors in our single center. Most NWRTs can readily be distinguished using a range of immunohistochemical markers. Molecular genetic profiling has allowed much progress in the understanding of this group of tumors, making diagnosis and classification less difficult. The mainstay treatment of malignant NWRTs, including MRTK, CCSK, RCC and PNET, is comprehensive treatment. The mainstay treatment of benign NWRTs, including RAML, CN, ORTI, CMN, metanephric tumors, and renal hemangioma, is surgical resection alone and when the tumor diameter is smaller than 7 cm and the tumor locates in one pole, NSS can be performed. Pediatric renal tumors account for approximately 6–7% of childhood solid neoplasms. Wilms tumor is the most common renal tumor in children and represents approximately 80%-90% of all pediatric renal tumors. Pediatric non-Wilms renal tumors (NWRTs), which comprise a small proportion of renal tumors, are a heterogeneous group of neoplasms with variable malignant potential, mortality, and response to treatment . We retrThis study was carried out in the Department of Urology at a comprehensive tertiary pediatric hospital in China. Following institutional review board approval, the medical records of children (under18 years of age) with renal tumors in our hospital between January 2008 and October 2019 were retrospectively analyzed. The patient inclusion criteria required NWRTs that were first diagnosed and treated in our hospital and that the patients’ medical records were complete. Patients who were first treated in other hospitals or had no pathological diagnosis in our hospital were excluded from our study.All patients were regularly monitored with surveillance for the recurrence and metastasis using ultrasonography, X-ray examination and computed tomography. Overall survival (OS) was calculated from the date of diagnosis to the date of last follow-up or disease-related death. SPSS23.0 statistical software was used for data collection and analysis. OS were estimated using the Kaplan–Meier method.From January 2008 to October 2019, a total of 729 cases of pediatric renal tumors were reviewed, of which 590 (80.9%) were Wilms tumors and 139 (19.1%) were NWRTs.The mean age of the 139 children with NWRTs was 3.5 years (range: 7 days–15 years and 2 months). The age distribution according to tumor histology is summarized in Table Clinical presentations included 50 cases with hematuria, 37 cases detected incidentally by ultrasound examination, 34 cases with abdominal mass, 10 cases with abdominal pain, 4 cases with hematuria and abdominal pain and 4 cases with vomiting and abdominal distension. The clinical presentations according to tumor histology are shown in Table Preoperative imaging, including ultrasound (US) and computed tomography (CT), was performed in all patients. The tumor sizes ranged from 2.0 × 1.5 × 1.5 to 17.7 cm × 12.8 cm × 19.1 cm, with an average of 7.5 cm × 6.1 cm × 6.9 cm. There were 100 cases (71.9%) of solid tumors and 28 cases (20.1%) of cystic tumors  , renal cell cancer (RCC) , clear cell sarcoma of the kidney (CCSK) , congenital mesoblastic nephroma (CMN) , cystic nephroma (CN) , metanephric tumors , renal angiomyoliporma (RAML) , renal primitive neuroectodermal tumor (rPNET) , renal hemangioma, inflammatory myofibroblastic tumor (IMT), ossifying renal tumor of infancy (ORTI) .Among the 26 patients with RCC, 20 (76.9%) had Xp11.2 translocations/TFE3 gene fusion, 3 had papillary carcinoma, 2 had clear cell carcinoma, and 1 had chromophobe cell carcinoma. All 26 RCC patients underwent TFE3 immunohistochemical assays, and 20 were positive for TFE3 , II (n = 16), III (n = 29), and IV (n = 11).Neoadjuvant chemotherapy was offered to 9 patients, including CCSK (n:4), MRTK (n:2), RCC (n:1), renal hemangioma (n:1) and rPNET (n:1) which were considered for preoperative diagnosis of Wilms' tumor.Nephron sparing surgery (NSS) was performed in 28 patients. In patients who underwent NSS, the