Endometrial receptivity and its management in assisted reproduction is now a significant focus of research interest. Endometrial receptivity tests, which analyze different panels of gene expression, are usually offered in fertility clinics to determine the women's individual ‘window of implantation’, providing a personalized timing for embryo transfer. However, there are still no definite indications on whether its inclusion in the study of the infertile couple or the study of patients with repeated implantation failure is essential. Endometrial receptivity (ER) and its management in assisted reproduction technology (ART) is now a significant focus of research interest. ER tests (ERts), which analyze different panels of gene expression, are usually offered in fertility clinics to determine the women's individual “window of implantation” (WOI), providing a personalized timing for embryo transfer. However, there is still no definite indication of whether its inclusion in the study of the infertile couple or the study of patients with repeated implantation failure (IF) is essential.In this discussion article, based on a strengths, weaknesses, opportunities, and threats analysis, the different aspects of the application of ERt  in reproduction are evaluated following Oxford criteria (Oxford Centre for Evidence-Based Medicine).ERts were developed as an attempt to identify the optimal ER status (WOI) in a more precise way than traditional endometrial dating, not only reaching this objective (evidence 2b)The first study on ER was published 10 years ago using the first developed ERt, the ERA (evidence 2b).ERts have been suggested to improve cumulative pregnancy rate (cPR) as it was shown in a multicenter, prospective, randomized controlled trial (RCT) that included 458 patients undergoing their first and second IVF cycles, which compared the efficacy of performing the embryo transfer guided by either the results of the ERA test or according to the standard clinical care. No benefit was observed in the intention-to-treat analysis; however, when results were analyzed per protocol, the pregnancy rate (PR) and cPR at 12 months improved when guided by ERA (evidence 1b).Test-based technology is rapidly evolving. Microarray and polymerase chain reaction-based clinical tests are being replaced by next-generation sequencing, a more precise technique in quantifying global gene expression profiles and more comprehensive in coverage.Moreover, a remarkable strength in the application of these tests is that endometrial transcriptomics has proven more objective and concordant with ER than serum progesterone (P4) (evidence 2c),One of the main limitations of these tests might be the need for more solid evidence to support the intra- and intercycle reproducibility within the same patient. The human endometrium is a dynamic tissue that experiences molecular and morphologic modifications throughout the menstrual cycle. Because of this extensive intercycle variation in reproductive physiology, the receptive status of the endometrium when the test is performed might differ from that of the embryo transfer cycle. ERA is one of the few ERts that has demonstrated reproducibility in published data on 7 patients (evidence 4),However, regarding other ERts, there are data on the reproducibility of ERMap from a double analysis of biopsies in 29 patients showing good reproducibility (evidence 3b).The methodological heterogeneity  and the lack of agreement on the genes to be studied limit the standardization of ERt (evidence 2b).Another important limitation is that most ERt studies—at least those showing a positive result—are sponsored by the companies selling the test.,In addition, bearing in mind the published literature, there is still no demonstrated evidence regarding the usefulness of these tests in any specific group of patients with infertility. For example, in patients with RIF, pET using ERA does not seem to improve gestational outcomes compared with standard ET (evidence 2a).Of note, endometrial sampling for the ERt is considered to be an invasive procedure.Another limitation of the published studies is not considering embryo quality. Among all the available studies, none describes embryo policy regarding transfer, and there might be a possibility that receptivity tests perform differently in good vs poor embryo quality.Finally, the risk of overdiagnosis and overtreatment is also present as the transcriptomic signature of the endometrium may exhibit more monthly variability than previously reported, leading to inaccurate and harmful recommendations for progesterone exposure.Transcriptomics offers the opportunity to obtain further molecular information that permits distinguishing new patterns of endometrial pathology or processes, thereby providing new endometrial taxonomies (evidence 2b)The expansion of genes implicated in different biological terms  could favor its implementation and its use in a correct patient profile  where these tests might provide information that could be crucial.A Grading of Recommendations Assessment, Development, and Evaluations–based assessment recommended a careful study of ER in ART cycles, distinguishing between couples undergoing ART for the first time and couples suspected to have RIF, even in the presence of a morphologically normal uterus. Furthermore, developing appropriate guidelines specifying the indication of ERt is a priority, and the treatment offered should be evidence based and designed to improve ER.Personalized treatment for endometrial factors has taken off, altering the clinical practice of more than 4000 reproductive clinics in more than 90 countries worldwide (evidence 5).ERts are very limited in addressing other causes of IF and are considered a laboratory-developed test intended for use only in the laboratories in which they were created. In addition, most of the signatures were published for molecular investigation, but they have been translated to clinical practice with no previous validation (evidence 2b).P4 exposure has been assumed to be able to better or postpone the endometrial maturity by precisely 12-hour intervals.In addition, comorbidities may distort test results  as they may have negative effects on the individual WOI, leading to embryo-endometrial asynchrony; therefore, the diagnosis and treatment of these conditions, such as CE, should be performed before ERA testing (evidence 2b).Finally, although there is limited supporting evidence for this add-on treatment in IVF, some patients with infertility assume that they are not receiving the most advanced care if a test is not offered or performed. Therefore, there is substantial demand from patients, physicians, and the biotechnology industry (evidence 3b) with limited information regarding their regulation for clinical use and a significant ethical difficulty in validating these studies.Prevailing evidence does not support the use of ERt daily. Additional well-designed research is required to ascertain the efficacy of ERt before achieving wider usage.


