V2, an engineered voltage-gated Na+ channel harboring a selenocysteine in its inactivation motif, as a non-photonic, sensitive, gateable, and reversible sensor for membrane-delimited reactive species. roNaV2 allows for the assessment of chemical modification induced in fluorescence microscopy settings with high sensitivity and time resolution and it demonstrates the usefulness of ion channels as highly sensitive reporters of membrane processes.Photonic experiments are of key importance in life sciences but light-induced side effects are serious confounding factors. Here we introduce roNa Therefore, there is a clear demand for sensitive probes to precisely monitor cellular RS with spatio-temporal resolution to understand the multifaceted role of cellular RS in cell physiology. Recently, mutants and fusions of genetically encoded GFP (green fluorescent protein), for example, reduction and oxidation sensitive variants roGFP2These fluorescent reporters offer several useful features, such as the choice of localized sub-cellular targeting, real-time RS detection, and ratiometric observation that overcome artifacts arising due to photo-bleaching and inhomogeneous distribution of the sensors. However, there is the inherent problem of phototoxicity that can lead to lasting irreversible destruction of cellular structures and may even result in immediate alteration of molecular function. Despite the obvious detrimental consequences of visible light in cell physiological studies, the issue of phototoxicity has been only infrequently considered in life science research891112+ (NaV) channel with a cysteine residue in its inactivation domain  as a non-photonic RS sensor delimited to the cell membraneV channels activate (open) and inactivate (close) rapidly in response to membrane depolarization, thus yielding transient Na+ inward current. Rapid and essentially complete inactivation of the NaV channel, which is crucial for maintaining cellular excitability, is mediated by the inactivation motif “IFM” (Ile-Phe-Met), located in the intracellular linker between domains III and IV of the NaV proteinV1 exhibits an RS sensitivity comparable to roGFP2, including a relatively slow response to changes in the cellular redox milieuPreviously, we have reported a genetically engineered voltage-gated NaV1 response is limited by the kinetics by which the cysteine residue harbored in the inactivation motif of the channel is modified. We therefore thought to create a second-generation genetically engineered NaV channel containing selenocysteine (Sec or U) in its inactivation motif, also referred as roNaV2. As outlined below, it may serve as a non-photonic tool for monitoring RS delimited to the cell membrane – in particular those generated by light exposure – in a switchable and ratiometric manner.The speed of the roNaV1, a voltage-gated NaV channel with a cysteine residue in its inactivation domain, responds to local RS by diminished fast inactivation  at position 1305. Selenocysteine, the 21st natural amino acid, differs from cysteine by the single substitution of selenium for sulfur. Selenium is a better nucleophile than sulfur and, under physiological conditions, selenocysteine residues are ionized whereas cysteine residues are typically protonated16V1.4 channels with a TGA stop codon at the position equivalent to M1305, and followed by a selenocysteine insertion sequence (SECIS) element downstream of the channel gene  in HEK 293 cells, currents were measured in the whole-cell mode of the patch-clamp technique. The peak amplitude at −20 mV for roNaV2 was much smaller  than for roNaV1  and wild-type rat NaV1.4 . Supplementation of the cell culture medium with sodium selenate , however, increased the functional expression of roNaV2 about 8-fold , while the mean current amplitudes of roNaV1  and NaV1.4  were unaffected  or high concentrations (25 mM) of glucose as to affect endogenous RS production. The inactivation time course of roNaV2 was noticeably slower in the cells cultured in high glucose, while no difference was detected for roNaV1 (I) was about 2.5-fold larger in high glucose compared to low glucose. Furthermore, treatment of the cells with the mitochondrial uncoupler BAM15 (5 μM), which does not depolarize the plasma membraneV1  was described with single-exponential functions. While the oxidant chloramine T  had a negligible effect on roNaV1 within 10 min, roNaV2 responded rapidly and robustly ; roGFP2 was intermediate (218 ± 2 s). Even 1 μM H2O2 application could be readily detected, whereas the response of roGFP2 was about 100-fold slower  of a single amino-acid residue, either Cys (roNaV1) or Sec (roNaV2), located in the inactivation motif of the channel, i.e. a site right at the interface of membrane protein and cytosol. In particular roNaV2 may thus serve as a physiologically important and convenient tool for investigating real-time oxidative cellular events as exemplified for the response to mitochondria uncoupling  resulted in progressive loss of inactivation, characterized by a mean time constant of 0.377 ± 0.042 s (n = 27), compared to 549 ± 112 s for roNaV1 and 5010 ± 610 s for the wild-type NaV1.4 channel  and GFP mutant S147C (i.e. lacking the partner for forming a disulfide bridge with C204 as present in roGFP2) was even slower . Even insertion of a selenocysteine at position 147 (eGFP:147U) showed a very weak and slow response , and in all three cases no reversibility was obtained. Only eGFP mutant 147U-204C showed a fast reaction on blue-light illumination , which was fully reversible  and hence comes close to the performance of roNaV2  by the membrane voltage allowing for complex experimental settings in which membrane-delimited occurrence of reactive species has to be monitored. Complementing other methods of RS determination in living cellsV2 adds a non-photonic variant with strict membrane delimitation and demonstrates the power of recombinant selenoproteins as sensitive sensors for redox processes.In summary, roNa+ channel construct used in this study was based on rat NaV1.4  downstream sequence and inserted after the original stop codon of the sequence of rNaV1.4, as shown in V1.4 construct.The wild-type Na2 incubator at 37 °C. Cells were trypsinized, diluted with culture medium, and grown in 35-mm dishes. HEK 293 cells were transfected with the respective plasmids using the Roti®-Fect transfection reagent  following the instructions of the supplier. Grown cells were supplemented with sodium selenate  for 12 hours from the day of transfection. After 12 hours, transfected cells were maintained in fresh cell culture media without sodium selenate. Electrophysiological experiments were performed 1–5 days after plating. Cells not expressing a fluorescence protein were cotransfected with CD8 to identify transfected cells by means of anti-CD8-coated beads .HEK 293 cells  were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) mixed 1:1 with Ham’s F12 medium and supplemented with 10% fetal calf serum in a 5% COWhole-cell voltage-clamp experiments were performed as described previouslyp/4 method with a leak holding voltage of −120 mV. Currents were low-pass filtered at 5 kHz and sampled at a rate of 25 kHz. Most experiments were performed at constant temperature of 19–21 °C; 32 °C were chosen for the assessment of H2O2 effects on HEK cells.A patch-clamp amplifier EPC10 was operated by PatchMaster software . Holding potential was −120 mV. Leak and capacitive currents were corrected with a 2, 1 MgCl2, 10 HEPES (pH 7.4 with NaOH).The patch pipettes contained (mM): 35 NaCl, 105 CsF, 10 EGTA, 10 HEPES (pH 7.4 with CsOH). The bath solution contained (mM): 150 NaCl, 2 KCl, 1.5 CaClm = 3 activation gates and a single-channel conductance according to the Goldman-Hodgkin-Katz equation.Channel activation and inactivation was assessed by depolarizing pulses to −60 through 60 mV in steps of 10 mV at an interval of 2 s from a holding potential of −120 mV. The peak current-voltage relationships were fit according to a Hodgkin-Huxley formalism with m is the voltage of half-maximal gate activation and km the corresponding slope factor. Γ is the maximal conductance of all channels and Erev the reversal potential.VSteady-state inactivation was measured from a holding potential of −120 mV with conditioning pulses of 500 ms at voltages ranging from −120 to −45 mV in steps of 5 mV. Subsequently, peak current was determined at −20 mV and normalized to a control peak current measured before conditioning. The repetition interval was 10 s. The normalized peak current plotted versus the conditioning voltage was described with a two-component Boltzmann function:h, and the corresponding slope factors, kh, which indicate the voltage dependence of inactivation.with the half-maximal inactivation voltages, V5) or 10 ms (I10) after the depolarization onset and the peak current (Ip) elicited by pulses to −20 mV (RI). 5 ms was chosen for cases were a direct comparison with wild-type channels and roNaV1 was intended because the inactivation of those channels is faster that that of roNaV2. The remaining non-inactivating remaining current fraction, RI(t), was plotted as a function of time (t) and fit with the following function:The time course of oxidation-induced loss of inactivation was monitored by measuring the ratio of current at 5 ms . Groups of data were compared with a two-sided Student’s t-test or ANOVA followed by a post hoc Bonferroni correction when appropriate.Data were analyzed with FitMaster (HEKA Elektronik) and IgorPro . If not stated otherwise, averaged data are presented as mean ± s.e.m. (2O2) were diluted in the respective bath solution immediately before application. The oxidant was applied extracellularly with a fine-tipped application pipette. In some experiments the reducing agent TCEP (tris(2-carboxyethyl) phosphine hydrochloride) was added to the internal patch pipette solution at 1 mM concentration with readjustment of the pH. BAM15 was from TimTec .Chemicals. Chloramine-T (ChT) and hydrogen peroxide . Excitation light of 400 and 470 nm (width about 15 nm) from a PolyChrome-V light source (TILL Photonics) was applied for 10 ms each. Fluorescence ratio (F400/F470) was formed based on the mean fluorescence values of the trailing 5-ms intervals. Filter set: 492/SP, FT 495, HC 520/35 .RS were generated by epifluorescence excitation light. This was achieved with light from the monochromator set to 470 nm, using a 63x/1.4 oil objective and the GFP filter set as mentioned above.How to cite this article: Ojha, N. K. et al. Non-photonic sensing of membrane-delimited reactive species with a Na+ channel protein containing selenocysteine. Sci. Rep.7, 46003; doi: 10.1038/srep46003 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


For some time, estrogen has been suspected to play a negative role in anterior cruciate ligament (ACL) fibroblast biosynthesis; however, reports on this issue have been controversial. In a recent study, our group demonstrated a negative combined effect of estrogen and mechanical loading on the gene expression of major extracellular matrix component molecules in ACL fibroblasts.