malignant tumor diameter was less than 6 cm in all 8 cases, including 7 cases of RCC, 1 case of MRTKFig. , and theAmong the 30 MRTK patients, 4 patients gave up adjuvant chemotherapy, 26 patients were treated with chemotherapy of VDC and ICE, and 4 patients were treated with radiotherapy simultaneously. All 24 CCSK patients were treated with chemotherapy of I regimen which NWTS-5 recommended, and 22 patients were treated with radiotherapy. The 2 patients with PNET were treated with chemotherapy + radiotherapy, alternating between chemotherapy VDC and IE, and the radiotherapy dose was 36 Gy. One patient with RCC underwent targeted cancer therapy.A total of 123 children (88.5%) were followed up for an average of 42 months (range 13 months to 72 months), and 16 children were lost to follow-up. Tumor-free survival was observed in 94 children. One patient who suffered from RCC is currently receiving targeted therapy and survives with the tumor. Twenty-eight children (22.8%) died.The follow-up of malignant NWRTs included ultrasound and chest X-ray examination every 3 months to 5 years postoperatively. Patients with MRTK had the highest mortality rate, and only 5  patients survived with an average follow-up of 24 months. The patient with MRTK who underwent NSS survived more than 6 years. The overall tumor-free survival was 73.9% (17/23) for CCSK with an average follow-up of 49 months. Twenty-one of 26 patients with RCC were followed up with an average of 37 months. Nineteen patients  survived, including 1 patient who underwent targeted cancer therapy and 7 patients who underwent NSS. One patient with chromophobe cell carcinoma suffered from regional lymph node metastasis 6 months after undergoing NSS. Therefore, an approximately 2.5 cm × 1.8 cm × 2.4 cm mass near the left renal hilum was completely removed during repeat surgery. With a follow-up of 3 years 10 months after resection, the patient had no recurrence or metastatic disease identified. (q24:q12) resulting in the EWS-FLI fusion gene. PNET is1;22(q24:The treatment principle of malignant NWRTs is the need for comprehensive treatment, including surgery, chemotherapy, and, if necessary, radiotherapy . SIOP reThe mainstay of treatment in most benign NWRTs is surgery and when the conditions permit NSS can be undertaken. But we suggest that NSS should be highly selective. When the tumor diameter is smaller than 7 cm and the tumor locates in one pole, NSS can be performed. CMN generally follows a benign course even with local spillage. Radical nephrectomy alone is usually sufficient; 95% of patients do not relapse, and most of the 5% who do relapse have the cellular variant of this disease . HoweverThe prognosis of malignant NWRTs is poor. For MRTK, age is an important prognostic indicator, as younger patients have a worse prognosis than older patients . SurvivaThere were several limitations to this study. First, the current present study is a retrospective, single-center study. Therefore, larger sample sizes with populations are necessary to validate the clinical characteristics of NWRTs. Second, the follow up of the current present study is an average of 42 months. A longer follow-up duration may help us to better understand the treatment, and survival of NWRTs.In conclusion, pediatric NWRTs comprise 19.1% of all renal tumors in our single center. NWRTs can occur in children of all ages, but the peak age of different tumor types is distinct. Clinical and imaging modality characteristics may support a particular diagnosis, but the final histopathologic results with immunohistochemical staining and cytogenetics should remain the gold standards for diagnosis. The treatment principle of malignant NWRTs requires comprehensive treatment, including surgery, chemotherapy, and, if necessary, radiotherapy. The mainstay of treatment in most benign NWRTs is surgery and when the tumor diameter is smaller than 7 cm and the tumor locates in one pole, NSS can be performed.


