We are familiar with the clinical signs that animate us to administer intravenous (IV) fluids in sepsis and septic shock. Fundamentally, these features—hypotension, oliguria, confusion, skin mottling, prolonged capillary refill, elevated lactate, etc.—are surrogates for hypoperfusion, that is, low blood flow , 2. Hencflow and vascular resistance. Therefore, for a given resistance, increasing flow from the heart raises pressure and from this relationship we leverage blood pressure to adjacently inform us on blood flow. But what about the not-infrequent scenario where IV fluid is given and blood pressure changes little, if at all? Is this because blood flow did not budge? Or did flow indeed rise, but in the face of falling resistance?[To be more concrete, let us consider blood pressure. In a simplified model of the circulatory system, blood pressure is the product of blood sistance? Without sistance?—to IV flsistance?, 6 and opersistently fluid refractory across the entire 72-hour investigation. Similar observations were made by Chen and Kollef [But how common is the heart refractory to IV fluids in sepsis and septic shock? With the available evidence, the answer is that it is surprisingly common. In the landmark ANDROMEDA-SHOCK investigation , which id Kollef  who repod Kollef , 6, 8.not received 30 mL/kg at randomization, there was likely overrepresentation of fluid responsive patients; in this subgroup, those randomized to the liberal arm trended towards lower mortality. Of course, these observations are hypothesis-generating at best. Nevertheless, the aforementioned FRESH [less IV fluid and had fewer fluid-related complications as compared to usual care. Ostensibly, this occurred because cryptic fluid refractory states were detected and IV fluid restricted at the appropriate time [Where does this leave us within the context of ‘liberal’ versus ‘conservative’ fluid management approaches? While both the CLASSIC  and forted FRESH  study giate time , 6.We should look forward to the forthcoming results of ANDROMEDA-SHOCK 2 and others who attempt resuscitation based on hemodynamic phenotypes. Much as we tailor antimicrobials to culture and sensitivity, bespoke IV fluid founded upon cardiac preload sensitivity should shape septic resuscitation strategy in the 21st century.


Fluorescence lifetimemultiplexing requires fluorescent probeswith distinct fluorescence lifetimes but similar spectral properties.Even though synthetic probes for many cellular targets are availablefor multicolor live-cell fluorescence microscopy, few of them havebeen characterized for their use in fluorescence lifetime multiplexing.Here, we demonstrate that, from a panel of 18 synthetic probes, eightpairwise combinations are suitable for fluorescence lifetime multiplexingin living mammalian cell lines. Moreover, combining multiple pairsin different spectral channels enables us to image four and with thehelp of self-labeling protein tags up to eight different biologicaltargets, effectively doubling the number of observable targets. Thecombination of synthetic probes with fluorescence lifetime multiplexingis thus a powerful approach for live-cell imaging. One such property is fluorescencelifetime, which has been used for multiplexing via fluorescence lifetimeimaging microscopy  to furtherincrease the number of simultaneously observable targets.10Fluorescencemicroscopy is anindispensable tool to noninvasively investigate dynamic processesin living cells. Such experiments often require imaging multiple biomoleculesand cellular compartments simultaneously. This is generally achievedby spectrally resolved detection using fluorophores with distinctexcitation and emission spectra 1A. Howevpy FLIM, 1B.7,8 Re11 They do not require genetic engineering  ofthe target cell and can therefore be applied to a wide variety ofcell types. Additionally, probes for different targets can easilybe combined, while the simultaneous expression of multiple taggedproteins can be challenging.12 Indeed,synthetic probes with distinct spectral properties were successfullycombined for multiplexing.15 However, they have only foundlimited use in fluorescence lifetime multiplexing, and their fluorescencelifetimes are often not characterized.17 Proof-of-concept studieswere restricted to fixed cell applications10 and/orused probes with both differences in fluorescence lifetime and emissionspectrum.16 We recentlydemonstrated the combined use of synthetic probes and self-labelingprotein tags for fluorescence lifetime multiplexing.17 However, our study was limited to only four probes andthus only partially exploited the potential of synthetic probes forfluorescence lifetime multiplexing.Synthetic probes based on small-moleculefluorophores for live-cellmicroscopy of various subcellular targets such as lysosomes, mitochondria,or filamentous actin (F-actin) are available.18 Combinationsof live-cell compatible, synthetic probes for biomolecules or cellularcompartments could find applications in live-cell fluorescence lifetimemultiplexing. Ideally, each probe should show a homogeneous and narrowfluorescence lifetime distribution, and spectrally similar probesshould show differences in fluorescence lifetimes to enable theirseparation.Here, we investigate iffluorophores of different classes and chemicallyidentical fluorophores targeted to different subcellular localizationsshow differences in fluorescence lifetime. Indeed, internal and externalfactors including vibrational and rotational freedom, viscosity, polarity,or the presence of quenching moieties can influence the fluorescencelifetime of fluorophores.Supporting Table S1).20 Four probes were previously used for fluorescence lifetime multiplexing,17 and another two were used in spectral FLIM (S-FLIM).10 For initial screening purposes we choose to assess the fluorescencelifetime properties by phasor analysis22 as it allowsrapid and visual screening of probes for differences in fluorescencelifetime without the need for fitting . Specifically, we labeled living U-2 OS cells with all18 probes individually and acquired FLIM images. Phasor plot analysisthen revealed narrow and homogeneous distributions for all actin,microtubule, and DNA probes. Probes for lysosomes had slightly broaderdistributions but were still homogeneous. However, only two of thefour probes for mitochondria displayed satisfying properties (MitoTracker-Greenand rhodamine B). The other two, MitoTracker-Red and MitoTracker-Orange,showed multiple fluorescence lifetime populations in their phasorplots . This mightresult from the probes accumulating in multiple organelles as previouslyreported for MitoTracker-Orange in fixed cells .9 While these differencesin fluorescence lifetime might be used to separate the specific mitochondriasignal from the unspecific signal in the endosome and the nucleoli,these probes cannot be combined with other spectrally similar probesfor further fluorescence lifetime multiplexing, and hence they werenot further investigated.We therefore characterized the spectral and fluorescencelifetimeproperties of 18 commercially available cell permeable probes. Theseencompassed popular rhodamine and BODIPY based probes in five differentspectral channels targeting DNA, F-actin, microtubules, mitochondria,and lysosomes  and MitoTracker-Green(0.96 ± 0.03 ns) followed by SPY555-DNA (3.00 ± 0.02 ns)and SPY555-Actin (1.79 ± 0.09 ns) in the orange channel.Supporting Figure S1). Generally, probes based on different fluorophore scaffolds  showed the biggest differences in fluorescencelifetime. The excitation and emission spectra of probes within onespectral region were generally highly similar, except for LysoTracker-Red,which shows a 20 nm hypsochromic shift in comparison to the two SPY620probes . LysoTracker-Redwas therefore not considered for multiplexing experiments. Furthermore,we demonstrated that most probes’ fluorescence lifetime showedonly little variation between different cell lines . An exception are the lysosomeprobes, which generally showed broad fluorescence lifetime distributions and therefore highervariability in average intensity weighted fluorescence lifetime withinone cell line as well as between different cell lines. This variabilitymight stem from differences in the intralysosomal pH.23 Further, we characterized the crosstalk for different probecombinations using the images from individually labeled U-2 OS cells. Most probe combinations showed less than 10% crosstalk into theother species’ lifetime channel. The combination BioTrackerOrange Lysosome (11.7 ± 4.7%)–SPY555-DNA (14.2 ±1.6%) had the biggest crosstalk.These two pairs are hence ideally suited for fluorescence lifetimemultiplexing 1C–E. DifSupporting Figures S4, S5). Moreover, average photon arrivaltimes as reported in the FastFLIM image could be used to distinguishthe two species if they are spatially separated and to identify overlappingregions (pixels with intermediate fluorescence lifetime). This allowsfor quick inspection during image acquisition without the need forfitting or phasor analysis, greatly simplifying the use of fluorescencelifetime multiplexing. As the probes’ fluorescence lifetimesshowed little variation between cell types (Supporting Table S2), multiplexing could also be performed in living HEK293 or HeLa cells .We then performed fluorescencelifetime multiplexing using pairwisecombinations of two probes in each of the four spectral regions : 670 nm excitation, 710–760nm emission). As predicted by the differences in average intensityweighted fluorescence lifetime 1D–G, mulin cellulo brightness of synthetic probes is not only determined by their molecularbrightness but can also be adjusted through the degree/density oflabeling and hence the labeling concentration. Specifically, mitochondriaand lysosome probes accumulate in their respective organelles. Thelabeling concentration and hence the brightness can be varied overa broad range. The brightness of DNA, actin, and tubulin probes, onthe other hand, cannot be varied to the same degree as their numberof binding sites is limited.In general, larger differencesin fluorescence lifetime and narrowfluorescence lifetime distributions of the individual species facilitateseparation especially if the two species exhibit overlapping regions.We therefore recommend the use of pairs of probes with a fluorescencelifetime difference of around 0.5 ns. Species with smallerdifferences can still be separated, but crosstalk becomes more important, andit is hence important to choose probes with narrow fluorescence lifetimedistributions. Separation is furthermore influenced by the brightnessof the two species: to obtain comparable signal-to-background ratiosfor both species after separation, similar photon numbers should becollected. Otherwise, the crosstalk of the brighter species into thedimmer species lifetime channel will approach the dimmer species’signal intensity. Acquiring similar photon numbers in FLIM can bechallenging when working with probes of different brightness as thefluorescence of both probes is acquired simultaneously using onlyone excitation wavelength. However, the 24 or SNAP-tag,25 react with fluorophores bearing a chloroalkane(CA) or benzylguanine (BG) ligand, respectively. Through fusion ofthe SLP tag to proteins of interest (POI), one can therefore localizecell-permeable fluorophores to different subcellular localizations.We previously demonstrated the use of SLP-tags in fluorescence lifetimemultiplexing and characterized the average fluorescence lifetime ofdifferent HaloTag7 fusion proteins conjugated to MaP555-CA (2.4 ns)or MaP618-CA (3.1 ns).17 We hence performeda similar characterization for SNAP-tag localized to different subcellularlocalizations and labeled with MaP555-BG in living U-2 OS cells . SNAP-tag-MaP555 showed little variation in fluorescencelifetime averaging around 2.5 ns. The MaP555 substrates for SNAP (BG)and HaloTag (CA) should therefore be multiplexable with the SPY555probes  as they have a fluorescence lifetimedifference of around 0.5 ns. To test this, we expressed HaloTag7-SNAP-tagas a fusion with the endoplasmic reticulum (ER) marker calreticulin/KDEL in U-2 OS cells and labeled them with either MaP555-BGor MaP555-CA. When combined with SPY555-Actin or SPY555-DNA, two speciescould be separated on the basis of their fluorescence lifetime information. On the other hand,the fluorescence lifetimes of Rhodamine B and BioTracker Orange Lysosomeare too similar to the corresponding SLP probes and therefore didnot allow multiplexing. MaP618-CA (3.1 ns) can be used for multiplexingwith SPY620-DNA but not SPY620-Actin .The herein tested probes allowedsimultaneous imaging of combinationsof mitochondria, lysosomes, nucleus (DNA), F-actin, or microtubules.To access alternative targets for which no synthetic probes are availableor for which the probes do not show a difference in fluorescence lifetime,the self-labeling protein (SLP) tag strategy can be employed. SLPtags, such as HaloTag7Supporting Figure S9). Alternatively, SPY555-Tubulin could be replaced by SPY555-Actinrevealing the F-actin network. Instead of combining the green andthe orange channel, two red probes (SPY620-DNA and SPY620-Actin) canalso be combined with the two probes in the green channel . More than four species can be accessed combining synthetic probeswith SLP-tags. We hence expressed HaloTag7-SNAP-tag in the ER of U-2OS cells and labeled them with LysoTracker-Green, MitoTracker-Green,MaP555-CA, SPY555-Actin, SPY700-DNA, and SiR700-Tubulin, allowingus to acquire six species’ images. Acquisition of all threechannels and separation of the two lifetime species in each of themrevealed lysosomes, mitochondria, the ER, F-actin, the nucleus, andmicrotubules . Inaddition, we were able to perform a time course experiment by repeatedFLIM measurements allowing us to follow the movement of all six speciesover 6 min . Furthermore,by combining the SNAP-tag with two HaloTag variants ),17 eight species could be imaged. We labeled cells expressing HaloTag7in the ER, HaloTag11 in the Golgi, and SNAP-tag at the plasma membranewith LysoTracker-Green, MitoTracker-Green, MaP555-BG, SPY555-Actin,MaP618-CA, SPY700-DNA, and