Fiducial markers are used in correlated light and electron microscopy (CLEM) to enable accurate overlaying of fluorescence and electron microscopy images. Currently used fiducial markers, e.g. dye‐labelled nanoparticles and quantum dots, suffer from irreversible quenching of the luminescence after electron beam exposure. This limits their use in CLEM, since samples have to be studied with light microscopy before the sample can be studied with electron microscopy. Robust fiducial markers, i.e. luminescent labels that can  withstand electron bombardment, are interesting because of the recent development of integrated CLEM microscopes. In addition, nonintegrated CLEM setups may benefit from such fiducial markers. Such markers would allow switching back from EM to LM and are not available yet.2O3:Eu3+ particles. Robustness is studied by measuring the luminescence of (single) NPs after various cycles of electron beam exposure. The gold‐core rhodamine B‐labelled silica NPs and QDs are quenched after a single exposure to 60 ke− nm–2 with an energy of 120 keV, while Y2O3:Eu3+ NPs are robust and still show luminescence after five doses of 60 ke− nm–2. In addition, the luminescence intensity of Y2O3:Eu3+ NPs is investigated as function of electron dose for various electron fluxes. The luminescence intensity initially drops to a constant value well above the single particle detection limit. The intensity loss does not depend on the electron flux, but on the total electron dose. The results indicate that Y2O3:Eu3+ NPs are promising as robust fiducial marker in CLEM.Here, we investigate the robustness of various luminescent nanoparticles (NPs) that have good contrast in electron microscopy; 130 nm gold‐core rhodamine B‐labelled silica particles, 15 nm CdSe/CdS/ZnS core–shell–shell quantum dots (QDs) and 230 nm YLuminescent particles are used as fiducial markers in correlative light and electron microscopy (CLEM) to enable accurate overlaying of fluorescence and electron microscopy images. The currently used fiducial markers, e.g. dyes and quantum dots, loose their luminescence after exposure to the electron beam of the electron microscope. This limits their use in CLEM, since samples have to be studied with light microscopy before the sample can be studied with electron microscopy. Robust fiducial markers, i.e. luminescent labels that can withstand electron exposure, are interesting because of recent developments in integrated CLEM microscopes. Also nonintegrated CLEM setups may benefit from such fiducial markers. Such markers would allow for switching back to fluorescence imaging after the recording of electron microscopy imaging and are not available yet.− nm–2 with an energy of 120 keV, while lanthanide‐doped inorganic NPs are robust and still show luminescence after five doses of 60 ke− nm–2. In addition, the luminescence intensity of lanthanide‐doped inorganic NPs is investigated as function of electron dose for various electron fluxes. The luminescence intensity initially drops to a constant value well above the single particle detection limit. The intensity loss does not depend on the electron flux, but on the total electron dose. The results indicate that lanthanide‐doped NPs are promising as robust fiducial marker in CLEM.Here, we investigate the robustness of various luminescent nanoparticles (NPs) that have good contrast in electron microscopy; dye‐labelled silica particles, quantum dots and lanthanide‐doped inorganic particles. Robustness is studied by measuring the luminescence of (single) NPs after various cycles of electron beam exposure. The dye‐labelled silica NPs and QDs are quenched after a single exposure to 60 ke The use of integrated CLEM is relatively new and is mainly used to investigate biological samples  and integrated CLEM systems that allow CL detection . Up to now, the robustness of fiducial markers for combined light and electron microscopy has not been studied in detail.Robust fiducial markers, i.e. luminescent labels that can withstand bombardment with electrons, are interesting, especially in view of recent developments in integrated CLEM systems (Zonnevylle 3+) ions incorporated into an inorganic host are promising as robust fiducial marker. Inorganic NPs have a high stability and the luminescence of lanthanide ions is insensitive to local structural changes as the luminescence involves optical transitions within the 4fn shell. The 4f orbitals are shielded by filled outer 5s and 5p orbitals. As a result, the influence of the environment on the intraconfigurational 4fn transitions is small, which gives Ln3+ ions several advantages as luminescent marker. First, the chemical and temperature stability of Ln3+ luminescence is high  lifetime. As a result, the lanthanide luminescence can be discriminated from autofluorescence by performing time‐gated detection  by measuring the luminescence intensity of (single) NPs after exposure to various electron doses. The luminescence of 130 nm gold‐core rhodamine B‐labelled silica NPs and 15 nm CdSe/CdS/ZnS core–shell–shell QDs is quenched after exposure to a single electron dose of 60 ke− nm–2 with energy of 120 keV. In contrast, the Y2O3:Eu3+ NPs are robust and show luminescence after five electron doses of 60 ke− nm–2. To further investigate the stability after electron beam exposure, the luminescence intensity of Y2O3:Eu3+ NPs is investigated as function of electron dose at various electron fluxes. The luminescence intensity initially drops, but rapidly reaches a constant intensity of ∼20% of the initial intensity. The luminescence intensity loss is not dependent on the electron flux, but on the total electron dose. The 200 nm diameter Y2O3:Eu3+ NPs retain their bright luminescence after electron doses as high as 66 Me− nm–2 at a level that allows single NP detection. The results indicate that Y2O3:Eu3+ NPs are suitable as robust fiducial marker, down to the single particle level.In this report, the robustness of lanthanide‐doped inorganic NPs, 230 nm Yet al., et al.  were synthesised according to the Turkevich method  with a diameter of 3 nm were prepared using a method described by Montanarella , et al.  and Mont  was diluted with water. Next, the dispersion was sonicated for 10 min and 25 μL of the dispersion was placed on a Formvar coated copper TEM grid. After evaporation of the solvent, the grid was washed with water. The as‐synthesised QD dispersion was diluted 10 000 times with cyclohexane to a concentration of 0.01 nM and dropcasted on a Formvar coated copper TEM‐grid. The Yet al. (–2 on sample) and an Omicron 100 mW 532 nm diode laser (25 W cm–2 on sample) as excitation sources. The laser light was guided to the sample through a Chroma ZT532rdc single dichroic and a Nikon CF IC EPI Plan ELWD 0.55 NA, 50x air objective. Various bandpass filters were used to select the emission from the samples: a Chroma ET585/65m filter for the gold‐core rhodamine B‐labelled silica NPs; a ET645/75m filter for CdSe/CdS/ZnS core–shell–shell quantum dots; and a Semrock FF01‐610/5m filter for the Y2O3:Eu3+ NPs. The three‐part number in the filter code represents the wavelength in the middle of the band and the last number indicates roughly the band width. The emission was detected with a PCO sCMOS camera PCO 4.2 edge. For typical measurement shown in the figures, 20 frames with a frame time of 3 s were accumulated. The data was analysed using the ThunderStorm Fiji plugin , i.e. 133 ± 6 nm gold‐core rhodamine B‐labelled silica NPs, 15 ± 2 nm CdSe/CdS/ZnS core–shell–shell quantum dots (QDs) and 228 ± 23 nm Yλem = 580 nm) of the gold‐core rhodamine B‐labelled silica NPs shows a band centred at 555 nm corresponding to the singlet–singlet (S0–S1) transition, see Figure λexc = 532 nm) shows a broad band originating from the reverse transition, see Figure et al., The excitation spectrum (glet S0–S transitiglet S0–S transitiλexc = 405 nm) is shown in Figure et al.  of Y2O3:Eu3+ NPs is shown in Figure 6 levels are observed. The excitation lines are assigned to 7F0,1 → 5H3, 5H6 (300–330 nm), 7F0,1 → 5L6, 5D3 (360–420 nm), 7F0,1 → 5D2 (460–480 nm) and 7F0,1 → 5D1 (520–540 nm) transitions. The emission spectrum (λexc = 260 nm) shows several sharp emission peaks between 580 and 630 nm, see Figure 6 transitions, are assigned to 5D0 → 7F0 (580 nm), 5D0 → 7F1 (585–600 nm) and 5D0 → 7F2 (605–630 nm) transitions. The spectral positions of the luminescence lines of Y2O3:Eu3+ NPs agree well with values reported previously in literature  NPs, the NPs were excited at 532 nm and the emission was collected from 607.5 to 612.5 nm using a narrow band filter. A typical luminescence image of the NPs is shown in Figure 5D0 → 7F2 emission of Eu3+ ions incorporated in the Y2O3 NPs. Next, the sample was exposed to an electron dose of 60 ke− nm–2 and the luminescence image of the NPs was recorded again. Figure 2O3:Eu3+ NPs survives direct electron beam exposure. To further investigate the robustness of the Y2O3:Eu3+ NPs, the NPs were exposed to four additional electron doses of 60 ke− nm–2. In between the electron exposures luminescence images were recorded. Figures 2O3:Eu3+ NPs is still observed after an electron dose of 300 ke− nm–2. The intensity of the NPs after the various electron doses was calculated using the ThunderSTORM plugin in ImageJ software. This software is typically used to determine the position of single luminescent molecules, but it also calculates the intensities and background levels. The results are shown by the blue dots in Figure 2O3:Eu3+ NPs show good robustness compared to the gold‐core rhodamine B‐labelled silica NPs and QDs. This can be in part related to the size of the Y2O3:Eu3+ NPs, 228 ± 23 nm, which is significant larger than the 133 ± 6 nm gold‐core rhodamine B‐labelled silica NPs and the 15 ± 2 nm CdSe/CdS/ZnS quantum dots. As a result, the electron beam has to travel a longer distance through the Y2O3:Eu3+ NP to fully penetrate the NP. Monte Carlo simulations (Casino v3.3) were performed to estimate the penetration depths of 120 keV electrons. Simulations obtained considering a 230 nm Y2O3 sphere with density 5.01 g cm–3 and threshold displacement energy of 50 eV . In addition, the interaction volume and penetration depth of the electron beam is dependent on the material properties. For example, the interaction volume increases for materials with a lower density  than atoms inside the NP , was investigated by recording the luminescence of (single) NPs before and after various cycles of electron bombardments. The luminescence of the rhodamine B dye and QDs was completely quenched after a single exposure to 60 ke− nm–2 with energy of 120 keV, while the Y2O3:Eu3+ NPs are robust, i.e. luminescence is observed after exposure to 120 keV electrons, even after an electron dose of 66 Me− nm–2. The intensity of Y2O3:Eu3+ NPs initially decreases, but rapidly reaches a constant value. Approximately 20% of the original intensity is maintained. The intensity loss is independent on the electron flux and determined by the total electron dose. The cause of the drop in intensity is probably the creation of defects in the surface layers of the NP or the interaction with carbon deposits accumulating on the NPs during electron beam exposure.The robustness of various types of nanoparticles (NPs), namely gold‐core rhodamine B‐labelled silica, CdSe/CdS/ZnS core–shell–shell QDs and Y2O3:Eu3+ makes the NPs promising as fiducial marker in correlative light and electron microscopy. In addition, the sharp and characteristic line emission can be used to create a variety of luminescent labels by changing and combining the type of dopant ion. The various labels can be detected down to the single particle level by recording the emission spectra of these labels.The robust character of the Y