These two statements need some discussion based on personal and other recent data about the complex relations between cognitive impairment and neuropathology in LB diseases. Ad (a): In a personal autopsy series of 330 PD patients, only 3.2% of demented ones  had Braak LB stages 2–3 with severe AD co-pathology (neuritic Braak stages V and VI), while 35.5% of demented PD patients revealed LB stages 4 or 5 with superimposed neuritic Braak stages V and VI. More than 50% of them showed a strong relationship between the severity of αSyn and tau pathologies, particularly in the limbic system. LB pathology with moderate or high grade AD lesions was seen in 40% of PDD patients, while one third of cases with diffuse LB pathology and mild AD lesions, restricted to Aβ plaques of limbic tau pathology, did not show considerable cognitive impairment2. PDD patients had significantly lower brain weight than non-demented ones, while significantly more severe Alzheimer neuropathological changes were present in PDD than non-demented (ND) PD patients3. In another study of 60 ND-PD and 110 PDD patients, the latter were significantly older than ND-PD ones—83.9 vs. 77.8 years; p < 0.01), PDD showed only slightly higher Braak LB scores (mean 4.2 vs. 4.0), but significantly higher neuritic Braak stages (mean 5.2 vs. 4.4), Thal Aβ phases (mean 3.0 vs. 2.3), and both significantly higher CAA frequency and severity (50% vs. 21.7% and mean 0.72 vs. 0.26). In conclusion, association between cortical Aβ load, generalized CAA and tau pathology are the morphological basis of cognitive decline in PD4.Menšíková et al.5, more severe cortical tau pathology (Braak 5.1 ± 0.7 vs. 4.2 ± 0.4), and higher CAA frequency and severity , the latter mainly in occipital and less in frontal lobes6. Most frequent and highest CAA scores were seen in cases with APOE ε4 7. Cortical tau pathology was also more frequent in DLB, the incidence of negative cases being 70% vs. 82% in PDD, also supporting the notion that the morphological distinction between the two phenotypes is not restricted to Aβ deposition, cortical LB and tau pathologies8. Further differences are more severe αSyn load in hippocampal subareas CA 2/3 and entorhinal cortex (EC) implicating the role of the EC-CA circuitry in the pathogenesis of DLB9. There is different involvement of substantia nigra, with more severe neuronal loss in ventrolateral cell groups in PD, but predominant damage of dorsolateral ones in DLB10, causing less severe postsynaptic dopaminergic upregulation, while significantly higher 5-HT1A receptor-binding density in cortex is seen in DLB compared to PDD11. DLB showed worse prognosis than PDD , which was linked to both increased tau and CAA pathologies distinguishing both disorders. The reasons for these clinical and pathological differences between the phenotypes, PD, PDD and DLB(+AD) are not clear, but recent studies suggest a close interaction of the various pathological proteins, in particular between αSyn, Aβ and tau12 within the spectrum of LB diseases (or diseases with LBs)13. Cerebrovascular co-pathologies, in particular cerebral microbleeds, showed a similar prevalence in PDD, DLB and AD14, although recent studies suggested a synergistic interaction between cerebrovascular disease and Lewy pathologies, which possibly extends to other highly concomitant pathologies in LB disorders15. In conclusion, it should be emphasized that other co-pathologies may influence the clinical features and progression in both PDD and DLB16. The impact of all of them, however, needs further elucidation.Ad (b): There is general agreement that both PDD and DLB share neuropathological features, with a variable mixture of LB and AD-related co-pathologies. However, recent studies have shown some essential morphological differences between PDD and DLB, that is, more frequently increased cortical and striatal Aβ load