SiR700-Tubulin and acquired all four spectralchannels . As the labeling withmultiple synthetic probes raises concerns of cell viability, we investigatedwhether the labeling influenced the percentage of living cells 20h post labeling, and indeed no significant difference was found . The combination of fluorescencelifetime multiplexing with spectrally resolved detection hence allowsto double the number of species imaged.We then combined fluorescence lifetime multiplexingwith spectrallyresolved detection. First, four probes were multiplexed in two spectralchannels, each containing two probes separable in fluorescence lifetime3A: LysoT17 This could openup the door to not only double but even triple the accessible species.However, this is currently limited as the phasors of suitable probesneed to form a triangle, ideally an acute triangle , in phasor space. None of the combinationsof the 18 synthetic probes fulfilled these criteria. Separation ofmore than three species requires transformation into higher harmonicsor the use of additional information.26 As more and more probes based on a variety of differentfluorophores become available, we believe that it should be possibleto expand the number of multiplexable species further and to accessmore species without genetic engineering. The use of synthetic probestargeting different biomolecules or cellular compartments is hencea straightforward strategy to generate fluorescence lifetime contrastand should facilitate the use of fluorescence lifetime multiplexingin living cells.In summary, the fluorescence lifetimesof 18 synthetic fluorescentprobes were characterized, and eight pairwise combinations of probessuitable for fluorescence lifetime multiplexing were identified. Incombination with spectrally resolved detection, these synthetic probesallow the doubling of the number of species that can be imaged, givingaccess to two and four species images and up to eight species whencombined with SLP-tags. Fluorescence lifetime multiplexing via phasoranalysis does not stop at pairs of probes but can technically alsoseparate three species.15 by B. Réssy or D. Schmidt (MPI-MR). All other probeswere purchased from commercial vendors (Supporting Table S1), or obtained from Spirochrome. Fluorophores wereprepared as stock solutions in dry DMSO and diluted in imaging mediumsuch that the final concentration of DMSO did not exceed 1% v/v.MaP555-BG, MaP555-CA, and MaP618-CAwere prepared according to literature proceduresSupporting Table S6. The microscope was equipped with a CO2 and temperature controllable incubator .Confocal fluorescence microscopywas performed on a Leica SP8 FALCON microscope (Leica Microsystems)equipped with a Leica TCS SP8 X scanhead, a SuperK white light laser,Leica HyD SMD detectors, and a HC PL APO CS2 40 × 1.10 waterobjective. Emission was collected as indicated in Supporting Methods.Fluorescence excitationand emissionspectra of synthetic probes were measured in living U-2 OS cells.For SNAP-tag and HaloTag7 probes, U-2 OS cells were transiently transfectedwith HaloTag7 or SNAP-tag . Settings can befound in the Supporting Table S6.FLIM was performedon a Leica SP8 FALCON microscope (as described above) at a pulse frequencyof 80 MHz unless otherwise stated. Emission was collected as indicatedin 2 < 1.2).For determinationof average intensity weighted fluorescence lifetimes of differentprobes cells were imaged, collecting 500 photons per pixel. The acquiredimages of cells were thresholded to remove the background signal fromempty coverslip space. Mean fluorescence lifetimes were calculatedin the LAS X software (Leica Microsystems) by fitting a mono-, bi-,or triexponential decay model  to the decay .27 No threshholding was applied.Structural images (species separation) were acquired as indicatedin For dynamic experiments, multiple images were acquired with a timedelay of 2 min between the start of the first and the start of thesecond image.Data Availability. Plasmids encodingSNAP-tag fusionswere deposited on Addgene. Correspondence and reasonable requestsfor materials should be addressed to K.J.


Introduction: It is possible that social media use can boost not just articles’ social impact but the number of citations and academic influence as well. If a positive correlation between Twitter usage and citation metrics exists in the ophthalmology literature, it is important to broadcast this information to the ophthalmology community so they can use Twitter to increase academic engagement with their research. There has also been an increase in the number of articles available as open access. Therefore, it is important to evaluate the presence of an open-access citation advantage in the field of ophthalmology. This study aims to evaluate the relationship between Twitter mention and open access status on citation metrics in the ophthalmology literature.Methods: We conducted a retrospective cross-sectional study comparing article citation metrics to Twitter mentions and open access status. We gathered data on ophthalmology research articles from the six highest-ranked ophthalmology journals published as part of a January 2019 issue. Data were collected in April 2022, 38 months after online publication. Data on citations for each article was based on Google Scholar and Scopus websites. The Altmetric Bookmarklet extension was used to determine the amount of social engagement each article received. The open-access status of each article was based on the status listed in its corresponding journal. Two-tailed t-tests were used to compare social media engagement and open access status with the number of Google Scholar and Scopus citations.Results: A total of 102 original research articles were analyzed. 89 (87.3%) articles received a Twitter mention. Articles tweeted at least once had a significantly higher Google Scholar score (27.2 +/- 4) compared to articles not tweeted . Likewise, the average Scopus score was significantly higher for tweeted articles (18.6 +/- 2.6) compared to articles not tweeted . Articles listed as open access had a significantly higher number of Twitter mentions (11.8 +/- 1.8) compared to articles that were not open access . Open-access articles also had higher citation scores compared to articles that are not open access, but this relationship was not statistically significant.Conclusion: This is the first study to evaluate the relationship between article Twitter mention and citation score in the field of ophthalmology. It demonstrates a significant positive correlation between the article Twitter mention and citation score and provides further evidence that social media engagement can be beneficial to the dissemination of academic information. Further studies on the relationship between social media engagement and article dissemination are warranted in the field of ophthalmology. The academic impact of scientific research articles has traditionally been gauged through the number of citations for a said article in peer-reviewed journals . Given tIt is possible that social media use can boost not just articles’ social impact but the number of citations and academic influence as well. There has been documented evidence that an author’s Twitter usage can be used as a predictor for citation metrics for publications . FurtherSince each specialty has embraced social media usage at different rates, the relationship between social media and citation numbers should be evaluated for each specialty ,10. If aMoreover, there has been an increase in the number of articles available as open access in recent years . Open-acPrevious studies in the field of ophthalmology have evaluated the relationship between social media and journal impact ,14. HoweGiven that social media usage (including Twitter) has been increasing among ophthalmologists , we believe there exists a positive relationship between Twitter mentions and ophthalmology citation metrics -17. We bWe conducted a retrospective cross-sectional study comparing article citation metrics to Twitter mentions and open access status. The analysis of the association between Twitter mentions and citation metrics has previously been outlined in studies in urology and otolaryngology ,5. SinceArticle selectionData on ophthalmology research articles from six different journals published as part of a January 2019 issue were reviewed. This date was selected to provide enough time for dissemination and citation of the articles across media to take place. A previous study in the field of otolaryngology also used the January 2019 issue of journals for article selection . AnotherData selectionData were collected in April 2022, 38 months after online publication. Data on citations for each article were based on Google Scholar and Scopus websites. Article titles were searched on each website to determine the number of citations each article had received. The Altmetric Bookmarklet extension was used to determine the amount of social engagement each article received. We gathered data on Twitter mentions for each article. Further, we recorded how long it took for an article to be mentioned on Twitter after it was either published online or published in print. Categories for a timeline from online publication included < 1 week online, < 1 month online, and 1 month to print date.Categories for a timeline from print publication included < 1 week in print, < 1 month in print, and < 1 year in print. Twitter mentions which occurred after one year were not reported to maintain consistency with similar studies that have demonstrated no increase in Twitter mentions after one year of print publication ,5.Cross-references of Twitter handles with authors to determine if a Twitter mention was from an author of the paper mentioned were done. Articles tweeted by one of its authors were listed as an author's self-tweet. The open-access status of each article was based on the status listed in its corresponding journal.Data analysisData were analyzed using SPSS software . Significance was set at p-value <0.05%. Two-tailed t-tests were used to compare the presence or absence of Twitter mentions with the number of Google Scholar and Scopus citations. Levene’s Test for Equality of Variances was used to test for homogeneity of variance and the corresponding t-test result was used. The same analysis was implemented with regards to the presence or absence of author self-tweet. A two-tailed t-test was used to compare the number of citations and Twitter mentions between articles that were listed as open access and those which were not open access.A total of 102 original research articles were reviewed. Table The median Google Scholar score (IQR) and the median Scopus score (IQR) were 16.5 (15.5) and 10.5 (12), respectively. Eighty-nine (87.3%) articles received a Twitter mention. The median number of Twitter mentions was 5 (6). The journal with the highest median Google scholar and Scopus scores was Ophthalmology with scores of 29 (40.5) and 23 (26.5), respectively. The journal with the highest median number of Twitter mentions was JAMA Ophthalmology with 15 (4).Figure Table Table We evaluated the relationship between a Twitter mention and citation score over a 38-month period. We identified a positive relationship between Twitter mentions and citation scores across both citation metrics (Google Scholar score and Scopus score). The increase in citation score among tweeted articles was about the same for both Google Scholar and Scopus scores (1.7 and 1.6-fold increase respectively). This finding is in accordance with a similar study in the field of otolaryngology which also found a 1.6-fold increase in Google Scholar score for articles with a Twitter mention . A similThe timeline of Twitter mentions was evaluated. Most Twitter mentions occurred within one week of online publication (62%). This is in accordance with the previously mentioned studies in the fields of urology and otolaryngology which also found most Twitter mentions occurred within one week of online publication ,5. This The relationship between open access status and citation scores and Twitter mentions was also evaluated. Articles listed as open access had a significantly higher number of Twitter mentions than articles that were not open access. This suggests that articles that are open access and therefore more accessible may reach a wider audience resulting in a higher number of Twitter mentions. Previous studies on the relationship between open access status and citation metrics have yielded mixed results with respect to the existence of an open access advantage . MultiplFinally, we sought to evaluate the relationship between the presence of author-self tweets and citation scores. No significant correlation between the presence of author self-tweet and citation score was found. This fact contrasts with previous studies which found a significant increase in citation scores for articles that had a Twitter mention by an author ,5.Limitations of our study include the inability to demonstrate a causal analysis as well as a small sample size for author self-tweet. Perhaps more data could be gathered on these metrics by including a larger number of articles in the analysis. Moreover, only top-ranked journals and original research articles were selected for analysis. This decision was made to maintain consistency with previously reported methodology and compare our findings to other surgical subspecialties while limiting the presence of other confounding variables ,5. HowevThis is the first study to evaluate the relationship between article Twitter mention and citation score in Ophthalmology. Our study demonstrates a significant positive correlation between article Twitter mention and citation score. It provides further evidence that social media engagement can be beneficial to the dissemination of academic information. It also suggests that the open-access status of an article may increase Twitter mentions and help increase an article’s social media engagement. Further studies on the relationship between social media engagement and article dissemination are warranted in the field of ophthalmology.