Because it is a binary system, it is possible to determine how much the orbit of the satellite changed, as part of a test of what is necessary to deflect an asteroid that might threaten Earth with an impact. In nominal cases, pre-impact predictions of the orbital period reduction ranged from roughly 8.8 to 17 min (refs. 3). Here we report optical observations of Dimorphos before, during and after the impact, from a network of citizen scientists’ telescopes across the world. We find a maximum brightening of 2.29 ± 0.14 mag on impact. Didymos fades back to its pre-impact brightness over the course of 23.7 ± 0.7 days. We estimate lower limits on the mass contained in the ejecta, which was 0.3–0.5% Dimorphos’s mass depending on the dust size. We also observe a reddening of the ejecta on impact.On 26 September 2022, the Double Asteroid Redirection Test (DART) spacecraft struck Dimorphos, a satellite of the asteroid 65803 Didymos Optical observations of Dimorphos, a satellite of the asteroid 65803 Didymos, before, during and after the impact of the DART spacecraft, from a network of citizen science telescopes across the world are reported. Four Unistellar eVscopes captured observations of Didymos during the Double Asteroid Redirection Test (DART) impact into Dimorphos on the night of 26 September 2022. Of all telescopes that observed the DART impact, from the ground and space, the eVscopes were among the smallest with apertures of 112 mm. Three eVscopes were located on Réunion Island and one in Nairobi, Kenya. Figure Of the four eVscope data sets that included the moment of impact, three were suitable for photometric analysis because the observers recorded dark frames for image calibration. Thus, we conducted aperture photometry on these three data sets through a circular aperture radius of 13.6′′ or 750 km at the distance of Didymos The resulting apparent magnitudes measured over time are shown in Fig. 1, itself coming 38 s after the actual time of impact on the spacecraft due to light-travel time (private communication with J. Bellerose). Before the impact, we use the observations taken with the eVscope located in L’Étang-Salé, Réunion to measure an apparent visual magnitude, mv = 14.48 ± 0.11 and a minimum magnitude (maximum brightness) mv = 12.18 ± 0.03. As the fast-moving ejecta moved out of the photometric aperture, the magnitude increased to mv = 12.96 ± 0.04. At a geocentric distance of 0.076 AU, a heliocentric distance of 1.05 AU and a phase angle of 53.28° at the time of impact, these correspond to absolute visual magnitudes of Hv = 18.12 ± 0.11, 15.83 ± 0.03 and 16.61 ± 0.04, respectively.Using data from the eVscopes located in L’Étang-Salé and Saint-Paul, Réunion, we calculate an impact time of UTC 23:15:02 ± 4 s on 26 September 2022, which agrees with the reported Earth-observed impact time of 23:15:02.183 UTCHv = 15.83 ± 0.03 and 16.61 ± 0.04, and assuming an average density and albedo for Dimorphos. This is further detailed in the We estimate the effective mass in the fast-moving ejecta by calculating the change in effective cross-sections corresponding to magnitudes 3 km on the plane of the sky roughly 113.7 h after impact as shown in Fig. 4 km on the plane of the sky 2 days after impact4. Our measurement is shorter because of the smaller collecting area of the eVscope and its lesser sensitivity to low surface brightness. We measure a 3σ limiting magnitude of 17.01 ± 0.03 in this image stack consisting of 1,205 4-s exposures.To begin with, the particles must be small as evidenced by the long tail that developed in the antisolar direction several days after impact. We approximate an antisolar tail length of roughly 7 × 105. From this, average dust radii spanning a ≅ 1–104 μm along a power-law distribution with an index q = 3.5 (refs. 7).We then refer to active asteroid (596) Scheila, whose activity was probably caused by an impact, and whose average ejected dust size is predicted to be small on the basis of the observed effect of solar radiation sweepingmfe ≅ (7.0 ± 1.2) × 103 kg contained in the fast-moving ejecta plume, respectively. We measured the speed of this fast-moving ejecta on the plane of the sky through the photometry aperture and find this fast-moving ejecta has a speed of vfe ≅ 970 ± 50 m s−1. The resulting mass then corresponds to a kinetic energy of KEfe ≅ (3.3 ± 0.6) × 109 J. The relative kinetic energy of the DART spacecraft at the time of impact is 1). This indicates that the observed fast-moving ejecta plume carried away roughly 30 ± 6% of the kinetic energy delivered by the DART spacecraft. This is comparable to impact simulations, which have shown that kinetic energy can be transferred from the impactor to the ejecta on the order of tens of percent10. Average particle sizes an order of magnitude larger than this would