An electric shaver was used as the source of the BLDC motor. The following states of the BLDC motor were analyzed: Healthy BLDC motor (HB), BLDC motor with one faulty shaft (1FSB), BLDC motor with two faulty shafts (2FSB), and BLDC motor with three faulty shafts (3FSB). A new method of feature extraction named PNID  was presented. Deep neural networks were used for the analysis of thermal images of the faulty shaft of the BLDC motor: GoogLeNet, ResNet50, and EfficientNet-b0. The results of the proposed technique were very good. PNID, GoogLeNet, ResNet50, and EfficientNet-b0 have an efficiency of recognition equal to 100% for four classes.A technique of thermographic fault diagnosis of the shaft of a BLDC (Brushless Direct Current Electric) motor is presented in this article. The technique works for the shivering of the thermal imaging camera in the range of 0–1.5 [m/s Electric motors are used in many areas of industry, for example, mining and oil industries. Electric motors differ in their design, operating principle, and power. They can be used for various applications: Washing machines, dusters, mixers, industrial machines, cars, and trains. In many areas, it would be difficult for us to live without motors. Some examples of where the electric motor is used are as follows: In cars as a starter, escalators, production lines, lathes, and grinders, and even in home appliances such as printers and electric toothbrushes. Electric motors do not use any fuel. They do not emit exhaust or other gases. Therefore, they can work in closed halls, garages, and even in very small spaces. Moreover, electric motors are produced in sealed housings. Thus, they do not generate sparks, meaning they are useful in hazardous areas.Electric motors are not immune to malfunctions and gradual deterioration. They need regular maintenance to avoid motor failures. Fault detection of electric motors is essential to reduce maintenance costs. The maintenance costs of electric motors can be high if the machines are not diagnosed on time. Faulty electric motors draw additional electricity to operate. Permanently damaged motors should be replaced with new ones. Replacing an electric motor is expensive. Moreover, the failure of an electric motor during operation can stop the production line in the factory. This is a financial and energy loss for the factory. Fault diagnosis of motors can improve their performance. With continuous use, electric motors slowly degrade over time. An increase in the equipment lifespan of a motor can be also performed using fault detection techniques.Fault diagnostics of electric motors include various faults, e.g., a faulty shaft, faulty bearings, faulty gear, a faulty fan, an imbalance of the shaft, broken rotor bars, and shorted coils. A technique of thermographic fault diagnosis of the shaft of a BLDC (Brushless Direct Current Electric) motor is presented in this article. A faulty shaft can cause an imbalance. Imbalance causes premature failure in electrical motors. It usually causes noise, heat, and vibration. If the damaged shaft is not repaired, the motor will be damaged again. The author’s motivation is to develop new methods of diagnosing electric motors.2]. The following states of BLDC motors were analyzed: HB, 1FSB, 2FSB, and 3FSB. PNID , GoogLeNet, ResNet50, and EfficientNet-b0 were used for the analysis of thermal images of the faulty shaft of the BLDC motor. The obtained efficiency E and MeanE were equal to 100%. PNID, GoogLeNet, ResNet50, and EfficientNet-b0 work very well for the analysis of thermal images. Verification was carried out using GoogLeNet, ResNet50, and EfficientNet-b0. A new method of feature extraction, PNID , was presented. Deep neural networks were used for the analysis of thermal images of the faulty shaft of the BLDC motor, namely, GoogLeNet, ResNet50, and EfficientNet-b0.The main contribution of the paper is the implementation and analysis of the technique of thermographic fault diagnosis of the shaft of the BLDC motor. The technique works for the shivering of the thermal imaging camera in the range of 0–1.5 . The obtained thermal images were different. Thermal images were measured for an emissivity coefficient ε = 0.8. The emissivity coefficient ε = 0.8 was accurate for analyzed thermal images , F = 50 Hz (frequency), I = 0.2 A (electric current), M = 0.19 kg (weight), and very quiet operation—up to 48 dB. The parameters of the Philips S5587/30 electric