The governing equations are solved numerically by using regularization methods in the context of the overset meshing technique. The results indicate that there is a good comparison between the experimental data and the simulations. The boundary layer thicknesses are predicted within the uncertainties of the measurements. The simulations indicate strong sensitivities to the rheological properties of the fluid.In this paper, we study the boundary-layer flow of a Herschel–Bulkley fluid due to a moving plate; this problem has been experimentally investigated by others, where the fluid was assumed to be Carbopol, which has similar properties to cement. The computational fluid dynamics finite volume method from the open-source toolbox/library OpenFOAM is used on structured quad grids to solve the mass and the linear momentum conservation equations using the solver “ Constitutive modeling of complex fluids , sometimn , to thel & Noll , the terl & Noll ,16. Cemel & Noll ,18. In tl & Noll ). In thel & Noll ,21; withl & Noll ,23; and 2 underground storage where supercritical gas tends to leak through microcracks in wellbores res (see ), where res , ChevalThe response of a yield-stress fluid, for example, a Herschel–Bulkley fluid , due to n, and 0.38, respectively, for shear rates ranging between 10−2 s−1 and 102 s−1. The plate is 25 cm long .The conservation of the angular momentum indicates that the stress tensor is symmetric when there are no couple stresses, that is: Looking at the above equations, we can see that before we can solve any problems, we need a constitutive relation for These conservation equations are supplemented with boundary and initial conditions, both at the walls and at the free-surface boundaries. The no-slip BC is prescribed at the plate and the walls such that:For many complex fluids, yield stress is an important rheological parameter ,45,46,47w/c, etc.w/c is the water-to-cement ratio. In a recent review article, Tao et al. [m < 0) or shear-thickening effects (when m > 0). This model potentially is capable of exhibiting normal stress effects through the terms In general, it can be assumed that the (Cauchy) stress tensor o et al. , proposer’s idea  for the er study .G is the shear modulus, indicating that below the yield stress, the material behaves as a linear elastic solid, obeying the Hooke’s Law, and where For the yield stress part, historically, Oldroyd  derived As Denn  indicateNewtonian regime rather than a Hookean one, and thus one can use a two-viscosity (bi-viscous) model, such as: Macosko  (p. 96) In this paper, we ignore the micro-structure of the cement, i.e., the size and the shape of the particles, and the impact of the porosity and how the volume fraction affects the motion of the fluid. Thus, we represent the cement suspension as a viscoplastic fluid modeled as a Herschel–Bulkley fluid, given by Equations (23)–(25).overInterDyMFoam” customized with non-Newtonian viscosity libraries. In our numerical scheme, we use the regularization methods. Indeed, to avoid the numerical implementation challenges associated with the discontinuity (singularity) in the stress tensor field between the unyielded and the yielded regions, the regularization method is used where the stress tensor The computational fluid dynamics finite volume method from the open-source toolbox/library, OpenFOAM , is usedastasiou  by:(27)ηTwo other commonly used regularization methods are also employed to study the sensitivity of the solution to such viscosity approximations; these two methods are the “simple” algebraic procedure  and theA detailed examination of the convergence challenges and issues associated with the regularized solutions are discussed, for example, in Frigaard & Nouar  and SaraSubstituting Equations (23)–(25) in Equation (6), we have the basic equations, which need to be solved numerically:at the moving plate:at the container’s walls:The dimensionless forms of the equations are presented in The grid of the computational domain is generated relying upon the overset mesh technique, which in this work consists of a uniform grid (in each direction) of the background mesh of the entire flow domain, supplemented by a fine grid around the downward moving plate. With this fine (or overset) mesh, the grid in the normal direction to the plate is clustered using nonuniform spacings according to a geometric series with a rational stretching factor; this would better capture the gradients as the plate moves through the fluid. The convective term in the momentum equations is discretized using the second order “linear” scheme. Spatial gradients are also discretized using the second order “linear” scheme . The simulations are performed while discretizing the unsteady terms with a backward Eulerian scheme. The coupling between the background and the overset grids at these mesh boundaries is accomplished for the resolved fields . The mesh densities along with the resolutions are summarized in The sensitivity of the solution to the mesh and the regularization method are examined below for the plate velocity of 1 mm/s. The sensitivity to the mesh resolution is performed using Papanastasiou’s approximation  (see ):(38)u=UIn Although Boujlel et al.  carefullWe now look at the boundary-layer flow for different properties of a yield-stress fluid modeled as a Herschel–Bulkley fluid. The results for the velocity profiles show a systematic slight mismatch of the predictions against the measurements in the yielded region behaving as a solid-like material. In For different values of the power-law exponent (cies see . Howevercies see b,c, whicThe velocity distribution in the vicinity of the plate plotted in The boundary layer experiments of Boujlel et al. , where a


HT1080 fibrosarcoma-bearing severe combined immunodeficient CB17 mice. 10 ± 1 days after cancer cell inoculation, positron emission tomography (PET) was performed applying [68Ga]Ga-DOTAGA-cKNGRE for tumour verification. On the 7th, 8th, 10th and 12th days the treated group of tumourous mice were intraperitoneally administered with 4.68 ± 0.10 MBq [213Bi]Bi-DOTAGA-cKNGRE, while the untreated tumour-bearing animals received 150 μL saline solution. In addition to body weight (BW) and tumour volume measurements, ex vivo biodistribution studies were conducted 30 and 90 min postinjection (pi.). The following quantitative standardised uptake values (SUV) confirmed the detectability of the HT1080 tumours: SUVmean and SUVmax: 0.37 ± 0.09 and 0.86 ± 0.14, respectively. Although no significant difference (p ≤ 0.05) was encountered between the BW of the treated and untreated mice, their tumour volumes measured on the 9th, 10th and 12th days differed significantly (p ≤ 0.01). Relatively higher [213Bi]Bi-DOTAGA-cKNGRE accumulation of the HT1080 neoplasms (%ID/g: 0.80 ± 0.16) compared with the other organs at 90 min time point yields better tumour-to-background ratios. Therefore, the therapeutic application of APN/CD13-affine [213Bi]Bi-DOTAGA- cKNGRE seems to be promising in receptor-positive fibrosarcoma treatment.Since NGR-tripeptides (asparagine-glycine-arginine) selectively target neoangiogenesis-associated Aminopeptidase N (APN/CD13) on cancer cells, we aimed to evaluate the in vivo tumour targeting capability of radiolabelled, NGR-containing, ANP/CD13-selective [ Angiogenesis/neo-angiogenesis has a central role in tumour development, tumour progression and metastatic spread ,2. Sever64Cu), gallium-68 (68Ga), lutetium-177 (177Lu) or rhenium-188 (188Re) together with the high-quality and non-invasive imaging modalities, a rising number of research studies have been centred around the evaluation of APN-directed radio-appended peptide compounds in the recent years Bi-labelled, NGR-motif containing radiopharmaceutical in CD13/APN-upregulated HT1080 fibrosarcoma-bearing mice. Prior to its therapeutic administration, positron emission tomography (PET) with 68Ga-labelled CD13-targeting compound was applied for tumour verification.In the present study we aimed at evaluating the therapeutic performance of a newly synthesised, Ga-DOTAGA-cKNGRE was purified by solid-phase extraction (SPE) applying an Oasis HLB 1cc cartridge. Analitical radio-HPLC was applied for the quality control (QC) of the purified, 68Ga-labelled complex. Analytical radio-HPLC was conducted using a KNAUER HPLC system, connected to an ATOMKI CsI scintillation detector (eluent A: 0.1% TFA in water and eluent B: 0.1% TFA in 95% ACN). The radiochemical purity (RCP) of the [68Ga]Ga-DOTAGA-cKNGRE was higher than 95%.The radiolabelling of DOTAGA-cKNGRE with a et al. . This mea et al. ,33,34. O213Bi]BiI4−/BiI52− ion was eluted from 225Ac/213Bi generator ) using 0.6 mL of a mixture of 0.1 M HCl and 0.1 M NaI (1:1). Then the [213Bi]BiI4−/BiI52− eluate was added to a vial containing 135 μL 2 M TRIS buffer, 50 μL 20% ascorbic acid and 30 μL of 6 mM DOTAGA-cKNGRE solution. The reaction mixture was incubated at 95 °C for 15 min. The 213Bi-labelled complex was purified with SPE using Oasis HLB 1 cc cartridge pre-conditioned with 5 mL EtOH and 10 mL water. After loading the reaction mixture, the cartridge was rinsed with water (1 mL). The 213Bi-labelled complex was eluted with a mixture of water and ethanol  and diluted with physiological saline . For quality control, the RCP of the purified radiotracer was determined by applying instant thin-layer chromatography (iTLC) by measuring the activity of the 440 keV gamma emission of 213Bi using miniGITA TLC scanner. For radio-iTLC, glass microfiber chromatography paper impregnated with a silica gel (iTLC-SG paper) as a stationary phase and 0.5 M citric acid (pH 5.5) as an eluent were used. Free 213Bi moves with the solvent front, while [213Bi]Bi-DOTAGA-cKNGRE remains at the bottom of the iTLC paper. RCP of the [213Bi]Bi-DOTAGA-cKNGRE was found to be above 99% and the molar activity was 0.5 MBq/nmol.For radiolabelling, n-octanol as well as water was determined by Gyuricza et al. in order to define the LogP value of [68Ga]Ga-DOTAGA-cKNGRE Ga-DOTAGA-cKNGRE.The distribution of the radioactivity in A-cKNGRE . To haveLogP value we used 205/206Bi isotopes as surrogates which were produced by GE PETtrace cyclotron using the method described by Lagunas-Solar et al. [4OAc buffer (pH 4) and 15 µL of 6 mM DOTAGA-cKNGRE aqueous solution were added to 50 µL [205/206Bi]BiCl3 in 0.1 M HCl. The labelled complex was purified as described above. Then 10 µL of the purified [205/206Bi]Bi-DOTAGA-cKNGRE in physiological saline  was diluted with 0.49 mL water. It was then mixed with 0.5 mL n-octanol in a vial and shaken for 5 min. After centrifugation  the radioactivity of the sample from both the aqueous and n-octanol phase was measured with a Perkin Elmer Packard Cobra gamma counter. The LogP value was −2.59.For the determination of r et al. . The rad68Ga]Ga-DOTAGA-cKNGRE dissolved in physiological saline  to 50 μL of mouse plasma, the mixture was incubated at 37 °C. With the application of radio-HPLC we