exceed the kinetic energy introduced by the DART spacecraft, so we do not further consider larger particles sizes as making up a substantial portion of the observed fast-moving ejecta plume. The lower end of the dust size estimations based on solar radiation pressure, mfe ≅ (7.0 ± 1.2) × 102 kg, corresponding to roughly 3.0 ± 0.6% of DART’s kinetic energy. These values probably represent underestimates on ejecta mass, given the assumed average dust radii, as only the particles with velocities high enough to escape Dimorphos (ν > ve ≅ 0.087 ± 0.01 m s−1) will contribute to the measurable increase in cross-section12.We first examine an average particle radius vce ≅ 0.37 ± 0.01 m s−1. We use an approximated impact relation  × 107 kg. Considering the change in effective cross-section before impact and after the fast-moving ejecta dissipated, equation  × 107 kg, in good agreement with our estimate above.We also estimate the particle radius in the observed coma by relating solar radiation pressure to the turn-around distance of the particles in the coma along the comet–Sun line, and the bulk velocity of the particles, In all scenarios, we find that whereas the impact led to a significant increase in apparent magnitude, the overall mass loss in the observed fast-moving ejecta plum or slower-moving ejecta in the coma created by DART’s impact into Dimorphos was not totally disruptive.15.We also measure the colour before, during and after DART’s impact into Dimorphos as seen in Fig. 16. Some of these redder observed surface colours may be due to irradiation of organics17, which can efficiently mantle the surface of bodies such as the Kuiper belt objects19. Furthermore, in general, the highest velocity ejecta from an impact is sourced from the material closest to the site of that impact20. The fast-moving ejecta plume may be more representative of the surficial material of Dimorphos than the material in the slower-moving coma. Whereas much of the fine-grain surface regolith on Dimorphos has probably been depleted through processes such as electrostatic removal21, it is possible that the remaining small particles have experienced some irradiation. Spectra indicate the presence heterogeneity of surface materials on Didymos, with various concentrations of probably hypersthene with a grain radius a < 25 μm, and olivine with a grain radius a < 45 μm (ref. 22). Laboratory simulations of space weathering showed that irradiation can cause significant reddening of olivine’s reflectance spectrum23. We emphasize that these posed interpretations are a few of possibly many.If the reddened colour is interpreted as a proxy for material composition, we must consider this in the context of the colours of active and inactive small bodies in the Solar System. Typically, active bodies appear bluer in colour on average than their inactive counterparts, such as short-period comets versus Kuiper belt objectsThe measurements in this work are derived from 23,240 images taken by 17 different eVscopes in the Unistellar network of eVscopes. At the time of this study, the Unistellar fleet of eVscopes contained three variations: the eVscope 1, eVscope eQuinox and the eVscope 2. All eVscopes have 112-mm diameter primary mirrors. The eVscope 1 and eQuinox both use a Sony IMX224LQR sensor and have a 37′ × 28′ field of view and pixel scale of 1.72′′ per pix, whereas the eVscope 2 uses a Sony IMX347LQR sensor and has a 45′ × 34′ field of view and pixel scale of 1.33′′ per pix. The detectors have RGB photosensors arranged as Bayer filter matrices. Exposure times of individual images varied between observers from 1.0 to 4.0 s but remained constant throughout each individual data set. Images were taken consecutively over ranges of 10–80 min. In this work, we report observations taken from 26 September to 31 October 2022 UT. Over this time range, the observing parameters of Didymos ranged from geocentric distances of 0.076 to 0.109 AU, heliocentric distances of 1.046 to 1.018 AU and phase angles of 53.2° to 73.8°. Figure mV. These magnitudes are plotted in Fig. To measure the magnitude of Didymos in the images, we first reduce the data submitted by Unistellar citizen astronomers. Unistellar citizen astronomers submit science images with their chosen parameters (as listed above) as well as dark frames for calibration that are recorded immediately after the science observations. The dark frames are median combined and subtracted from the science images. The Unistellar network is not prompted at this point to take flat frames to further calibrate the data. In general, we find the errors on photometric measurements are dominated by photon noise and sky rather than the lack of a flat-field correction. We also aim to keep Didymos in the middle of the detector when conducting observations to further mitigate