Background: Non-binary and genderqueer (NBGQ) people are those who do not identify within the gender binary system , not falling exclusively in man/male or woman/female normative categories. A higher proportion of NBGQ people is usually found within young persons. This population is marginalized and, as such, is at risk of stigmatization and of developing negative health outcomes. As literature on the health of NBGQ people is sparse, this study aims at systematically review the limited studies on this field.Methods: The research questions which guided the systematic review were: (1) What are the differences in the health levels between NBGQ and binary transgender (BT) individuals? (2) What are the differences in the health levels between NBGQ and cisgender individuals? (3) Which medical and psychological interventions are most suitable for improving NBGQ health? According to PRISMA guidelines, a systematic search was conducted in PubMed, PsycInfo, Web of Science, and Google Scholar.Results: Eleven studies met the inclusion criteria for the current systematic review. Among them, 9 were focused on the health differences between NBGQ and BT individuals, 4 of the latter and 1 individually were focused on the health differences between NBGQ and cisgender individuals, and 1 was focused on the evaluation of health outcomes related to medical procedures. No studies assessed psychological interventions aimed at improving health in NBGQ individuals. All studies were cross-sectional, did not generally recruit a large sample of NBGQ individuals, and used non-probability sample design. Results related to the difference in health between NBGQ and BT were mixed; indeed, some found a better health status while others a worse one. Results related to the differences in health between NBGQ and cisgender highlighted higher health needs in NBGQ than in BT individuals. The only study analyzing the effects of medical interventions on health found that NBGQ female-assigned at birth individuals improved their quality of life after chest surgery.Conclusions: Although scholars are starting to pay attention to the NBGQ health, research needs to be expanded both in terms of methodology and research contents. Clinical, health-related social policies, and research recommendations in this field are reported. Transgender is an umbrella term referring to individuals who have a gender that differs from that normatively expected of their assigned sex [American Psychological Association (APA), non-binary and genderqueer (NBGQ) refers to individuals who have a gender identity that does not fall exclusively in man/male or woman/female normative categories. NBGQ individuals identify themselves with a neither exclusively masculine nor feminine gender, and their gender identity is situated beyond the gender binary, fluctuates between genders, or rejects the gender binary  of 14.320 transgender respondents identified themselves as NBGQ  What are the differences in the health levels between NBGQ and BT individuals? (2) What are the differences in the health levels between NBGQ and cisgender individuals? (3) Which medical and psychological interventions are most suitable for improving health in NBGQ population?A systematic review was performed to identify published papers on the health of NBGQ individuals using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines , qualitative studies, mixed-method studies, and longitudinal studies were considered eligible. Reviews, meta-analyses, letters to the editor, books or book chapters, commentaries, and abstracts were excluded.Two reviewers (CS and FM) extracted the relevant data and assessed titles and abstracts identified in the literature search. They also excluded duplicates from the dataset. Disagreements between the two reviewers were solved through the involvement of two additional reviewers (PV and RV). All studies which matched the inclusion criteria were reviewed by the authors (CS and FM) and any disagreement was settled through a discussion involving two other reviewers (PV and RV).n = 1); 2) no research designs were performed (n = 4); (3) the article did not specifically address health outcomes (n = 11); (4) the journal was not peer-reviewed (n = 1), and (5) the identity of participants was not clearly defined  (n = 2).The initial search identified 218 records, as shown in the PRISMA flow diagram . Only 2 The 11 papers included in the current systematic review are listed in N = 7), while 2 were conducted in UK, 1 in Spain, and 1 in Canada. Among the studies selected, 9 (81.81%) were focused on the health differences between NBGQ and BT individuals (first research domain), 4 of the latter and 1 individually (45.45%) were focused on the health differences between NBGQ and cisgender individuals (second research domain), while only 1 (9.09%) was focused on the evaluation of health outcomes related to medical procedures . No studies assessed psychological interventions aimed at improving health in NBGQ individuals.Overall, studies were published between 2016 and 2019 and all of them used a cross-sectional design with quantitative methods, except for one study which supported quantitative data with focus groups. Considering the number of NBGQ individuals within the general sample recruited, the NBGQ subsample size was not generally large, as it ranged from 28 to 380 participants. However, considering the cross-sectional nature of the studies, some of them seem to have acceptable subsample size  were NBGQ, all FAAB. Among them, 56% (N = 33) completed the post-operative questionnaire. NBGQ participants agreed that surgery improved their quality of life, comfort with exercise, sex life, and comfort in physical appearance with and without clothes. Thus, this study shows the benefits that also NBGQ individuals might experience from undergoing medical procedures. As reported before, no studies addressing specific psychological interventions  for the improvement of health in NBGQ individuals were found.Only one study addressing health outcomes related to medical interventions for NBGQ individuals was found. Among 458 patients undergoing gender-affirming chest surgery, Esmonde et al.  found thThe aim of this study was to systematically review the literature related to the health of NBGQ individuals, exploring in particular 3 domains: (1) difference in health between NBGQ and BT individuals; (2) difference in health between NBGQ and cisgender individuals; and (3) medical and psychological interventions addressed to the improvement of NBGQ health. A total of 11 studies met the inclusion criteria and were used for this systematic review.As regards the characteristics of the selected studies, papers were recently published (from 2016), prevalently used a cross-sectional design with quantitative methods, did not generally recruited large NBGQ subsample . This is particularly noteworthy as it seems that NBGQ individuals analyzed in the studies selected, except for the NBGQ subsample recruited by Jones et al.  whose meAs regards the differences in health between NBGQ and BT individuals , studies selected reported mixed findings, some finding a better health status  are quite clearer than the previous ones. Indeed, although Warren et al.  did not Finally, the only study analyzing the effects of medical interventions on health of NBGQ individuals  found that NBGQ FAAB individuals, contrary to the belief that they would not need to medically affirm their gender, improved their quality of life and health after chest surgery , From a health-related social policy perspective, our findings shed light on the urgency of implementing policies to address health needs of NBGQ people, overcoming discriminatory practices and promoting health equity. First, NBGQ identities should be recognized by healthcare service systems and providers as existing and healthy identities. To this end, healthcare providers could benefit from specialized training aimed at improving the knowledge on NBGQ identities, as well as the related specific health needs  NBGQ identity development and specific challenges, matching theoretical perspectives on gender diverse population  with theoretical frameworks of developmental psychology ; (b) causal relationships between stigmatizing processes , protective factors  and health outcomes across different life stages;To expand studies to geographical contexts different from USA, UK, Canada, and Spain, as well as to deepen the role of socio-economic status in health disparities in order to build culturally competent studies on NBGQ population;To make every effort to recruit probability samples to allow generalization of the results to the specific population from which the sample was recruited. Although recruiting probability sample in LGBT community is a challenging task ;To build randomized controlled trial experiments to assess the efficacy of specific psychological interventions  in improving health in NBGQ people.This systematic review shows that the scientific interest on NBGQ health is growing but, at the same time, needs to be expanded both in terms of methodology and research contents. Indeed, it was hard to obtain a clear picture of the NBGQ population in terms of health, as all selected studies used a cross-sectional design and reported data on non-probabilistic samples. Notwithstanding these limitations, studies provided valuable information on the health of NBGQ people, starting to run an innovative research field which unhooks transgender population from a gender binary system that is often reproduced in scientific research. Thus, this review may represent one of the references for future studies, which could hopefully follow the research recommendations to increase the knowledge on NBGQ health building an increasingly relevant research from both the cultural and methodological point of view, as well as informing affirmative clinical practice and social policies to reduce the health equity gap.CS, FM, and RV conceived and designed the study and drafted the manuscript. CS and FM managed the literature searches and analyses. NM, MB, and VB refined the literature search. PV and RV solved disagreements between two main reviewers (CS and FM). All authors revised the article critically and read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The “F-words in Childhood Disability”  are an adaptation and an attempt to operationalize the World Health Organization’s (2001) International Classification of Functioning, Disability and Health (ICF) framework. Since the paper was published (November 2011), the “F-words” have attracted global attention . Internationally, people have adopted the “F-words” ideas, and many families and service providers have expressed a need for more information, tools, and resources on the “F-words”.This paper reports on the development and pilot evaluation of a Web-based knowledge translation (KT) resource, the “F-words” Knowledge Hub that was created to inform people about the “F-words” and to provide action-oriented tools to support the use of the “F-words” in practice.CanChild Centre for Childhood Disability Research collaborated to develop, implement, and evaluate the Knowledge Hub. A pilot study design was chosen to assess the usability and utility of the Web-based hub before implementing a larger evaluation study. Data were collected using a brief anonymous Web-based survey that included both closed-ended and open-ended questions, with the closed-ended responses being based on a five-point Likert-type scale. We used descriptive statistics and a summary of key themes to report findings.An integrated research team of families and researchers at From August to November 2017, the Knowledge Hub received >6,800 unique visitors. In 1 month (November 2017), 87 people completed the survey, of whom 63 completed the full survey and 24 completed 1 or 2 sections. The respondents included 42 clinicians and 30 family members or individuals with a disability. The majority of people visited the Knowledge Hub 1-5 times (n=63) and spent up to 45 minutes exploring (n=61) before providing feedback. Overall, 66 people provided information on the perceived usefulness of the Knowledge Hub, of which 92% (61/66) found the Knowledge Hub user-friendly and stated that they enjoyed exploring the hub, and a majority (n=52) reported that the Knowledge Hub would influence what they did when working with others. From the open-ended responses (n=48), the “F-words” videos (n=21) and the “F-words” tools (n=15) were rated as the best features on the Knowledge Hub.The “F-words” Knowledge Hub is an evidence-informed Web-based KT resource that was useful for respondents, most of whom were seen as “early adopters” of the “F-words” concepts. Based on the findings, minor changes are to be made to improve the Knowledge Hub before completing a larger evaluation study on the impact at the family, clinician, and organizational levels with a wider group of users. Our hope is that the “F-words” Knowledge Hub will become a go-to resource for knowledge sharing and exchange for families and service providers. The “F-elopment . The inielopment . Since iCanChild Centre for Childhood Disability Research focused on disseminating the “F-words” into practice. At that time, several parents  had learned about the “F-words,” liked the ideas, and were interested in how to share the “F-words” message with more families. Recognizing the potential impact of an integrated approach to this work , we partnered with family stakeholders to develop and evaluate knowledge translation (KT) strategies tailored to meet the families’ needs and preferences.In 2014, based on considerable interest in the paper, we formed an integrated research team at The first project involved the development, dissemination, and evaluation of a three-minute awareness video . A videoCanChild website was identified by 65.7% of respondents (90/137) as the most popular strategy for sharing further information on the “F-words” concepts. A complete report of our findings and the lessons learned from this project are published [We evaluated the video by tracking its reach and asking viewers to complete an anonymous Web-based survey. In the first 2 months, there were 715 views and 137 survey responses. Overall, we learned that 97.8% (134/137) of people “extremely liked” or “liked” the “F-words” ideas, 87.5% (120/137) indicated they would share the video, and 92.7% (127/137) wanted to learn more. The The awareness video was only the first step toward moving the “F-words” into practice. By January 2015, we had given >30 international presentations and the “F-words” ideas had continued to spread over social media. We were gratified by the uptake of these ideas around the world and were excited to see the imaginative ways in which people were adapting and adopting the “F-words” to local contexts. We were also learning a great deal about the application of the “F-words” by connecting and working with families and other stakeholders such as service providers and health care administrators around the world. Therefore, as a research team, we were acting as “knowledge brokers”  by workiFrom our conversations with the families and service providers, it was evident that there was significant interest in having more information on the “F-words” as well as action-oriented resources and tools to assist with the application of the “F-words” into practice. Furthermore, as the “F-words” ideas continued to spread, we recognized the need (and opportunity) to compile and share all that was being done on the “F-words” ideas by building a centralized Web-based community for knowledge sharing and exchange. Therefore, in 2015, our research team decided to develop, implement, and evaluate the usability and utility of a Web-based KT resource: a website called “The ‘F-words’ in Childhood Disability Knowledge Hub.”CanChild’s website [CanChild website is world-renowned in the field of childhood disability and receives over 12,000 unique visitors each month from over 205 countries [The purpose of the “F-words” Knowledge Hub was to inform families and service providers about “F-words”/ICF concepts and to provide action-oriented tools to support the uptake and use of the “F-words” in practice. The Knowledge Hub is currently hosted on  website  and is mountries .In the last several years, there has been increasing interest in using the internet as a platform for KT and the use of Web-based KT resources as a strategy for disseminating health research evidence in the field of childhood disability -10. LevaWhile the current evidence base for Web-based KT strategies is limited, some studies have shown promising findings ,13; howeaction cycle: “select, tailor, and implement the intervention,” “monitor knowledge use,” and “evaluate outcomes.” This study was part of a larger research program that had already addressed the earlier stages of the action cycle [This paper reports on the development process, usability, and utility of the Knowledge Hub. The Knowledge-to-Action (KTA) framework was used as the guiding theoretical underpinning for this research . KT theoon cycle .We implemented a formal integrated knowledge translation (iKT) strategy to develop, implement, and evaluate the Knowledge Hub. iKT involves the collaboration of researchers and knowledge users  throughout all stages of the research or KT process  and has All team members were involved in each stage of the project: (1) participating in initial planning stages; (2) providing feedback on the content and design of the Knowledge Hub; (3) creating and sharing tools/resources; (4) assisting with evaluation; and (5) disseminating the hub across their social networks. During the initial planning stages, team meetings were held by teleconference. We initially planned to develop an “F-words” Tool Kit as a paper-based resource designed to share knowledge and provide tools/resources to support the use of the “F-words” in practice; however, after extensive conversations with stakeholders and a review of the literature, we turned toward Web-based KT strategies . AC led the development of the hub, but feedback was sought and received from all team members throughout the development process. Most team correspondence was done through email.CanChild students we developed draft tool templates that could be distributed to all team members for feedback. When all team members had approved the tools, they were then posted on the Knowledge Hub.An area in which all three family stakeholders were heavily involved was the creation of the “F-words” tools; the “F-words” agreements, photo collage, goal sheet, and profile. Many of the ideas for the tools came from family stakeholders’ personal experiences of working with service providers and their perceptions of how the “F-words” could be used in practice. As an integrated research team, we discussed the purpose and goals for each tool, and then with the support of To help with the planning and development of the Knowledge Hub, we used Levac et al’s  best praThe purpose of the Knowledge Hub is to have a single site where people can go to learn about and share ideas for utilizing the “F-words” concepts in practice. The Web-based hub  includesCanChild’s social networks. The recruitment poster is provided in A pilot study design was used to assess the usability and utility of the Knowledge Hub, and to make any necessary changes, before implementing a larger evaluation study. Usability was measured with “usefulness” questions  and utility was measured using “use” questions . Usability and utility testing is a critical component to the success of KT interventions ,23. VisiThe survey included both closed-ended and open-ended questions. The closed-ended responses had a five-point Likert-type scale that evaluated the visitors’ prior familiarity with the “F-words”, the perceived usefulness, and reported or intended use of the Knowledge Hub. Adaptive questioning was used  to reduce the complexity of the survey. There were 37 questions in the survey. Google analytics evaluated the reach by tracking the number of visitors to the hub over a four-month period. Descriptive statistics were used to analyze the quantitative information, and descriptive content analysis was used to identify and synthesize the key themes from the open-ended questions. Ethics approval was obtained from Hamilton Integrated Research Ethics Board (Project# 2017-0977).CanChild newsletters featuring the Knowledge Hub) and spread of the Knowledge Hub by people who liked and were sharing it within their communication channels and social networks.Over the four-month evaluation period (August-November 2017), there were over 6,800 unique visitors to the Knowledge Hub, with the number of visitors increasing each month . This coThe survey went live on November 3, 2017, and data were collected for 1 month. A total of 87 respondents provided information, with 63 completing the full survey and 24 partially completing the survey , providing a completion rate of 72%. Most people visited the Knowledge Hub 1-5 times (n=63) and spent up to 45 minutes exploring the hub (n=61) prior to providing feedback. The following results were based on the survey data.Just under half the respondents that completed the survey lived in Canada . The only other country with >10 respondents was the United States . The remainder of respondents came from 13 countries. Respondents were asked to state the perspective from which they were viewing the Knowledge Hub . Of the 87 people who completed the survey, 42 were clinicians and 30 were family members (n=20) or individuals with a disability (n=10). There was a wide distribution of perspectives with many respondents (n=36) falling into >1 stakeholder category .The majority of people  had heard of the “F-words” prior to visiting the Knowledge Hub and either “extremely liked the ideas”  or “liked the ideas” . Of the 62 people who were familiar with the “F-words”, 43 (69%) felt confident identifying and explaining the “F-words” ideas, 37 (60%) had shared them with others, and 35 (56%) indicated that they had used or applied them in practice prior to exploring the hub. To understand how people were using or applying the “F-words,” we asked for open-ended feedback. The majority of people who provided written responses were clinicians, researchers, people with disabilities, or family members. Depending on the stakeholder group, the use of the “F-words” concepts varied. Examples of how the “F-words” concepts have been used by each stakeholder group are shown in To evaluate the usefulness of the Knowledge Hub, respondents were asked to rate their overall satisfaction. Of the 87 people who started the survey, 66 people completed this section. Therefore, the following data are based on these 66 responses. Overall, 86% (57/66) of respondents felt the purpose was clear, 92% (61/66) found the Knowledge Hub user-friendly, and 92% (61/66) and 94% (62/66) perceived the content to be meaningful and relevant for families and service providers, respectively. The average scores ranged from 4.23 to 4.39 out of 5 for each category .Respondents were also asked to indicate which sections of the Knowledge Hub they liked and what could be improved. A total of 65 people answered this question, all of whom indicated they liked at least one section of the Knowledge Hub, 57% (37/65) indicated that they liked all sections and 45% (29/65) indicated they had no further suggestions for improvements. The survey also collected open-ended feedback to gain a better understanding of what were perceived to be the best features of the Knowledge Hub (48 respondents) and which areas needed improvement (25 respondents). The best features and areas for improvement were categorized into two aspects: content and format or design of the Knowledge Hub. The key themes within these areas were then identified based on the number of responses. The final section of the survey explored the use or intended use of the Knowledge Hub and the “F-words” concepts. Among the people who started the survey, 72% (63/87) completed this final section. The following data are based on responses from these 63 people .Overall, 97% (61/63) people indicated that they either “extremely liked”  or “liked”  the “F-words” concepts, 92% (58/63) people reported that the hub increased their understanding of the “F-words”, and 78% (49/63) people reported that the hub influenced their thinking about the “F-words”. We were also interested in participants’ confidence in identifying and explaining the F-words after exploring the Knowledge Hub. Overall, 90% (57/63) people indicated that they were either “extremely confident”  or “confident” . When asked whether the Knowledge Hub would be useful to them, 83% (52/63) people reported that it would influence the way they did things when working with others.Lastly, respondents were asked to rate the Knowledge Hub as a KT tool for sharing information with families and service providers. Overall, 90% (57/63) people rated it 4 or 5  as a KT tool for families, 98% (60/63) people rated it as 4 or 5 as a KT tool for service providers, and 97% (58/63) people planned to share the Knowledge Hub.From the beginning, it was important to us that the Knowledge Hub be cocreated with stakeholders. While our integrated team of families and researchers led the development process, many stakeholders outside of our research team were involved. For example, we worked with clinicians and health care administrators, who we knew were applying the “F-words” to share examples of how they were using the “F-words” in their organizations. These examples then served as examples of application for other service providers.We believe early stakeholder involvement was crucial not only to the development of a meaningful and relevant resource but also to the dissemination of knowledge regarding the Knowledge Hub. Individuals interested in the Web-based hub were more likely to share it with their own communities, thus increasing its reach and potential impact as it was spread through broad communication channels and social networks . The impAnother key feature of the Knowledge Hub was its promotion of knowledge sharing and exchange . In compCanChild’s resources  to design and maintain the Knowledge Hub. Creating and collating content for the Knowledge Hub also took a lot more time than initially expected. The development process involved iterative rounds of feedback from various stakeholders. We did not follow a structured system or timeline for collecting feedback, which led to a longer process. In the future, we would recommend the use of a structured process tailored to collecting feedback from a diverse group of stakeholders [One common barrier reported in the literature was the time and resources needed to develop and implement KT interventions ,25,30. Weholders .A key facilitator for this project was the use of theory and best practice guidelines to inform the KT intervention ,31. The The main aim of the Knowledge Hub is to inform families and service providers about the “F-words”/ICF concepts and to provide action-oriented tools to support the uptake and use of the “F-words” in practice. As such, the goal of this pilot evaluation was to evaluate the usability and utility of the Knowledge Hub. The findings from this study revealed that these self-assigned goals were attained. Overall, the respondents reported that the Knowledge Hub was informative and useful and the “F-words” tools were one of the best features of the Knowledge Hub.perceived usefulness and potential use. While mixed-model analyses between groups were not completed, the high ratings given by all participants implied that the Knowledge Hub was perceived to be a meaningful resource for both service providers and families. This finding was consistent with earlier research from CanChild that found that when educational materials were clearly written and user-friendly, they were useful and impactful for multiple target audiences  [In general, the hub received high ratings with regard to both its oviders) ,33. Furtoviders) ,35.early adopters— those who are already using the “F-words”—to the late adopters— those to whom the “F-words” are new) [We recognize that prior to exploring the Knowledge Hub, over 70% of people who completed the survey had previously heard of the “F-words.” Of these respondents, the majority felt confident identifying and explaining the “F-words” ideas, and about half of them indicated that they had used or applied the “F-words” in practice. Despite many respondents already being familiar with the “F-words” concepts, the majority stated that the Knowledge Hub increased their understanding of the “F-words” ideas. This is an important finding as it implies that the Knowledge Hub can increase perceived knowledge even if individuals have prior familiarity with the concepts. This probably occurred because the resources provide tangible materials that move beyond simple concept familiarity. Unfortunately, due to the low response rate from people for whom the “F-words” concept was new, it is not possible to say whether the Knowledge Hub is useful across all adopter categories  .early adopters of the “F-words” concepts) and what was needed to broaden the applicability of the Knowledge Hub to a wider audience . These findings will both inform and complement future evaluations of the Knowledge Hub. Recognizing that experimental evaluations only identify whether an intervention is effective, process evaluations such as this are recommended to understand the reasons why interventions are (or are not) effective [Conducting a pilot evaluation of the usability and utility of the Knowledge Hub is an important step toward ensuring its overall impact and sustainability ,23. Thisffective ,37.Based on the respondents’ positive feedback, we anticipate that the Knowledge Hub will be a useful resource for both families and service providers. A limitation to this work is that feedback was gained from only a small sample of the people who visited the hub during this period. It is important to remember that the majority of people who provided feedback were those who were already familiar with the “F-words” concepts and also liked the “F-words” ideas. Thus, their potential biases must be recognized.In order to reach a broader audience, more time is needed to actively disseminate the Knowledge Hub. While the preliminary findings after a one-month evaluation were reported here, in order to overcome selection bias , the evaluation will remain posted on the Knowledge Hub and further feedback will be monitored. The hope is that over time more people  will complete the survey.The next step is to evaluate the impact of the Knowledge Hub and “F-words” concepts at the family, clinician, and organizational levels. As active implementation strategies are useful in supporting the dissemination and uptake of educational materials, we plan to combine the Knowledge Hub intervention with tailored invitational outreach visits to local children’s treatment centers (CTCs). Once again, this is a stakeholder-driven strategy as CTCs have contacted us and expressed a need for in-person educational training on the “F-words” concepts. Based on our positive experiences of working with families and service providers to develop the Knowledge Hub, this project will continue to be informed by an iKT strategy.early adopters  [early majority), further active implementation strategies are needed.Working with families and service providers, we designed a theory-informed and evidence-informed Web-based KT resource that was perceived to be relevant and meaningful to families raising children with disabilities and to service providers working in the field. To date, the Knowledge Hub has mainly reached rmation) ; therefoKT is not only the doing but also the studying of the KT process and outcomes. From the evaluation of the usability and utility of the Knowledge Hub, we now have a good understanding of what was done well and what can be improved. Based on the findings from this pilot study, we intend to make minor changes to the Knowledge Hub before conducting a larger evaluation study of the impact at the family, clinician, and organizational levels. Knowledge gained from this study is transferrable to other KT initiatives involving families and service providers. We hope that the findings and lessons learned from this integrated KT project will assist others in advancing iKT science and practice in other areas of childhood disability research.


P‐value of .05, the distribution of TIICs in 73 PCa tissues and 11 normal tissues was illustrated. Activated CD4+ T cells and M0 macrophages account for a high proportion in PCa tissues, while neutrophils and monocytes were found at a high density in normal tissues. Further results showed that the density of plasma cells, Treg cells and resting mast cells were associated with advanced PCa. Additionally, M2 macrophages affected the RFS of PCa patients, and AR was also involved. In the current study, we first evaluated the immune infiltration among PCa and revealed that M2 macrophages could predict the prognosis of PCa patients. Meanwhile, based on the LASSO regression analysis, we established a novel nomogram to assess the recurrence risk of PCa based on immune cell proportions and clinical features.Prostate cancer (PCa), a severe health burden for males, accounts for the second frequent cancer and fifth tumor specific death cancer around the world. Several studies on tumor‐infiltrating immune cells (TIICs) have shown inconsistent and controversial results to PCa. We downloaded a gene expression matrix and clinical information from TCGA, and CIBERSORT was used to identify the proportion of TIICs. Wilcoxon's Sign Rank Test evaluated different gene expression levels in PCa and normal tissues. Kaplan‐Meier curves were used to evaluate the associations of TIICs and recurrence‐free survival (RFS). Finally, based on the preset  This effect was accompanied with increased expression of PD‐L1 from tumor cells. Philippe et alTumor‐infiltrating immune cells (TIICs) are essential components of the tumor microenvironment and can alter the immune status of the tumor. Several studies have demonstrated therapeutic strategies against tumors by targeting TIICs.Due to the limitations of methods and techniques, prior studies focused on finite areas of the immune response, whereas the full view of the immune response is based on the infiltration of different TIICs. Alternatively, the immunohistochemistry (IHC)‐based labels of TIICs are not precise, and several TIICs may express the same makers on the membrane, leading to the inaccurate measurements of TIIC density in PCa tumor tissues. In the present study, the proportion of 22 TIICs in PCa tissues was illustrated by a newly developed deconvolution algorithm, CIBERSORT, which can predict the density of TIICs through the expression of 547 key genes, and revealed the potential effect of TIICs in PCa prognosis.22.1In the current study, TCGA database of PCa tissues and control tissues were enrolled. We downloaded the overall gene expression profiles and clinical information of patients from TCGA up to June 31, 2019. Finally, the gene expression profiles of 499 PCa samples and 52 controls were prepared for subsequent analysis. We performed the stable multi‐array average algorithm to preprocess the recorded raw data. The mRNA sequencing data of TCGA database were first converted to a microarray‐similar result using the voom method . Then, we annotated the gene ID, and the mRNA sequencing results were normalized with the “limma” package of R, to average the data of repeated genes and delete results that were not available. In Figure 2.2+ T cells, naïve CD4+ T cells, resting memory CD4+ T cells, activated memory CD4+ T cells, follicular helper T cells, regulatory T cells (Treg cells), gamma delta T cells, resting NK cells, activated NK cells, monocytes, M0 macrophages, M1 macrophages, M2 macrophages, resting dendritic cells, activated dendritic cells, resting mast cells, activated mast cells, eosinophils, and neutrophils, with the preset signature matrix at 1000 permutations. After using the CIBERSORT program, the distribution of 22 subtypes of TIICs was presented, along with the results of correlation coefficient, P‐value and root mean squared error (RMSE), which can evaluate the accuracy of the results in each sample. The P‐value ≤ .05 reflects a statistical connotation of the results of deconvolution across all cell subsets for each sample and is useful for excluding results with less accuracy. Finally, 73 PCa samples and 11 control samples were selected for later analysis because they met the required P‐value.The analytical tool called CIBERSORT was used in the study, which can quantify the percentage of different types of TIICs accurately, under the complex “gene signature matrix” based on 547 genes.2.3http://www.r-project.org). ROC and AUC were completed to assess the discriminatory ability of the nomogram, while decision curve analysis was also performed to evaluate the usability of the nomogram. STRING, an online tool that could reveal the functional protein association networks, was employed to identify androgen receptor associated proteins.P < .05 was considered statistically significant.For each type of TIICs, we assessed the different levels in patients and controls with Wilcoxon's Sign Rank Test and displayed the violin plot. Using the Pearson correlation coefficient, correlations between two immune cell subsets were evaluated. The different distribution of TIICs in several subgroups of clinical features were evaluated with Wilcoxon's Sign Rank Test in parameters divided into two groups and with the Kolmogorov‐Smirnov test in parameters separated into more than two groups. Kaplan‐Meier (K‐M) curves were constructed to analyze and evaluate the associations of immune cell infiltrate and corresponding recurrence‐free survival (RFS) using the log‐rank test. The median of the proportion of