evaluated the serum stability of the radiotracer 0, 30, 60 and 90 min post-incubation with mouse blood serum under predefined circumstances. [68Ga]Ga-DOTAGA-cKNGRE demonstrated metabolic stability in serum after 90 min since the sample taken from the mixture showed an RCP above 95%, which meets the requirements for 68Ga-labelled radiopharmaceuticals.Following the addition of 50 μL of [205/206Bi]Bi-DOTAGA-Bn-cKNGRE in physiological saline  was added to the solution of fifty μL of mouse plasma. The RCP of the samples was determined by radio-iTLC at 0, 30, 60 and 90 min time points using the same method as described above. After 90 min the [205/206Bi]Bi-DOTAGA-Bn-cKNGRE remained intact as the RCP of the sample taken from the mixture was above 95%, which meets the requirements for 213Bi-labelled radiopharmaceuticals.Fifty μL of Ga-DOTAGA-cKNGRE-based PET imaging 10 ± 1 days post tumour cell inoculation. To evaluate the organ distribution pattern of the 213Bi-labelled therapeutic peptide compounds, ex vivo biodistribution studies were also executed 10 ± 1 days following the HT1080 cell transplantation at an average tumour volume of 155 ± 20 mm3. Thereafter, tumour sizes based on the largest and the smallest tumour diameters were registered /2). We used calliper measurements for tumour growth determination.Experimental tumour induction was carried out under inhalation anaesthesia with a dedicated small animal anaesthesia device  applying 3% isoflurane , 0.4 L/min O68Ga]Ga-DOTAGA-cKNGRE 10 ± 1 days after tumour generation at a tumour bulk of approximately 150 mm3. MiniPET scans were acquired 90 min post radiotracer administration with the application of a MiniPET scanner . Isoflurane-based inhalation anaesthesia  was assured throughout the whole study employing the Tec3 Isoflurane Vaporizer small animal anaesthesia device . Three-dimensional ordered-subsets expectation-maximization line-of-response (3D OSEM-LOR) image reconstruction was performed to provide satisfactory PET images for interpretation. To determine the following uptake parameters, volume of interests (VOIs) were deposited around selected organs and tissues with the BrainCAD image analysis software: standardised uptake value (SUV), SUVmean and SUVmax. Calculated by the following formula, SUV was used as a relative measure of radiopharmaceutical accumulation: SUV = [VOI activity (Bq/mL)]/[injected activity (Bq)/animal weight (g)]. The average radioactivity within the VOI is reflected by the SUVmean value, while SUVmax refers to the highest radiotracer concentration of the region concerned.CB17 SCID mice bearing a HT1080 fibrosarcoma in their left shoulder area were intravenously (i.v.) injected with 9.36 ± 1.24 MBq [213Bi]Bi-DOTAGA-cKNGRE-treated group . Approximately 5 MBq [213Bi]Bi-DOTAGA-cKNGRE was intraperitoneally (i.p.) administered to the experimental mice of the treated cohort on the 7th, 8th, 10th and 12th experimental days. The control mice were i.p. injected with 150 μL saline solution applying the same time regime. Seven days after HT1080 cell inoculation the tumour-bearing animals were randomly classified into two subgroups: treatment-naïve group  and [213Bi]Bi-DOTAGA-cKNGRE, ex vivo investigations were performed. The HT1080 fibrosarcoma-bearing experimental mice were anaesthetised with Forane (5%) and were sacrificed at 30 and 90 min post administration of the radiopharmaceutical. After autopsy, selected tissues, organs and the tumour itself were removed, weighed wet and their radioactivity was measured on a calibrated gamma counter . Then, the activity was decay corrected to the time of the injection. The percent of administered dose per gram of tissue (%ID/g) was determined for all samples from the counts per minute values (CPM). We presented the ex vivo data as mean %ID/g ± SD.To assess the organ distribution of Ga-DOTAGA-cKNGRE 10 ± 1 days after s.c. tumour induction. Within the framework of the in vivo studies, the tumour targeting potential of the CD13-affine 68Ga-tagged peptide probe was also assessed. mean and SUVmax—as quantitative uptake parameters—were registered as well  was strengthened in CD13 overexpressing ES2 ovarian cancer bearing nude mice by in vivo microPET imaging Ga-NOTA-c(NGR) [68Ga]Ga-NOTA-c(NGR) to ANP/CD13 pos. ortho and heterotopic transplanted Ne/De tumours was observed Bi-DOTAGA- cKNGRE. The treatment-naïve study mice i.p. received 150 μL saline solution on the same experimental days. In a bid to assess the therapeutic efficacy of the investigated probe, body weights  and tumour volume (mm3 ± SD) of the small animals were registered. Calliper measurements were applied to evaluate tumour development.On the 7th, 8th, 10th and 12th days of the investigation at an average tumour volume of 26.56 ± 2.39 mmp ≤ 0.05). The BW in the untreated cohort was 19.84 ± 1.24, 19.99 ± 1.35, 18.82 ± 1.11, 18.31 ± 0.97 recorded on the 7th, 8th, 10th and 12th days, respectively. As for the [213Bi]Bi-DOTAGA- cKNGRE-treated subclass the following values were obtained 7, 8, 10 and 12 days after the tumour cell inoculation: 20.12 ± 1.04, 20.93 ± 0.85, 19.98 ± 0.97 and 19.11 ± 1.07, respectively. Since the weight of the tumourous animals remained relatively stable throughout the whole examination period, we may presuppose that the currently applied [213Bi]Bi-DOTAGA-cKNGRE treatment exerts no considerable effect either on tumour weight or BW. Hence, this alpha-emitting NGR-based anti-tumour molecule may constitute a novel, safe therapeutic alternative in the existing treatment avenue of fibrosarcoma. As the evolution of BW is dependent upon a myriad of factors; however, additional long-term studies are warranted to confirm our hypothesis.Rigorous measurements of BW took place on the 7th, 8th, 10th and 12th experimental days. As demonstrated on the follow-up curve of 3 measured on the 12th day in the untreated control and the [213Bi]Bi-DOTAGA-cKNGRE treated subgroups, respectively. We determined the subsequent tumour volumes in the treatment-naïve cohort: 24.88 ± 3.15 mm3 (on day 7), 40.67 ± 5.98 mm3 (on day 8), 63.00 ± 11.53 mm3 (on day 9), 155.43 ± 20.47 mm3 (on day 10) and 204.43 ± 28.36 mm3 (on day 12), whereas as for the treatment-administered subcategory tumour bulks of 28.25 ± 2.67, 23.33 ± 6.87, 35.36 ± 7.57, 58.33 ± 14.36 and 112.50 ± 18.42 mm3 measured on the 7th, 8th, 9th, 10th and 12th days, respectively represented continuous size incrementing.Tumour volumes of both small animal subgroups were identified and recorded on the 7th, 8th, 9th, 10th and 12th days of the investigation. The follow-up curves representing tumour progression are presented in p ≤ 0.01). Albeit, no remarkable disproportion was observed between the tumour progression of the two groups on the remaining experimental days . As no significant alterations of tumour bulk were assessed during the administration of the first two therapies, we may draw the conclusion that at least two treatment regimes are necessary to induce measurable changes regarding tumour growth. Given the relatively permanent weight of the tumourous animals during the investigation and the experienced remarkable difference between the tumour volume of the treated and the untreated mice on the 9th, 10th, and 12th experimental days, we may draw the conclusion that the reduction of the tumour mass was not as significant in terms of affecting the BW of the study animals. In addition, the fact that the consistency of the tumours might become more compact may underpin why the meaningful tumour size reduction did not impact tumour weight. Taking into account the above remarked findings, application of [213Bi]Bi-DOTAGA-cKNGRE appears to be an efficient tool in targeted fibrosarcoma treatment.A statistically meaningful disparity was observed between the tumour volumes of the untreated and the treated mice on the 9th, 10th and 12th days post-tumour cell implantation  also confirmed outstanding renal accumulation in a study evaluating the tumour-targeting potential of the mentioned probe in HT1080 fibrosarcoma with high APN density and CD13-negative HT-29 human colon adenocarcinoma-bearing mice an hour after the administration of 370 kBq of [68Ga]Ga-NOTA-G3-NGR Bi-DOTAGA-cKNGRE injection, respectively and 0.06 ± 0.02, 0.08 ± 0.05, 0.17 ± 0.24 and 0.08 ± 0.03 90-min uptake values, respectively. Similarly, modest muscular and osseous [68Ga]Ga-NOTA-G3-NGR tracer accumulation was recorded in a former biodistribution study conducted by Shao and co-workers Ga-NODAGA-NGR and [68Ga]Ga-NOTA-(NGR)2—also encountered fast elimination from the blood pool Cu-NOTA-RGD-NGR between a KCH genetically engineered mouse model bearing integrin αVβ3 and CD13pos. pancreatic ductal adenocarcinoma (PDAC) and healthy control counterparts with normal pancreas revealed insignificant radioactivity in the tumour naïve pancreas of the control cohort [213Bi]Bi-DOTAGA-cKNGRE accumulation in the lungs. Relatively high activities of the lungs with uptake figures of 2.09 ± 0.74 and 0.71 ± 0.44 measured 30 and 90 min p.i., respectively, refer to the pulmonary retention of the labelled probe. Based on the research findings of Hajdu et al. we may suppose that the prolonged dissemination of the hydrophilic [213Bi]Bi-DOTAGA-cKNGRE in pulmonary chambers with water content may underlie this notable tracer uptake Ga-NOTA-G3-NGR with a comparable contrast level to that of ours investigating CD13pos. HT1080 fibrosarcoma-bearing nude mice with static microPET imaging ethylenediamine-N,N′-diacetic acid (HBED-CC) in CD13pos. A549  and HT1080 preclinical tumour models Bi-DOTAGA-cKNGRE in HT1080 oncological models, it would be a promising therapeutic drug candidate for incorporation into standard-of-care fibrosarcoma treatment protocols. Beyond therapeutic purposes, concomitant gamma radiation of 213Bi together with satisfactory image quality make the radiometal applicable for diagnostic usage as well.Since the overexpression of pro-angiogenic cell surface molecules such as CD13 on neoplastic as well as tumour vascular endothelial cells has a central role in cancer-related neo-angiogenesis, radiolabelled APN peptide-targeted ligands could represent a landmark in directed anti-tumour treatment . Given t68Ga]Ga-DOTAGA-cKNGRE to APN/CD13-overexpressing neoplasms, this PET imaging probe serves as a silver bullet for the precise identification of receptor pos. primary malignancies as well as pertinent metastases. Our results fuel the view that alpha-emitting 213Bi-tagged, APN-targeting NGR-motif—[213Bi]Bi-DOTAGA-cKNGRE—represents a breakthrough in current oncological therapies, leading to the ultimate goal of the establishment of bespoke, directed theranostic cancer treatment in the foreseeable future.Given the adequate binding competence of [