shaver were pV = 100–240 V (voltage), pF = 50 Hz (frequency), pI = 0.36 A (electric current), pP = 9 W (power of the motor), and pM = 0.21 kg. The parameters of the Philips S1332/41 electric shaver were sV = 100–240 V (voltage), sF = 50 Hz (frequency), sI= 0.36 A (electric current), sP = 9 W (power of the motor), and sM = 0.19 kg.BLDC motors of electric shavers were used . The analyzed electric shavers were similar to each other. The parameters of the Prime3 Men’s Shaver SRS11 electric shaver were The following states of BLDC were analyzed: Healthy BLDC motor (HB), BLDC motor with 1 faulty shaft (1FSB), BLDC motor with 2 faulty shafts (2FSB), and BLDC motor with 3 faulty shafts (3FSB). Thermal images of the analyzed states of the BLDC motor (Prime3 Men’s Shaver SRS11) are presented . The theThe thermal images of the BLDC motor with 1 faulty shaft (1FSB) are shown in The thermal images of the BLDC motor with 2 faulty shafts (2FSB) are shown in The thermal images of the BLDC motor with 3 faulty shafts (3FSB) are shown in State HB has a large number of cold areas (blue/gray color). State 3FSB has a large number of hot areas (red/white color).Thermal images of the analyzed states of the BLDC motor (Philips S5587/30) are presented .Thermal images of the analyzed states of the BLDC motor (Philips S1332/41) are presented .Measurements of the BLDC motor states were carried out for operating motors. The temperature of the motors after 5 min of operation was similar to that of a few hours. These motors do not heat up much. For motors with higher powers, measurements should be made after 1 h of operation. A faulty electric motor gives a higher surface temperature. The higher surface temperature is detected and analyzed using the proposed fault diagnosis technique.The thermographic fault diagnosis technique consists of six steps of image processing: Thermographic measurement, conversion of the movie into thermal images, conversion of thermal images into the proper dimensions of 224  network . The movCreate training and test sets.diff_hb_1f1 = |hb1-1f1|,…, diff_hb_1f30 = |hb30-1f30|, diff_hb_2f1 = |hb1-2f1|,…, diff_hb_3f1 = |hb1-3f1|,…, diff_1f_2f1 = |1f1-2f1|,…, diff_1f_3f1 = |1f1-3f1|,…, diff_2f_3f1 = |2f1-3f1|,…, diff_2f_3f30 = |2f30-3f30|; hb—matrix of thermal image of class HB, 1f—matrix of thermal image of class 1FSB, 2f—matrix of thermal image of class 2FSB, 3f—matrix of thermal image of class 3FSB.Compute differences of thermal images: R, where R = diff_hb_1f1 + … + diff_hb_1f30 + diff_hb_2f1 + … + diff_hb_2f30 + diff_hb_3f1 + … + diff_hb_3f30 + diff_1f_2f1 + … + diff_1f_2f30 + diff_1f_3f1 + … + diff_1f_3f30 + diff_2f_3f1 + … + diff_2f_3f30.Compute matrix maxR—maximum value of matrix R.Compute M = R/maxR.Compute matrix Bij = Mij × Mij, Bij—new value of matrix B, Mij—the value of matrix M.Compute the value B.Compute matrix t threshold of binarization using Otsu’s method for matrix B. Compute the power of the computed threshold of binarization 2t.Compute the B using a new level of binarization (t2). Compute matrix A.Create binarization of matrix N = S + A, where S—matrix of the analyzed thermal image.Compute V=N−1.Compute V. Form the output matrix Z .Change values less than 0 to 0 for matrix The new method of feature extraction—PNID —is presented. The PNID method has the following steps:A flowchart of the PNID method is depicted in Z of the analyzed states for the PNID method are presented in t  were analyzed to test the neural network. In total, 240 training thermal images (60 for each class) were used for the training of the neural network.E) for one state of the BLDC is defined as follows (1):X1 is the number of recognized test images and X2 is the number of all test images.The parameters of neural networks were as follows: InitialLearnRate = 0.001, ValidationFrequency = 5, MaxEpochs = 500, MinBatchSize = 11, Solver—sgdm (stochastic gradient descent with momentum). The efficiency of recognition (E is expressed as follows (2):E1 is E of HB, E2 is E of 1FSB, E3 is E of 2FSB, and E4 is E of 3FSB.The arithmetic mean of E and MeanE are equal to 100% . Measurements will be taken at the different camera–motor distances.