each cell type was computed to divide the patients into high‐ and low‐density groups. The nomogram was constructed using the rms package in R (33.1P < .05). The flowchart of study design and the process of samples enrolled is shown in Figure + memory T cells were highly present in normal tissues (P = .029), as well as monocytes (P < .001), resting dendritic cells (P = .013), resting mast cells (P = .001) and neutrophils (P < .001)  and M0 macrophages (P = .004) was higher in PCa tissues compared to normal tissues , and neutrophils were linked with monocytes (R = 0.44). In contrast, the existence of resting CD4+ memory T cells was negatively associated with follicular helper T cells (R = −0.44), activated NK cells, activated CD4+ memory T cells (R = −0.38), resting CD4+ memory T cells and M0 macrophages (R = −0.38). Figure To determine the underlying relationships of different immune types, we evaluated the correlation between TIICs among PCa tissues. Figure s R = −0.8, restins R = −0.8, restin3.2P = .076), M2 macrophage in Gleason 9 + 10 group (P = .008) , T3 + T4 stage (P = .36). Treg cells were high in tumor tissues among severe stages, including the 9 + 10 Gleason score group (P = .009), T3 + T4 stage (P = .044), while resting mast cells showed a lower density among the 9 + 10 Gleason score group (P = .004), T3 + T4 stage (P = .027)  Figure A. Additi) Figure .P = .025) , while a lower proportion indicated a more prolonged RFS  Figure C. There 3.3To establish a convenient method to predict the recurrence risk of PCa, we designed a nomogram based on clinical information and density of TIICs. First, the subtypes of TIICs were selected by LASSO regression analysis, including M2 macrophages, follicular helper T cells and resting dendritic cells Figure . In addi3.4P < .001), after adjusting for tumor purity, and similar results were illustrated for NCOA2  and NCOA4 . In contrast, the expression of FOXA1 , KDM1A  and KLK3  were negatively associated with the distribution of macrophages in PCa tissues.AR plays pivotal roles in the biochemical recurrence and drug resistance of PCa. As a high proportion of M2 macrophage infiltration is positively associated with the shorter RFS, we evaluated the relationship of AR and M2 macrophages infiltration, as well as AR‐related genes. The five main AR‐related proteins were identified with STRING, an online website that illustrates the correlation between proteins. All of the information was based on curated databases, experimentally determined or text mining. Finally, we recorded KLK3, KDM1A, NCOA2, NCOA4, and FOXA1 Figure . FurtherP < .001), VSIG4 , MS4A4A , MRC1 , CCL24 , and CD209   Figure . These r4+ cells, and naïve CD4+ T cells, while Treg cells and NK cells increased. Regarding macrophages, Shimura et alImmune infiltration plays a critical role in tumor progression. Immune evasion is now regarded as a cancer hotspot. One of the critical mechanisms is the programmed cell death protein (PD‐1) receptor on cytotoxic T lymphocytes (CTLs) and its ligand (PD‐L1) on cancer cells or host TIICs. Immunotherapies targeting PD1/PD‐L1, which can reverse immune tolerance and yielded remarkable clinical responses, have been widely studied in tumors.+ T cells did not appear in any patient or control. We found that activated CD4+ T cells and M0 macrophages are found at high levels in tumor tissues, while neutrophil and monocyte levels are lower, and these results are consistent with previous studies.In previous studies, most researchers detected the density of TIIC proportion based on fewer specific markers. However, several papers demonstrated the overlap of molecular markers for TIICs.+ T cells are positively associated with activated CD4+ memory T cells, while resting CD4+ memory T cells are negatively associated with follicular helper T cells. Connie et al+ memory T cells, are required to enhance the primary activated process of CD8+ T cells in immune responses, while Edith et al+ T cells are required for secondary expansion and memory of CD8+ T cells. Follicular helper T cells are distinguished from CD4+ T cells, which function in the formation of germinal centres and promote the proliferation and differentiation of B cells. Hale et al+ T cell populations that commit to follicular helper T cell lineages, and CD4+ memory T cells can recall their previous lineage of effectors provided by follicular T cells.In the second part of the study, we illustrated the TIICs in PCa tissues and their internal correlation. The features of patients are divided into two parts with unsupervised hierarchical clustering, meaning that the different proportions of TIICs in PCa are associated with the clinical results. We also determined that CD8+ Treg cells was associated with advanced PCa tumor stage. Erlandsson et alSubsequently, the analysis of TIICs and clinical features was performed, and we found that the proportion of plasma cells and mast cells was downregulated in severe PCa, compared to mild PCa, while the density of Treg cells was upregulated. Lohr et alAfter assessing the association between TIICs and RFS of PCa patients, M2 macrophages were singled out. Patients whose M2 macrophage density is higher had a shorter RFS time. Although the K‐M curve of M0 and M1 macrophages showed a similar trend, no statistical significance was found. Feng et alIn summary, using the deconvolution algorithm of CIBERSORT, we first obtained the distribution of 22 TIICs in PCa patients based on the expression of 547 core genes. The proportion of immune infiltration was significantly different between PCa and control tissues. Plasma cells, Treg cells, and mast cells are involved in the progression of PCa, while M2 macrophages could predict the prognosis of PCa patients. Meanwhile, based on the LASSO regression analysis, we established a novel nomogram to assess the recurrence risk of PCa based on immune cell proportions and clinical features, of which need to be verified by a large size cohort in the feature.All authors declared no competing interests.JLM, MZ, and CZL conceived and designed the study. MZ, YL, and SF performed the data extraction. JLM, MZ, and SYG analyzed the data. YL, SF, and ZYH contributed materials and analysis tools. JLM, JZ, JZ, and CZL wrote the manuscript. All authors have read and approved the final version of the manuscript and agreed with the order of presentation of the authors. Click here for additional data file.


Single-anastomosis duodenoileal bypass with sleeve gastrectomy (SADI-S) is a simplified biliopancreatic diversion. The objective of this study was to develop a reproducible animal model for SADI-S. We used three techniques for duodenal exclusion and duodenoileal anastomosis: (a) surgical clip and side-to-side anastomosis, (b) ligation and side-to-side anastomosis and (c) sectioning the duodenum, closing the duodenal stump and end-to-side anastomosis. We recorded the surgical technique and complications for each method. Twenty-five of 31 rats survived to the end of the study period. One death occurred from accidental anaesthesia overdose and the others from anastomosis leak. Four duodenal exclusions had repermeabilised at necropsy. Our murine model of SADI-S can be consistently reproduced. Sectioning the duodenum is preferable to avoid repermeabilisation of the duodenum. Single-anastomosis duodenoileal bypass with sleeve gastrectomy (SADI-S) was proposed by Sànchez-Pernaute A. et al.  as a modThirty-one adult male Winstar rats (12–16 weeks old) were included in this study. All animals were housed in individual cages under constant ambient temperature (23 °C) and humidity, and were maintained in a 12-h light/dark cycle room.The night before surgery, the rats were kept on raised wire platforms to avoid coprophagy and fasted for approximately 12 h; only water was allowed ad libitum. An isoflurane anaesthetic chamber was used for anaesthesia induction, which was changed to nose cone flow for maintenance of anaesthesia during the procedure. Enrofloxacin 2.5%, 25 mg/kg, was administered intramuscularly for antibacterial prophylaxis, and 20 mL of normal saline solution was given subcutaneously for hydration (10 mL before surgery and 10 mL postoperatively).linea alba, a midline incision was made in the abdominal wall using an electrical scalpel. The abdominal cavity was then exposed using a surgical retraction system .Following induction, the abdomen was shaved with electric hair clipper from sternum to groin, and the rat was placed on a heating pad in dorsal recumbency. The skin was disinfected using an aqueous iodophor solution, and after identifying the xiphoid process, a 5-cm midline abdominal incision was made using a #11 surgical scalpel. After identifying the We performed a complete gastrectomy of the greater curvature using surgical scissors. The gastrosplenic ligament was sectioned after ligation with 4.0 absorbable sutures . We continue with dissection of the duodenum Fig. . Next, wLigation of the duodenum 5 mm from the pylorus using 3.0 nonabsorbable suture  followed by side-to-side duodenoileal anastomosis using a continuous pattern with absorbable 6.0 suture .Ligation of the duodenum 5 mm from the pylorus using a nonabsorbable surgical clip  followed by side-to-side duodenoileal anastomosis using a continuous pattern with absorbable 6.0 suture .Sectioning the duodenum 5 mm from the pylorus and closure of the duodenal stump in a continuous pattern with nonabsorbable 6.0 suture  followed by end-to-side duodenoileal anastomosis in a continuous pattern with absorbable 6.0 suture . It is interesting to note that duodenal sectioning can be an alternative method of gastric tube calibration.Three methods were used for duodenal exclusion and duodenoileal anastomosis:Following the anastomosis Fig. , we retuImmediately postoperatively, each rat recovered under a heat lamp with continuous monitoring until regaining consciousness. After recovery, the animals were transferred to a cage in isolation, kept on a raised wire platform, and fasted for the first 24 h. Liquid diet  was provided for the next 3 days. Antibiotics (enrofloxacin) were administered for 72 h. Full solid diet was administered on day 7 with a transition time of 3 days during which we provided liquid and solid diet simultaneously. At the end of the study period, the rats were sacrificed by decapitation after exploratory laparotomy under isoflurane anaesthesia. The brain was removed for immunohistochemistry studies measuring neuronal activity (quantification of cfos which is a marker of activated neurons).Operative time and anaesthetic death were recorded, and the study period, as our goal was to validate the technique in rats, was 2 weeks in duration. Body weight was recorded before surgery and every postoperative day, with each death investigated to identify complications. After 2 weeks, an exploratory laparotomy was performed to identify fistula, intestinal occlusion, or repermeabilisation of the duodenal exclusion. Data analyses are expressed as the mean ± standard deviation.p < 0,001) , and the mean postoperative weight was 261 ± 42 g (range 195–391 g) , and 4 cases of repermeabilisation of the duodenal exclusion .p = 0.34). To decrease duodenal stump complications, we performed duodenal exclusion with either suture ligation or surgical clip ligation. However, four cases of repermeabilisation of the duodenal exclusion were seen, and these occurred with duodenal exclusion without sectioning the duodenum. In contrast, we encountered no duodenal stump complications when we sectioned the duodenum.To our knowledge, ours is the first study evaluating the feasibility of an animal model for SADI-S. We chose this strain of Wistar rats because it is the strain most used in preclinical studies focused on obesity and diabetes. This strain is also highly sensitive to weight gain on hyperlipidic diet and quickly becomes insulin resistance. We experienced low overall mortality  and seven anastomotic leaks: four in the side-to-side anastomosis group and three in the end-to-side anastomosis group (To create a larger gastric tube, we used an 8-FR calibration catheter Fig. , contrarIn the original technique described by Sanchez-Pernaute , a 200-cIn conclusion, this study represents the first report of a preclinical model for SADI-S. This technique will allow researchers to evaluate postoperative metabolic homeostasis and compare their findings with other standard surgical bariatric techniques.