N-acetylglucosamine transferase (GnTase). In humans, mutations in ALG13 or ALG14 lead to severe neurological disorders with a multisystem phenotype, known as ALG13/14-CDG . How these mutations relate to disease is unknown because to date, a reliable GnTase assay for studying the ALG13/14 complex is lacking. Here we describe the development of a liquid chromatography/mass spectrometry-based quantitative GnTase assay using chemically synthesized GlcNAc-pyrophosphate-dolichol as the acceptor and purified human ALG13/14 dimeric enzyme. This assay enabled us to demonstrate that in contrast to the literature, only the shorter human ALG13 isoform 2, but not the longer isoform 1 forms a functional complex with ALG14 that participates in LLO synthesis. The longer ALG13 isoform 1 does not form a complex with ALG14 and therefore lacks GnTase activity. Importantly, we further established a quantitative assay for GnTase activities of ALG13- and ALG14-CDG variant alleles, demonstrating that GnTase deficiency is the cause of ALG13/14-CDG phenotypes.The second step of eukaryotic lipid-linked oligosaccharide (LLO) biosynthesis is catalyzed by the conserved ALG13/ALG14 heterodimeric UDP- Asparagine (N)-linked glycosylation is a posttranslational modification that directly affects the structure and function of glycoproteins, thus influencing a multitude of essential biological processes . ProteinALG genes are required for N-glycosylation in the ER (ALG13 and ALG14 are unique in that the enzymes they encode interact to form a heterodimeric UDP-N-acetylglucosamine transferase (GnTase). Unlike other ALG GTases which may function alone, ALG13/14 GnTase only possesses activity when they interact with each other (N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to GlcNAc-pyrophosphate-dolichol (Gn-PDol) (S. cerevisiae. Yeast Alg13 (yAlg13) is a small, soluble cytosolic protein that contains the GnTase catalytic subunit, while Alg14 (yAlg14) is a membrane protein that recruits soluble yAlg13 to the cytosolic face of the ER. Both yAlg13 and yAlg14 are essential for viability -based techniques have been used for developing in vitro quantitative assays to study the enzymatic activity of ALG GTases (Fourteen n the ER . Among tch other . This diGn-PDol)  (Bickel Gn-PDol) . ALG13/1iability  and yAlgiability . BecauseG GTases , but as https://www.uniprot.org-ALG13, identifier: Q9NP73). Among them, isoform 1 (ALG13-iso1) and 2 (ALG13-iso2) are found in all mammals. ALG13-iso1 is the longest isoform of ALG13, consisting of 1,137 amino acids, while ALG13-iso2 is shorter, consisting of 165 amino acids. This short ALG13-iso2 shows more than 30% sequence similarity with animal, fungi and plant   (Pathogenic mutations in human hy (DEE) , while Ame (CMS) . Given tme (CMS) . Thus diE. coli and purified recombinant ALG13/14 complex from a membrane fraction. Using chemically synthesized Gn-PDol as acceptor substrate, we confirmed the GnTase activity of recombinant ALG13/14. We used this purified enzyme to study the kinetic properties of ALG13/14 GnTase with an in vitro quantitative assay utilizing LC–MS. Furthermore, this in vitro assay system was used to compare the GnTase activities of the different human ALG13 isoforms. Our results confirmed that ALG13-iso2, but not ALG13-iso1, forms a dimeric complex with ALG14 on the cytosolic face of the ER membrane and contributes to GnTase activity for LLO synthesis. We also applied this assay to demonstrate the GnTase activities of ALG13- and ALG14-CDG variants. These variants display severe enzymatic defects in vitro, thus providing evidence for a genetic link between GnTase activity and ALG13-related DEE or ALG14-related CMS.In this work, we co-expressed human ALG13 and ALG14 in The plasmids used in this study and their important features are described in 2 atmosphere at 37°C.Human embryonic kidney cells, HEK293, were cultured in DMEM supplemented with 10% FBS , penicillin and streptomycin (1 μg/ml) in a humidified 5% COE. coli Rosetta cells  harboring both pET26b-pelB-FLAG-ALG13-iso2 and pCDFDuet-6His-ALG14 plasmids were cultured in 200 ml of Terrific-Broth medium  at 37°C to an OD600 of 1.0 and then cooled to 16°C. After adding 0.1 mM isopropyl-β-d-thiogalactopyranoside , the cells were incubated for additional 18 h–24 h at 16°C to induce protein expression. The harvested cells were resuspended in 15 ml lysis buffer  and homogenized by sonication. The cell lysate was centrifuged at 4,000×g to remove debris, and the supernatant was collected (this supernatant was used as the “crude extract”). To obtain the membrane fraction containing ALG13/14 complex, the crude extract was further centrifuged at 12,000×g to collect precipitated membranes. Any remaining cytosolic ALG13 was removed by washing the membranes with 15 ml lysis buffer three times. This washed membrane fraction was then solubilized by resuspension in 5 ml lysis buffer containing 1% Triton X-100 and incubated on ice for 15 min to obtain the detergent extract. This detergent extract was used for purification of the recombinant ALG13/14 complex following the procedure described below. This detergent extract was also used for the co-immunoprecipitation experiment presented in E. coli cells co-expressing FLAG-ALG13-iso2 and His-ALG14 was used to purify the recombinant ALG13/14 complex. First, His-tagged ALG14 proteins, including the recombinant FLAG-ALG13-iso2/His-ALG14 complex in detergent extract, were trapped by a HisTrap HP affinity column . Their eluted solution was then incubated with anti-FLAG Affinity Matrix  to enrich the ALG13/14 complex and remove the free His-ALG14. After elution with FLAG peptide, the purified ALG13/14 complex was analyzed by native PAGE and SDS–PAGE followed by silver staining to confirm the purity. Native PAGE was performed using a Blue/Clear Native PAGE Electrophoresis Kit  according to the manufacturer’s instructions.The detergent extract prepared from the HEK293 cells were seeded in 6-well plate and transfection was performed when the cell density was 80%–90%. Transfection was performed using Lipofectamine 2,000  according to the manufacturer’s instructions. 4 μg plasmid  and 5 μL lipofectamine 2000 were diluted using 250 μl OPTI-MEM respectively, after incubated at room temperature for 5 min, then mixed and incubated for another 20 min. The mixed reagent was added in HEK293 cells which grown in 2 ml DMEM medium with 10% FBS. Changed the mixture to DMEM medium with 10% FBS after 8 h and harvested the cell after 72 h.6 cells were solubilized in HEPES buffer with 1% NP-40  and incubated on ice for 30 min. Cell lysates were centrifuged at 15,000×g for 10 min at 4°C. The resulting supernatants were collected as the protein extracts. To prepare the membrane fraction which contain endogenous ALG13/14, HEK293 cells (1 × 106 cells) were resuspended in 1 ml of hypotonic buffer , followed by incubation on ice for 30 min. Cells were then homogenized by passing them 36 times through a homogenizer. Nuclei and cell debris were pelleted by centrifugation at 2,500×g for 5 min at 4°C. The supernatant was collected and centrifuged again at 12,000×g for 30 min at 4°C. The obtained membrane fraction  was used as the enzyme fraction of ALG13/14 GnTase in studies that characterized the suitability of synthetic Gn-PDol (C95) as substrate, as presented in To prepare the protein extract, 1 × 10E. coli co-expressing FLAG-ALG13-iso2 and 6His-ALG14 were cultured, and crude extracts were prepared as described above. Ten micrograms of this crude extract was separated by 12% SDS–PAGE analysis and transferred to a PVDF membrane . Immunoblotting was performed with an anti-His mouse antibody  and anti-FLAG mouse monoclonal antibody  as the primary antibody and the anti-mouse IgG-horseradish peroxidase (HRP) conjugate  as the secondary antibody followed by chemiluminescence (ECL) detection . For co-immunoprecipitation, 6His-ALG14 or FLAG-ALG13-iso2 was immunoprecipitated with Ni-NTA His binding resin  or anti-FLAG Affinity Matrix  as described elsewhere  and anti-HA rabbit polyclonal antibody  as the primary antibody and the anti-mouse IgG-HRP conjugate or the anti-rabbit IgG-HRP conjugate  as the secondary antibody. Immunoreactive bands were visualized by ECL. For co-immunoprecipitation assays, protein extracts were immunoprecipitated with anti-HA rabbit polyclonal antibody followed by incubation with 40 μl Protein A + G Agarose  for 3 h to pull down the HA-tagged ALG13-iso1 or ALG13-iso2 protein. After washing 3 times with HEPES buffer, the immunoprecipitate was solubilized, separated by SDS-PAGE and analyzed by western blotting with anti-FLAG mouse monoclonal antibody to detect the co-immunoprecipitated FLAG-ALG14.6 cells) expressing HA-ALG13-iso1 or HA-ALG13-iso2 were harvested and the protein extract was prepared as described above. The protein extract was incubated with anti-HA rabbit polyclonal antibody and 40 μl Protein A + G Agarose for 4 h to enrich the HA-tagged ALG13-iso1 or ALG13-iso2 protein. After washing the agarose by PBS for 3 times, the agarose-bound immunoprecipitated proteins were divided into two parts. One part was immunoblotted with anti-ALG13 rabbit polyclonal antibody  to detect HA-ALG13 while the other part was employed to test the GnTase activity.For the purification of ALG13-iso1 and ALG13-iso2, HEK293 cells  and protein. After incubation at 37°C, the reaction was stopped by heating to 100°C. The specific amount of protein included and reaction times are described in detail in each section. The donor concentration using 2 mM, which was 50 folds than the substrate Gn-PDol concentration. ALG13/14 can only transfer one GlcNAc from UDP-GlcNAc to Gn-PDol, so the donor concentration was excess which could facilitate the reaction.After terminating the reaction, glycans were released by adding 50 μl of HCl (40 mM) to the reaction and shifting the temperature to 100°C for 1 h. The hydrolyzed glycans were purified as described . The des2+, Mn2+ and Mg2+, respectively) or 10 mM EDTA for depletion conditions. The apparent Km and kcat values for Gn-PDol (20 μM–100 μM) were calculated by nonlinear regression curve fitting . The donor specificity of ALG13/14 was confirmed using different donor  with same concentration (2 mM).Enzymatic assays were performed using purified protein with the final concentration as 0.3 ng/μl  or 0.2 nThe stereochemistry of the linkage between two GlcNAc residues in Gn2-PDol (C95), the product of ALG13/14 GnTase, was confirmed by using yeast Alg1 (yAlg1) MTase. Purified recombinant His-yAlg1ΔTM and GDP-Man were added to the recombinant ALG13/14 reaction system, followed by 1 h of incubation at 30°C. The products were analyzed by LC–MS as described above.∆alg13::His+5; ∆alg14::Kan+ and GAL1/10pr-3HA-yALG14/yALG13-FLAG::trp1), contains a deletion of ALG13 and ALG14 in the chromosome and a GAL1/10 promoter-driven yALG13 and yALG14. This strain was used to test for the ability of human ALG13 different isoforms and ALG14 to complement growth in the presence of glucose. The URA3-marked YEp352GAPII-3HA-ALG13-iso1 or YEp352GAPII-3HA-ALG13-iso2 plasmid was co-transformed with the LEU2-marked YEp351GAPII-ALG14-FLAG into XGY186 and selected on SG (-Ura-Leu) plates. Transformants were cultured in medium containing glucose, followed by growth on solid YPA medium  supplemented with 2% glucose (YPAD) or 2% galactose (YPAG) at 30°C for 2 days.The yeast strain XGY186  value are provided in the figure, figure legend and table footnote. Kinetic parameters were determined by nonlinear regression fitted to Michaelis-Menten equation in GraphPad Prism 8.0.in vitro assay system for quantitatively detecting UDP-GlcNAc transferase (GnTase) activity. We previously described an LC–MS-based quantitative assay for LLO mannosyltransferases (The human ALG13/14 (ALG13/14) complex catalyzes the addition of a β1,4-linked GlcNAc to Gn-PDol to produce Gn2-PDol in the DLO synthetic pathway. We aimed to establish an sferases  and thissferases , ALG13/1sferases . A polypsferases . The pro+, and the mass peak at m/z 222.12  is a suitable substrate for ALG13/14 GnTase, the HEK293 cells membrane fraction  was prepared and applied to a standard reaction mixture with a final volume of 50 μl, in which Gn-PDol (C95) and UDP-GlcNAc were used as the acceptor and donor substrates . Hydrochloric acid was added to hydrolyze the saccharide moieties, followed by purification and detection using LC–MS for quantitative analysis . Two groz 222.12  was hydrGn2-PDol . The twoGn2-PDol . The conalg13 or alg14 mutants, when co-expressed, human ALG13 (ALG13-iso2) and ALG14 can rescue the growth defect caused by either the loss of yeast ALG13 or ALG14  cells. After induction with IPTG, the expression of both FLAG-ALG13-iso2 and His-ALG14 proteins was confirmed using anti-FLAG or anti-His antibodies  and UDP-GlcNAc for 1 h, followed by detection of Gn2 product using LC–MS. The Gn2 product can be detected as two peaks at retention times of 7.39 min and 7.78 min (+ (data not shown). When extracts prepared from two different E. coli strains , which individually expressed ALG13-iso2 and ALG14 were mixed and assayed for activity under the same conditions, no product peaks were detected  prepared from ALG13-iso2 and ALG14 genes in E. coli can form an enzymatically active GnTase complex.To further confirm the stereochemistry of the GlcNAc-GlcNAc linkage in the newly formed product, we asked if it could be recognized as a substrate by yAlg1, the mannosyltransferase that catalyzes the reaction following ALG13/14 GnTase. yAlg1, adds β1,4-Man to GlcNAcGlcNAc-PDol (Gn2-PDol) with strict substrate specificity . To testE. coli harboring both pET26b-pelB-FLAG-ALG13-iso2 and pCDFduet-6His-ALG14 plasmids was purified as described in “Material and methods”. The purified protein was calculated as 2.6 mg/ml (0.1 ml), the concentration exhibiting a 500-fold compared with E. coli membrane extract. Native-PAGE analysis, in which the complex remains associated, revealed a single band with a molecular weight of approximately 45 kDa–66 kDa, while denaturing SDS–PAGE analysis revealed two bands, the individual ALG13-iso2 (25 kDa) and ALG14 (22 kDa) proteins . As shown in e extent . These rs–Menten .As the canonical human isoform, the longer ALG13-iso1 contains not only an N-terminal glycosyltransferase 28 domain, but also several other domains including an ovarian tumor deubiquitinase domain . Comparein vitro (6 cells) expressing HA-ALG13-iso1 were harvested and resuspended in 300 μl of HEPES buffer containing 1% Triton X-100 . After incubation for 30 min on ice to solubilize membrane proteins, HA-ALG13-iso1 was immunoprecipitated from the cell lysate with anti-HA antibody and Protein A + G Agarose beads. The collected agarose beads were resuspended in 40 μL of reaction buffer. One half of this mixture (20 µl) was incubated with our standard reaction mixture to detect GnTase activity while the other half was subjected to SDS–PAGE analysis and immunoblotted with anti-ALG13 rabbit antibodies to confirm the expression and enrichment of HA-ALG13-iso1 proteins (data not shown). In parallel, HA-ALG13-iso2 was also expressed in HEK 293 cells and immunoprecipitated with anti-HA agarose , which can be shut off by shifting to growth in medium containing glucose. To determine if ALG13-iso1 and ALG14 have in vivo activity, we measured their ability to rescue growth of this yeast strain in the presence of galactose versus glucose. As shown in in vitro results  ALG13 + WT ALG14, WT ALG13 + mutant ALG14 P65L or mutant ALG13 N107S and WT ALG14 . Membrane fractions containing the ALG13 N107S/ALG14 or ALG13/ALG14 P65L complex were collected and solubilized in 5 ml lysis buffer with 1% Triton X-100 . These detergent extracts were analyzed with anti-FLAG or anti-His antibodies to detect the expression of the ALG13/ALG14 complex  for ALG13 and ALG14. In this study, we tried to purify the active ALG13/14 complex from the E. coli membrane fraction by co-expression of ALG13 and ALG14. In contrast, GnTase activity was not detected by simply mixing the individual recombinant ALG13 and ALG14 proteins GlcNAc-PP-Dol (Gn2-PDol) production while the long isoform 1, which failed to interact with ALG14 and therefore lacked GnTase activity . These findings further support our conclusion that isoform 2 is the only ALG13 isoform required for N-glycosylation. This raises the question of what is the function of ALG13-iso1. Besides containing the N-terminal GnTase catalytic domain, human ALG13-iso1 also contains an ovarian tumor deubiquitinase domain with predicted active sites in its C-terminal region. It has been suggested that ALG13 ovarian tumor deubiquitinase domain may catalyze ubiquitination of certain protein targets, such as GTases in the LLO pathway  acceptor (in vitro assay we describe will open new avenues for better understanding N-glycosylation and its relationship to the pathogenicity and clinical severity of CDG.The ynthesis . Expressacceptor . By omitacceptor . Our semacceptor ; Table 2


One of the most important functions of the oral fluid is to maintain oral homeostasis. In-office teeth whitening systems are able to change the mineral metabolism and the activity of a number of enzymes in the oral fluid, but there are conflicting data in publications about this. The aim of this study was to compare the effect of Opalescense Boost, ZOOM Advance POWER, and ZOOM Phillips White Speed, which contain different percentages of hydrogen peroxide, on the performance of oral fluid. After the procedure of whitening teeth with the studied in-office systems, the concentration of calcium in the oral fluid increased, and the activity of alkaline phosphatase decreased. Calcium levels returned to baseline values after 30 days, and alkaline phosphatase activity returned after 14 days. There was no significant difference in the changes in calcium concentration and alkaline phosphatase activity between different tooth whitening systems. Chemical teeth whitening with the Opalescense Boost system caused the largest change in the activity of superoxide dismutase in the oral fluid compared to the ZOOM Advance POWER and ZOOM Phillips White Speed photocatalytic teeth whitening systems. An increase in the activity of superoxide dismutase by +75.5% was shown immediately after the procedure of teeth whitening with the Opalescense Boost system, which indicated an increase in the power of antioxidant defense mechanisms. To assess the effectiveness and safety of using various whitening systems, it is possible to study the dynamics of the activity of superoxide dismutase, which reflects the processes of antioxidant protection of the oral cavity. Oral fluid or mixed saliva is a secretion product of the salivary glands in the composition of epithelial cells, leukocytes, microorganisms, micro and gingival fluid. Saliva itself is a complex of fluids produced by specific glands and relies on a system of ducts into the oral cavity. This biological fluid has a role in diagnosing and monitoring the patient’s health. Oral fluid plays an important role in maintaining the homeostasis of the oral cavity: it takes part in the mineralization of the hard tissues of the tooth, exhibits antioxidant functions, reduces the activity of microorganisms in the oral cavity, and participates in moisturizing the oral mucosa. Almost all the chemical elements of the periodic table are found in human saliva, but calcium and phosphorus are of paramount importance, since they maintain the optimal composition of the components of dental tissue.Oral fluid is the least studied and most underestimated biological environment in the human body which may have a diagnostic significance. According to the results of numerous studies, peroxide compounds that make up teeth whitening gels can affect not only the chemical composition of enamel but also the indicators of mineral metabolism and the activity of enzymes in the oral fluid ,2,3,4, bThe procedure of teeth whitening is carried rather often due to the great interest of patients in this type of procedure. It brings very good results taking into account the aesthetic aspects. However, teeth whitening also causes great concern to patients and dentists due to its potential harmful effects on tooth tissue and the biochemical environment in the mouth cavity ,6. The pThis study was conducted at the Department of Therapeutic Dentistry of Samara State Medical University. The study was approved by the ethics committee of Samara State Medical University . This protocol was compliant with the Helsinki Declaration of 1975, as revised in 2000. The protocol was explained to all participants, and after answering every question, they signed the informed consent form before their saliva samples were taken for analysis. The study involved 32 patients aged 18 to 32 years. The inclusion criteria were the following: enamel pigmentation; aesthetic preferences of a patient; tetracycline-stained teeth; the dashed, spotty, chalky-mottled form of generalized fluorosis of the teeth; age-related changes of enamel color; and local enamel hypoplasia of mild severity. The exclusion criteria were the following: childhood and adolescence; enlarged dental pulp chamber; periodontal tissue diseases; the presence of orthopedic and/or orthodontic appliances; all types and stages of caries; hyperesthesia; the presence of tumors, bronchial asthma, and uncontrolled increase of blood pressure. Before the start of the whitening procedure, the oral cavity was sanitized. Three systems of teeth whitening were used—chemical whitening with the Opalescense Boost system , light catalytic whitening ZOOM Advance POWER (ZOOM 3) , and light catalytic whitening ZOOM Phillips White Speed (ZOOM 4) . Chemical whitening with the Opalescense Boost system was carried out on the basis of a gel with 40% hydrogen peroxide content. The teeth whitening system ZOOM Advance POWER included a gel based on 25% hydrogen peroxide activated by a halogen lamp emitting light in the range of 350–400 nm. Teeth whitening with the ZOOM Phillips White Speed (ZOOM 4) system was also carried out on the basis of a gel with 25% hydrogen peroxide, which