Substantial improvement of ULM is further demonstrated on a chicken embryo tumor xenograft model and a chicken brain, showing both morphological and functional microvasculature details at super-resolution within a short acquisition time . The proposed technique allows more robust MB localization and tracking at relatively high MB concentrations, alleviating the need for dilute MB injections, and thereby shortening the acquisition time of ULM imaging and showing great potential for clinical translation.Super-resolution ultrasound localization microscopy (ULM), based on localization and tracking of individual microbubbles (MBs), offers unprecedented microvascular imaging resolution at clinically relevant penetration depths. However, ULM is currently limited by the requirement of dilute MB concentrations to ensure spatially sparse MB events for accurate localization and tracking. The corresponding long imaging acquisition times  to accumulate sufficient isolated MB events for full reconstruction of microvasculature preclude the clinical translation of the technique. To break this fundamental tradeoff between acquisition time and MB concentration, in this paper we propose to separate spatially overlapping MB events into sub-populations, each with sparser MB concentration, based on spatiotemporal differences in the flow dynamics (flow speeds and directions). MB localization and tracking are performed for each sub-population separately, permitting more robust ULM imaging of high-concentration MB injections. The superiority of the proposed MB separation technique over conventional ULM processing is demonstrated in flow channel phantom data, and in the chorioallantoic membrane of chicken embryos with optical imaging as an  Contrast MBs can be considered as acoustic point scatterers, since they are substantially smaller than the typical ultrasound wavelength, but also provide strong acoustic backscatter, permitting the application of signal processing methods that can overcome the inherent compromise between ultrasound frequency and attenuation. ULM has been shown by numerous research groups18 to break the diffraction limit of conventional ultrasound, resulting in an approximately tenfold improvement in imaging resolution while maintaining imaging penetration depth. Furthermore, as an ultrasound-based imaging modality, ULM is safe, non-invasive, low-cost, and lacks ionizing radiation, thereby promising great clinical significance by providing capillary-level imaging resolution at acoustic-level imaging penetrations. As such, ULM has rapidly gained traction and is applicable to a wide spectrum of preclinical and clinical applications, including the diagnosis and characterization of cancer8, cardiovascular disease19, and the monitoring of neurological activity20.Super-resolution ultrasound localization microscopy (ULM), the acoustic analogue to optical sub-diffraction-limit techniques such as PALM5. The successful reconstruction of microvasculature via ULM requires an adequate number of MBs in circulation to fully traverse all vasculature of interest. However, MBs must also be sufficiently spatially isolated to allow for accurate and unequivocal localization of signal centroids—thus, poorly isolated MB centroids are typically excluded from analysis. This represents a challenging trade-off for imaging biologically relevant vascular beds: The imaging of small vessels, which typically have slower flow speeds, benefits from higher MB concentrations that increase the probability of MB traversal; however, this is at the expense of more MB signal overlap in larger vessels. Lowering the concentration of MBs by dilution will result in sparser signal events but will also significantly increase the data acquisition time needed to reconstruct a vascular bed. A long acquisition time is challenging to implement in vivo as both tissue- and operator-induced motion will degrade the final super-resolution image. The dilution method also requires a constant infusion of MBs or multiple bolus injections, both of which are challenging in a clinical setting. Phase-change droplets, which can change phase from liquid to gas when activated by ultrasound, have been utilized as a contrast agent for ULM and have recently demonstrated the potential for real-time super-resolution imaging in vitro18. Distinct from clinically used MBs, the droplet-based super-resolution method can sparsely activate droplets for localization, allowing for high droplet concentrations, and does not necessarily require frame-to-frame droplet flow for contrast-agent detection. However, further in vivo evaluation and validation may be required for this technique.However, ULM is currently challenged by technical and clinical difficulties including long data acquisition times, low temporal resolution, limited clinical dosages of contrast MBs, and inaccurate localization and tracking of MBs due to ultrasound noise and phase aberrationTo break this tradeoff between acquisition time and MB concentration, we propose leveraging the spatiotemporal characteristics of MBs in circulation to improve the performance of MB localization and tracking. Spatiotemporal filtering (e.g.: SVD filtering) is already extensively used in ultrasound blood flow imaging and ULM pre-processing, as a clutter filter to separate MB signals from tissue background. We propose an additional step of signal post-processing to separate MBs into sub-populations based on differences in spatiotemporal flow dynamics, such as movement speed, flow direction, and signal decorrelation. Although MB signals often spatially overlap over the course of an injection bolus, they may be still separable through the judicious use of spatiotemporal filtering. This paper focuses on the use of 3D conical filters in the Fourier domain for detecting and extracting signal differences to separate MB datasets into distinct subsets, each of which can be processed independently. We apply this technique to MB signal acquisitions taken from the chorioallantoic membrane (CAM) of chicken embryos and use optical imaging as validation. We posit that this type of filtering increases the possibility of observing isolated MBs for high-concentration injections and therefore alleviates the need for MB dilution; as a result, it also shortens the corresponding long acquisition time, which is critical for the clinical translation of ULM.The proposed MB separation method divides an original, densely populated MB dataset into subsets of data with a sparser effective MB concentration for more robust localization and tracking. This is demonstrated in the workflow diagram presented in Fig. Separate ultrasound datasets were acquired from a flow channel phantom running at a constant flow rate with a relatively low concentration of MBs . A total of four different flow rates  and two different flow directions were combined frame-by-frame to generate a synthetic ultrasound data set. This combined data set was used to test the performance of the MB separation technique, as the data set represents a complicated flow scenario where MBs are flowing at different speeds and directions simultaneously. To serve as a reference standard, each individual low-concentration MB dataset was processed using conventional ULM super-resolution processing  and more consistent flow direction. ULM was performed on each data subset individually, producing separate super-resolution images Sup Fig. . A finalIt should be noted that generating synthetic data by adding up different individual datasets to simulate high MB concentration does not represent a true physiological blood flow situation. For a more realistic situation, the proposed method was also applied to one individual flow channel data, where different flow velocities exist naturally along the radial direction. The results are shown in Fig. ex ovo CAM provides a thin vascular bed with complex branching patterns and a minimal amount of tissue motion  chicken embryo through intact skull bone; a stack of 5000 frames of data (corresponding to data length of 16.6 s) was used to generate super-resolved images. As with tumor imaging, conventional ULM produced vascular localization maps . Likewise, MB separation applied to CAM imaging revealed better delineation of a broader spectrum of vascular bed features than conventional ULM alone , as validated with optical imaging. Furthermore, MB separation could produce velocity maps that retained complex flow patterning, such as the counter-directional blood flow of arterial capillaries . Finally, the technique was applied to high-MB concentration contrast-enhanced ultrasound (CEUS) datasets acquired from multifaceted three-dimensional networks—namely the chicken CAM tumor xenograft model and chick brain. In both cases, MB separation produced well-defined microvascular features  while also retaining the highly perfused vessel lumen of feeding vasculature. Thus, the proposed technique offers a simple post-processing solution that alleviates the tradeoff between acquisition time and MB concentration for ULM imaging. This permits the widespread utilization of the method without the need to adjust experimental parameters and even allows for retrospective analysis of previously gathered datasets.We have demonstrated that the proposed MB separation technique, which leverages the spatiotemporal characteristics of MBs in circulation to allow for independent analysis of sub-populations of MBs, improves the performance of MB localization and tracking for ULM imaging at high MB concentrations. This technique was tested ies Fig. . Finallyk-ω domain). With these assumptions in mind, we designed a set of conical k-ω domain filters to isolate MB subpopulations that are flowing at different speeds and trajectories. Applying this set of 3D cone-shaped filters to MB k-ω domain data separates the MB signal space into multiple subsets (15 subsets for in vivo data in this study) containing spatially sparser MB subpopulations with less flow complexity. We posit that this MB separation technique allows for useful ULM imaging at relatively high MB concentrations and is therefore preferable to the clinically relevant solutions of MB where MB signals may be spatially overlapping. Given that Hingot et al.21 report that the long acquisition times for ULM imaging are dictated by rare MB flow events in the microvasculature, it follows that higher MB injection concentrations would allow for reduced acquisition times. We observed that MB separation improved the performance of established MB localization and tracking algorithms7 and thereby resulted in better super-resolution images of vasculature.An underlying assumption of the proposed MB separation technique is that MBs will follow physiologically relevant blood flow trajectories and will therefore be constantly changing their relative positions and directions to one another. However, groups of MBs that are flowing in large vessels will exhibit high flow speeds and complicated flow dynamics, resulting in a low degree of correlation. In contrast, MBs in capillary beds will flow more slowly and stably and will maintain a high degree of correlation in their motion. These differences in movement speed, flow direction, and signal decorrelation produce tangible shifts in the spatiotemporal flow dynamics of MBs, which then correspond to distinct energy concentrations within different regions of the 3D Fourier domain , but it should be noted that this number can be arbitrary, and the only requirement is to avoid significant MB signal smearing or distortion by using relatively large passbands. MB signals of each subset can be reviewed separately to empirically determine an appropriate passband for each filter. In general, a tradeoff exists between the number of separated datasets and computation cost. Although increasing the number of subsets can potentially provide a larger number of localized MBs, it also dramatically increases the computational cost. The optimal number of subsets and passband range would need to consider MB concentration(s), complexity of the flow dynamics for specific organ, flow speed range, and ultrasound center frequency, which would be varying from case to case.Ultimately, the goal of the proposed MB separation