was activated by an LED lamp operating in the range of 400–505 nm. Chemical teeth whitening was performed using the Opalescence Boost 40% (Ultradent) system, based on 40% hydrogen peroxide. The whitening gel was applied to the surface of the teeth three times, the exposure was 20 min.In catalytic bleaching, teeth whitening using systems ZOOM Advance Power and Zoom Phillips White Speed, based on which a 25% hydrogen peroxide whitening gel was applied to the surface of the teeth three times, the exposure time was 15 min. All patients were divided into 3 groups depending on the use of the whitening system. Patients were allowed to choose the bleaching system based on their personal preferences as to date there is no difference in the criteria of their use. Teeth whitening with the Opalescense Boost system was performed in the first group of patients (8 people), teeth whitening with the ZOOM Advance POWER system was performed in the second group of patients (8 people), and teeth whitening with the ZOOM Phillips White Speed system was performed in the third group of patients (16 people). The ZOOM 4 whitening system is the most popular today. The main difference between ZOOM 4 and ZOOM 3 is that it is considered to have a gentler effect on the teeth. A safer whitening by the ZOOM 4 system was a key factor why it was chosen by 50% of the study group.g for 5 min in an Eppendorf MiniSpin centrifuge.Oral fluid samples were taken before the tooth whitening procedure, immediately after, after 2 weeks, and after a month. Before sampling the oral fluid, all patients rinsed their mouths with drinking water. Samples were collected 30 min after a meal. Collection of oral fluid was carried out in a sterile test tube, followed by freezing at a temperature of −20 °C. The next step was defrosting the test tubes for 10 h at a temperature of +3 °C; after defrosting, the temperature of the samples was brought to room temperature. Afterwards, the oral fluid was aliquoted into Eppendorf tubes and centrifuged at 5000× The determination of indicators of the oral fluid was carried out on an Aquarius CE 7200 spectrophotometer . The concentrations of calcium, phosphorus, magnesium, and iron and the activities of acid phosphatase (EC 3.1.3.2), alkaline phosphatase (EC 3.1.3.1), and superoxide dismutase (EC 1.15.1.1) were determined. The calcium concentration was determined by the photometric method with o-cresolphthalein complexone , the magThe determination of the activity of phosphatases in the oral fluid was carried out using ready-made kits from Human . The determination of the activity of alkaline phosphatase was carried out by an optimized standard method according to the recommendation of the German Association for Clinical Chemistry with p-nitrophenyl phosphate  and in tThe statistical data analysis was performed using SPSS 25 software . Descriptive statistics used M—mean value, m—standard error of the mean, and Δ%—difference in percentage of sample mean values (Mtest/Mcontrol) × 100%).p < 0.05.Comparisons of independent groups were performed using a Kruskal–Wallis analysis (Pk–W) followed by pairwise comparisons using the Mann–Whitney test. Differences were considered statistically significant at values The study of the effect of tooth whitening systems showed that the concentration of some indicators of mineral metabolism and the activity of enzymes changed in the oral fluid. The concentration of calcium in the oral fluid is less than that in the blood serum and is normally 0.75–3.00 mM . In the Calcium levels returned to baseline values after 30 days. There was no significant difference in the changes in calcium concentration after the tooth whitening procedure between the patient groups.p = 0.026), and the phosphorus concentration returned to the initial value after 14 days. The ratio of calcium/phosphorus (Ca/P) in the oral fluid increased immediately after the tooth whitening procedure and returned to baseline values after 14 days, reflecting the change in the content of calcium and phosphorus in the oral fluid.The phosphorus content in the oral fluid reached 2.2–6.5 mM. In the third group of patients, the phosphorus concentration before the tooth whitening procedure was 4.31 ± 0.13 mM. There was a decrease in the concentration of phosphorus immediately after the tooth whitening procedure to 3.69 ± 0.22 , acid phosphatase (EU/L), and superoxide dismutase in the oral fluid were determined at different periods of observation. The activity of alkaline phosphatase significantly decreased after the whitening procedure in all three groups of patients and returned to baseline values after 14 days .Acid phosphatase activity (EU/L) remained unchanged in the three groups of patients. For Opalescense Boost, the indicator value before whitening was 0.47 ± 0.09 EU/L, and 30 days after whitening, it was 0.47 ± 0.06 EU/L. For ZOOM Advance POWER (ZOOM 3), the initial value was 0.50 ± 0.09 EU/L, while 30 days after whitening, the value was 0.50 ± 0.08 EU/L. For ZOOM Phillips White Speed (ZOOM 4), the baseline value before whitening was 0.48 ± 0.06 EU/L and the final value after 30 days was 0.48 ± 0.05 EU/L.In the control , a gradual increase in extinction was noted as a consequence of the formation of adrenochrome in the process of adrenaline autooxidation. In experimental tests , the increase in extinction slowed down as a consequence of the inhibition of adrenaline autooxidation under the action of superoxide dismutase. The SOD activity in the oral fluid in the three study groups increased immediately after the tooth whitening procedure; after 14 days, a significant change in SOD activity was preserved. The enzyme activity returned to its initial values after 30 days .During tooth whitening, hydrogen peroxide diffuses into the tooth enamel and dissociates to form free radicals, such as hydroxyl radicals, perhydroxyl radicals, perhydroxyl anions, and superoxide anions, which attack organic pigmented molecules . Some stAll in-office tooth whitening systems tested produced changes in various parameters of the oral fluid. The results obtained indicate an increase in the concentration of calcium in the oral fluid, which occurs as a result of the removal of ions from the enamel of the teeth. The concentration of phosphorus in the oral fluid did not change after the tooth whitening procedure. The calcium/phosphorus ratio is an important indicator of the process of remineralization of dental hard tissues. The results obtained show a change in the Ca/P ratio after the tooth whitening procedure toward an increase due to an increase in the concentration of calcium in the oral fluid. The Ca/P ratio returned to the initial values after a month, which is a favorable prognostic sign of tooth remineralization. According to Dourado P. et al., the use of teeth whitening systems with hydrogen peroxide leads to the leaching of calcium and phosphorus from the enamel of the teeth .There was a tendency toward an increase in the concentration of magnesium in the oral fluid in all groups of patients immediately after the tooth whitening procedure; the concentration of iron after tooth whitening in the study groups did not change at different periods of observation. In studies by Cakir F.Y. et al., it was shown that magnesium was one of the first elements removed during the reaction of peroxide from the tooth surface . The forAcid and alkaline phosphatases function to maintain the level of phosphorus in saliva. The activity of phosphatases in the oral fluid increases, as a rule, in inflammatory processes of the oral cavity. In the research of Sanches, it was shown that professional teeth whitening increased the activity of alkaline phosphatase, lactate dehydrogenase, aspartate, and alanine aminotransferases in the oral fluid, thereby affecting the homeostasis of the oral cavity by reducing the remineralizing potential of the oral fluid . Soares The study of the activity of alkaline phosphatase in the oral fluid after the tooth whitening procedure showed that the activity of alkaline phosphatase decreased slightly in the three groups of patients immediately after using the tooth whitening procedure and recovered after 14 days. No intergroup differences were found.p = 0.036) and between groups 1 and 3 (p = 0.02). The Opalescense Boost teeth whitening system caused an increase in superoxide dismutase activity by +75.5%, which indicated an increase in the power of antioxidant defense mechanisms. It was shown that chemical teeth whitening with the Opalescense Boost system had the greatest effect on SOD activity in comparison with the ZOOM Advance POWER and ZOOM Phillips White Speed systems.The system of antioxidant protection of the oral cavity includes superoxide dismutase. The mechanism of tooth whitening is based on the ability of hydrogen peroxide to generate free radicals, which causes an increase in the activity of superoxide dismutase in the oral fluid after the tooth whitening procedure. There was a significant increase in SOD activity after tooth whitening and a gradual return to baseline values after 30 days. The SOD activity in the oral fluid immediately after the bleaching procedure and 14 days after the bleaching procedure was the highest in the first group of patients. This whitening system uses a gel with 40% hydrogen peroxide content, and the lowest in the third group of patients. This whitening system uses gel with a 25% hydrogen peroxide content. Comparisons of independent groups using a Kruskal–Wallis one-way analysis of variance showed significant differences between groups 1 and 2 (There was no significant difference in oral fluid indicators when using various bleaching systems, with the exception of superoxide dismutase activity. To assess the effectiveness and safety of various whitening systems, it is possible to study the dynamics of the activity of superoxide dismutase, which reflects the processes of antioxidant protection of the oral cavity.The results obtained confirmed that teeth whitening procedures lead to calcium leaching from tooth enamel; however, these changes are reversible and can be eliminated by the use of remineralizing toothpastes.All teeth whitening systems cause an increase in the concentration of calcium in the oral fluid and a gradual return to the initial values after 30 days. The activity of alkaline phosphatase decreased in patients after using the teeth whitening procedure and recovered after 14 days in three study groups. There was a significant increase in SOD activity after teeth whitening and a gradual return to baseline values after 30 days for all teeth whitening systems used. There was no significant difference in oral fluid parameters when using different bleaching systems, with the exception of the activity of superoxide dismutase. The Opalescense Boost teeth whitening system caused an increase in superoxide dis-mutase activity by +75.5%, which indicated an increase in the power of antioxidant defense mechanisms.