algorithm is to produce multiple spatially sparse MB subsets from overlapping signals, thereby alleviating the need for dilution of indicator, rejection of overlapping MB signals, and long acquisition times. However, we acknowledge that our proposed method does not produce perfect MB separation and, indeed, such a goal may be impossible using only post-processing techniques. One salient concern with the proposed method is the risk of MB signal distortion resulting from splitting the MBs into two or more filters. To minimize the risk of MB signal distortion, we use relatively large passbands with smooth transitions and large overlaps between adjacent filters, which would avoid hard splitting of single MB signals. The remaining undesired signals can be further suppressed via intensity thresholding and correlation thresholding in the proceeding ULM processing steps (as detailed in Methods). As a result of filter overlap, MBs may be included within the passband of two or more filters and could therefore be localized and paired multiple times. This “MB doubling” effect would lead to an overestimate of vascular density value  but should not influence the imaging the vasculature structure or morphology. Instead, “MB doubling” might be beneficial for maintaining the intensity of the ULM density image at the big vessel lumen, where the MBs signals would have been discarded without MB separation  will be the subject of a future study.This study has some limitations that should be addressed. The synthetic flow channel phantom data were combined under the assumption of purely linear interference, whereas an actual high concentration MB injection may have more complex and nonlinear interference characteristics. The purpose of using such a synthetic data was to artificially generate an extremely complicated MB flow dataset that contains different MB velocities and directions, in order to validate the capability of the proposed MB separation method in the differentiation of different MB sources. However, the synthetic combination of flow channel data produces conditions that are physiologically impossible, such as simultaneous counter-directional flow in a single vessel and MB boundaries that are truly spatially overlapping. We thus applied the proposed method to an individual flow channel data, which represented a realistic flow situation for a large vessel, and the results Fig.  were in k-ω domain, increases the possibility of observing spatially sparse MB events and therefore alleviates the need for MB dilution. This method could substantially reduce the acquisition times required for the reconstruction of microvasculature, which is critical for the clinical translation of ULM.In this paper we demonstrate a substantial improvement in the performance of ULM at relatively high MB concentrations by using a post-processing technique to separate MB subsets based on their spatiotemporal characteristics. The application of the proposed MB separation algorithm, which consists of a series of 3D cone-shaped filters in the r. A MB moving in a straight line with a constant velocity v (and speed r0 at t = 0 can be described as:Assume B(k) is the Fourier transform of k-ω domain), where k-ω domain . Since MB signal is modulated by the center frequency (f0) of the transmitted ultrasound, its energy is centered at c is the sound speed, deviating from the baseband in k domain. This facilitates the reduction of overlap between MBs with different speeds. Therefore, by applying a bandpass cone-shape filter with frequency response:The spatiotemporal Fourier transform of the moving MB can then be described as:mentclass2pt{minimk-ω domain. Instead, we are extracting a group of MBs whose majority of spatiotemporal movement energies are concentrated inside the cone-shaped filter defined above, which may include energy from other MBs moving at other velocities. The hypothesis is that this undesired energy is suppressed inside the cone-shaped filter and, by subsequent post-processing like intensity thresholding and de-noising, this energy should be further reduced.It should be noted that we are not separating all MBs moving with all possible velocities, which is very difficult due to the theoretical overlap of MBs in k-ω domain also indicates the direction of moving MBs, with those moving towards or away from the transducer allocated in distinct quadrants. Therefore, MBs extracted from each cone-shaped filter  of the MB ultrasound data. It is hypothesized that MBs with different velocities correspond to different frequency components in the temporal direction . The amount of Doppler frequency shift is proportional to MB moving velocity along the ultrasound beam direction, where a positive frequency indicates the MB moving towards the transducer, and a negative frequency indicates the MB moving away from the transducer. Therefore, the MB signal within a certain speed range can be extracted by applying a bandpass filter with frequency response as follows:A custom-made flow channel phantom , with a 2-mm inner diameter straight flow channel which has an oblique angle of 30 degrees with respect to the surface of the phantom was used in this study. The outlet of the flow channel was connected to a syringe pump  that provided constant flow through the channel. Lumason MB suspension  was diluted with saline to approximately 1/2000 times the original concentration to provide a solution with adequately isolated MB signals. Ultrasound in-phase quadrature (IQ) data were acquired separately from the longitudinal view of the flow channel at four different flow rates  using a Verasonics Vantage ultrasound system  and a GE 9 L linear array probe . A 10-angle coherent compounding plane-wave imaging (step angle = 1°) was implemented for data acquisition with a center frequency of 6.25 MHz (single cycle pulse) for each transmit angle. The transmit voltage was lowered to 10 V (one-side voltage) to minimize MB destruction. The frame rate was 500 Hz, and for each flow rate setting, a stack of 6000 ultrasound frames (12 s) was acquired. For each flow rate, the flow direction was reversed by switching the outlet connected to the syringe pump, and the data acquisition was repeated. Thus, a total of 8 independent datasets were acquired (4 flow rates and two directions) at a relatively low MB concentration. All 8 IQ datasets were then linearly added frame-by-frame to generate a synthetic ultrasound dataset, which represents a complicated flow scenario at higher MB concentration, to validate the proposed MB separation method for ULM. The 8 independent datasets, each at low MB concentration, can also be processed separately to produce 8 ULM images, and the combination can be used as a reference standard.No IACUC approval was required for the chicken embryo experiments presented in this paper, as under NIH PHS policy avian embryos are not considered to be live vertebrate animals.oC, 5% CO2, humidified incubator.Renca cells (ATCC CRL-2947) were obtained from the American Type Culture Collection Inc.  and cultured in accordance with ATCC guidelines. Growth medium was RPMI 1640 media  supplemented with 10% FBS , non-essential amino acids (0.1 mM), sodium pyruvate (1 mM), and L-glutamine (2 mM). Cells were subcultured at a subcultivation ratio of 1:5 when >80% confluence. All cells were kept in a 37 29, with slight modification to facilitate co-registered acoustic-optical imaging. Fertilized chicken eggs were obtained from Hoover’s Hatchery  and placed into a turning humidified incubator . For some preparations, acoustic windows were opened in the plastic weigh boat with a razor blade and sealed with saran wrap. On the fourth day of embryonic development (EDD-04) egg shells were opened with a rotatory dremel tool and contents were carefully transferred into the prepared weigh boats. Embryos were then transferred into a second humidified incubator (Caron model 7003-33) until ultrasound imaging on EDD-18.We followed the CAM preparation and tumor engraftment procedures outlined in5 cells per 10 µL. This mixture was kept on ice until implantation into the CAM. On the engraftment day, EDD-09, a small disk of autoclaved Whatman No.1 filter paper was used to abrade the surface of the CAM, and 10 µL of the cell mixture was placed in the wound. Embryos were then returned to the incubator.Renca cells of high confluence (>80%) were trypsinized  and pelleted (centrifuged for 5 minutes at 300 g) on the day of engraftment (EDD-09). Matrigel (BD Bioscience) was combined with the cell pellet to obtain an inoculation dose of 4 × 109 MBs/mL.All CEUS imaging was performed using an intravenous injection of the commercially available Bracco Lumason microbubble solution. For this study, MBs were reconstituted with 1.0 mL sterile saline yielding a solution of approximately 1.8 × 10For CAM injection, an 18 G × 1.5-inch beveled needle tip was attached to 8 cm of Tygon R-3603 laboratory tubing. The open end of tubing was fitted with a glass capillary needle. With the aid of a dissecting microscope, the glass capillary needle tip was manually cannulated into a high-order vein on the CAM surface for contrast injection. A total volume of 70 μL of the MB solution was injected into each embryo.For CAM membrane and tumor imaging, a 15-angle compounding plane-wave imaging was performed (step angle = 1°) using a Verasonics Vantage ultrasound system equipped with an L35-16vX high-frequency linear array transducer  operating at a center frequency of 25 MHz. A two-cycle pulse was used for each transmit angle and the transmit voltage was set to 5 V (one-side voltage). The post-compounded frame rate was 500 Hz. For the CAM membrane, a total of 21 IQ datasets were collected from one bolus injection, with each dataset containing 720 ultrasound frames (corresponding to 1.44 seconds data length per dataset for the given frame rate). For each CAM tumor, a total of 5 acquisitions were gathered at 720 frames of IQ data each, for a total length of 3600 frames (7.2 seconds).Chick embryo brain imaging was performed using a Vevo 3100 high-frequency ultrasound system  equipped with an MX250S linear array transducer  transmitting at 21 MHz (single-cycle pulse). Line-by-line focused imaging was used to scan the chick brain through the intact skull bone with a 16 mm by 6 mm field-of-view (FOV) and one fixed transmit focus, leading to a frame rate of 301 frames per second  at 4x magnification, and images were digitized with an integrated Nikon DS-Ri2 digital camera (Nikon). Optical images were acquired from the top of the weigh boat housing the embryo, and an L35-16vX ultrasound transducer was placed to image the CAM surface through a lateral acoustic window in the side of the weigh boat  using a 2-D spline interpolation. Following interpolation, an intensity threshold was applied to the envelope of MB signals to remove the noisy background, which would also help suppress undesired energy leaking from other subsets of MBs. A point-spread function (PSF) specific to the imaging system and settings was derived from a multivariate Gaussian function. A 2-D normalized cross-correlation between each frame of the interpolated MB signal and the derived PSF was performed for MB localization. A threshold was applied to the cross-correlation map to remove pixels with correlation coefficient values lower than 0.6, which further suppresses unwanted signals from other MB subsets while preserving high confidence MB signals for the given subset. Then, MB centroids were localized using a regional maximum search of the cross-correlation peaks for each frame. A bipartite graph-based pairing and tracking algorithm was then applied to the identified MB centroids, with a minimum persistence of 7 frames22. A Kalman-filter-based algorithm was then applied to each MB trace to further reject unreliable MB tracks based on the MB movement acceleration and direction constraints, and improve in-painting of the MB trace and MB speed measurement22.A spatiotemporal SVD-based clutter filter was first applied to the ultrasound IQ dataset to extract moving MB signals by rejecting background tissue signals and stationary MB signalsSupplementary information.Supplementary Figure S1Supplementary Figure S2Supplementary Figure S3