Overall, our results indicate that letrozole PEG-protamine NPs alter the release profile of letrozole, which could be an excellent approach for overcoming letrozole resistance in human breast cancer.The objective of the study was to develop PEGylated protamine letrozole nanoparticles to combat human breast cancer by modifying the release pattern of letrozole. Breast cancer is amongst the most prevalent diseases in women due to overactivity of human epidermal growth factor receptor 2 (HER2). PEG-protamine letrozole nanoparticle formulation was designed and optimized to alter the release pattern of the drug. The size, morphology, and structure of PEG-protamine letrozole NP were characterized by FTIR, XRD, Zetasizer, and SEM analysis. The result showed the PEG-protamine letrozole nanoparticles were irregular in shape and have size ranging from 258 nm to 388 nm, polydispersity index 0.114 to 0.45, zeta potential of 11.2 mV, and entrapment efficiency 89.93%. XRD studies have confirmed that the crystal structure of letrozole has become amorphous. The drug release study maintained the prolonged release for 72 hours. Moreover, the PEG-protamine letrozole NPs displayed a strong anticancer action compared to MCF-7 cells with an IC50 70  Overexpression of HER2 protein is associated with tumor aggression, prognosis, and reactivity to hormonal and cytotoxic agents in breast cancer patients . HER-2 a. Overexp pathway . Aromatandrogens . It is vndrogens . Letrozoynthesis . Severalynthesis , 8. Incoynthesis . Nanoparynthesis , 11. RecLetrozole was donated by Rotex Pharmaceutical Industry, Islamabad, Pakistan. Sodium tripolyphosphate protamine sulphate, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC-HCL) were purchased from Sigma.Protamine and PEG (0.1%) solution was formulated using purified water. NHS and EDC-HCL solutions were blended with the solution of PEG, and solution of protamine was dropwise added to the PEG solution. The subsequent mixture was stirred at a speed of 800 rpm for at least 5 hours. Later, that mixture was added to the dialysis membrane of molecular cutoff value 1000 Dalton and left for 72 h for dialysis. The dialyzed solution was lyophilized, and freeze-dried powder of PEG-protamine complex obtained.PEG-protamine complex (0.3%) solution was prepared. Letrozole 05 mg, 10 mg, and 15 mg were dissolved in ethanol. Sodium tripolyphosphate (TPP) 0.4% solution was prepared and added slowly and drop by drop with continuous shaking at a speed of 500 rpm leading to formation of PEG-protamine complex. Letrozole was then added dropwise at baseline temperature into this mixture. The solution was then shaken for at least 5 hours at a speed of 800 rpm and centrifuged at 12000 rpm at a temperature of 15°C. Settled particles were then gathered while removing the supernatants.−1 [The PEG-protamine complex was characterized by FTIR, in which the pallets were prepared by KBr (1 : 08) using a pellet press. The spectrum was collected and interpreted accordingly (FTIR-8400 Shimadzu). Similarly, PEG-carboxylate, letrozole, PEG-protamine complex, protamine sulphate, blank nanoparticles, and drug-loaded PEG-protamine letrozole NPs were also characterized by FTIR ; spectra were logged in the wavelength scale of 4500-400 cm−1 .μl/ml was prepared for particle size evaluation. Dried out nanoparticles were distributed in deionized water and were sonicated for a minimum of 5 min for the proper blending of mixture [The nanoparticle size and zeta potential of the formulations were determined using Malvern Zetasizer ZS . The average diameter and polydispersity index (PDI) of nanoparticles in a homogenized mixture were defined by using the dynamic light scattering (DLS) technique , 14. A s mixture .Purity and crystal-like properties of nanoparticle powder were assessed with the help of SmartLab SE XRD . The sample was filled in the sample holder, glass slide was exploited for the smoothing surface, while, for the scanning purpose, copper was utilized (ranging from 10 to 70) with diffractive meter and peaks were noted .The nanoparticle surface properties were examined by SEM. The nanoparticles were lyophilized and spread over the aluminum slice, which was covered with gold about 300 Å thick. Surface morphology and properties were reported for the sample analyzed at 10 kV .The quantity of letrozole in the formulation was determined indirectly. The LTZ quantity from the supernatants was based on the absorbance of sample 240 nm. A standard curve was drawn, and absorbance of the sample was measured in 5 ml quartz cuvettes using Shimadzu UV-1700 Pharma spec UV-visible spectroscopy . SamplesQ0–Qt= K0t, where K0 stands for the rate constant, Qt is the amount of drug released at time t, and Q0 is the initial amount of drug in nanoparticles. First-order kinetics can be expressed mathematically in DQ/dt = −K1Q, whereK1represents the first-order kinetic rate constant and Q = logQ0–k1t/2.303. The Higuchi model sets out the drug release process as a dependent process based on Fickian diffusion from the polymer matrix. The Korsmeyer-Peppas model describes the administration of the drug from polymeric substances obtained from a simple relationship, according to the value of “n” which determines the release of the drug from the polymeric matrix [Drug release form nanoparticles was conducted by utilizing dissolution apparatus II. Sample amounts of 5 ml were withdrawn from the dissolution apparatus and replaced by fresh buffer solution at intervals of 5 min, 10 min, 20 min, 40 min, 1 h, 2 h, 4 h, 6 h, and 8 h. Various models including Higuchi, Korsmeyer-Peppas, and zero-order and first-order kinetics were utilized for dissolution data analysis. Pharmacokinetic characteristics of the drug can be concluded generally utilizing the zero-order kinetics mathematically represented as c matrix .w/w) percentage was concluded from the weight of the lyophilized dried powder acquired from all batches. The retrieved nanoparticles were weighted and then divided by sum of the initial materials [Weight by weight YielThe dried nanoparticles were dispersed in 100% DMSO (dimethyl sulfoxide) in an amount of 20 mg/ml; working solution of all the samples was prepared in DMEM at a concentration of 2 mg/ml which was further serial diluted.H–tetrazolium bromide- (MTT-) based cell sustainability assay. Cells were cultured for 48 hours to achieve the desired densities of the cells and finally treated with different concentrations of letrozole and PEG-protamine letrozole NPs (10-140 nM). Control cells were treated with 0.1% dimethyl sulfoxide (DMSO). The plates were kept for 24 h in an incubator, set for cell culture. Media was removed, and cells were treated with 20 μg/mile end concentration of MTT prepared in the media. The plate was kept in a CO2 incubator at 37°C for 4 h, and then, 100 μl DMSO was added to solubilize formazan crystals. Absorbance reading was taken by using a microplate reader  at 570 nm and 630 nm. Background reading (630 nm) was subtracted from 570 nm reading. The whole experiment was performed in triplicate, and the results are calculated as percent inhibition following the formula%cytotoxicity = 100 − [absorbance of sample/absorbance of untreated control × 100).The nanoparticles were tested against MCF-7 (human breast adenocarcinoma cells) for their cytotoxic activity by utilizing dimethyl–2–thiazolyl–2,5–diphenyl–2−1, 1623.44 cm−1, and 2320.73 cm−1 representing the typical peaks of protamine . SEM images of the prepared nanoparticles showed irregularities in the shape and size, which is evident from R2 was larger than 0.5 which showed that the drug release observed first-order kinetics and drug discharge was reliant on the quantity of the drug, which means that if the drug quantity in the polymeric matrix is high, then a large amount of drug will be released from nanoparticle complex. Moreover, the Higuchi model and Korsmeyer papers indicated that drug release from the polymeric nanoparticles is based on diffusion. During our study, drug release was noted at different pH ranging from 6.8 to 7.4; specifically, 70.2-83.9% of drug release was recorded at pH 6.8. Similarly, after 72 h dissolution, the maximum percent release at pH 6.4 and 7.4 was 89.93% and 81.95%, respectively . After measuring the cell capability by the MTT assay, it was observed that both the letrozole and PEG-protamine letrozole NPs inhibited MCF-7 cellular viability in a dose-dependent manner. μM). The substantial cytotoxic effects were shown by one-way ANOVA at 50, 70, and 90 μM at p < 0.0001 with a cell sustainability inhibition of 50%, 40%, and 20% separately as related to the control. The measured value for IC50 was 70 μM for letrozole and 50 μM for PEG-protamine letrozole NPs, which clearly indicates the effectiveness PEG-protamine letrozole nanoparticles.The cytotoxicity of PEG-protamine letrozole nanoparticles were tested against the MCF-7 cells. The cells were subjected to treatment for at least 24 h, with varying quantities of letrozole and PEG-protamine letrozole NPs (10-140 50 in MCF-7 cells. Future research work will be carried out to explore the mechanism leading to increased formulation efficacy and detailed in vivo study, such as typical live/dead cell staining, cellular uptake, and histological examination.Current studies on the PEG-protamine letrozole formulation have revealed a significant IC50 was 70 μM for letrozole and 50 μM for PEG-protamine letrozole NPs.Letrozole has been prepared as a nanoformulation for which the method ionic gelation was adopted to integrate drug into the PEG-protamine complex with the help of a TPP connecting agent. The letrozole-loaded PEG-protamine nanoparticles exhibited a potent cytotoxic effect against MCF-7 cell lines. The calculated value for IC