the lack of a flat frame calibration. Before conducting aperture photometry, the science images are aligned and median combined in time; however, the observations conducted during the DART impact shown in Fig. 24. We plot the resulting B-G, G-R and B-R colours, the B-G and B-R colours in Fig. To obtain the colours of the Didymos binary system, the colour channels of the detectors must be isolated. In a Bayer filter matrix of blue (B), green (G) and red (R) photosensors, the first 2 × 2 section of pixels is arranged as . This pattern repeats to fill the size of the detector. We isolate each colour channel on the basis of this known pattern of the photosensors and conduct aperture photometry as described above. We calibrate the instrumental fluxes using calibrator stars with known Gaia magnitudes in the background of each image. We measure the aperture fluxes of those calibrator stars in each colour channel and, combined with their known Gaia magnitudes, use them to convert the Didymos aperture fluxes in those channels to B, G and R magnitudesmV are converted to absolute magnitudes, HV, which represents the magnitude of the object with heliocentric and geocentric distance  of 1 AU and at phase angle, α = 0°. The correction isβα is the phase function that represents the dependence on sunlight scattering by the dust particle at an angle α in degrees. We assume a linear phase function with phase coefficient, β = 0.035, a typical value for S-type asteroids such as Didymos29. With this, our measured absolute magnitude before the impact of HV = 18.12 ± 0.11 agrees with past measurements of HV = 18.16 ± 0.04 of the binary system30. Absolute magnitudes are plotted in Fig. pv ≅ 0.15 ± 0.02 is the albedo of the Didymos system measured by the DRACO camera1, HV is the absolute magnitude we measure for the Didymos system and V⊙ ≅ −26.77 is the apparent magnitude of the Sun. For Didymos, we find an effective scattering cross-section Ce ≅ 0.53 ± 0.06 km2 before impact, Ce ≅ 4.35 ± 0.13 km2 at Didymos’ peak in brightness just after impact and Ce ≅ 2.17 ± 0.09 km2 after the fast-moving ejecta moved out of the photometric aperture. A cross-section Ce ≅ 0.53 ± 0.06 km2 indicates an effective radius re ≅ 411 ± 22 m, which is consistent with previous radar measurements of Didymos finding a volume equivalent radius of re ≅ 390 ± 15 m (ref. 11). The mass is related to the effective cross-section byρ ≅ 2,400 ± 250 kg m−3 is the bulk density of the Didymos system1, and we adopt 5. The change in effective cross-section measured at peak brightness and at the levelled brightness after the fast-moving ejecta dissipated then allows us to measure the change in mass, or the mass contained in the fast-moving ejecta plume with equation  and u is the initial velocity of the particles that we measure as u ≅ vce ≅ 0.37 ± 0.01 m s−1 from the fading rate of Didymos. B is the ratio of acceleration due to Solar radiation pressure to the acceleration due to Solar gravity expressed asK = 5.7 × 10-4 kg m−2 is a constant, Qpr is the radiation pressure coefficient often assume to be 1, ρ is the particle density and we examine the average dust a = 1. Assuming ρ ≅ 2,400 ± 250 kg m−3 gives B ≅ (0.24 ± 0.02)/ρ ≅ 3,480 ± 80 kg m−3, which is the average density of LL ordinary chondrite material31 as associated with S-type asteroid Didymos32, also results in millimetre-sized particles corresponding to masses within the range we estimate when assuming ρ ≅ 2,400 ± 250 kg m−3.Apparent magnitudes tc, of the particles in the photometric aperture. This is the time between the moment of impact and the moment the magnitude settled to mv = 12.96 ± 0.04. We determine the peak time and dissipation time of the fast-moving ejecta by analysing the magnitudes binned into rolling bins of five images to determine peak time and 15 images for the settling time. We then choose times when the residuals were within the respective measured errors on the peak magnitude and magnitude after fast-moving ejecta dissipation. We find a crossing time tc ≅ 775 ± 40 s over a photometric aperture radius of 13.67′′, which is equivalent to roughly 750 km at the distance of Didymos. Therefore, we obtain a speed of v ≅ 970 ± 50 m s−1.To estimate the energy carried by the mass in that fast-moving ejecta plume and slower-moving coma, we obtain their speeds. The fast-moving ejecta can be measured visually, following the plume on the detector over time. Furthermore, we measure the crossing time, HV = 18.12 ± 0.11. Then, assuming equal projectile and target densities, we make a simple estimation of the mass of the ejecta me by the impact relationUP ≅ 6,144.9 ± 0.3 is the impact speed of the DART spacecraft1, MP ≅ 579.4 ± 0.7 is the mass of the DART spacecraft on impact1, u ≅ vce ≅ 0.37 ± 0.01 m s−1 is the bulk velocity of the particles in the ejecta, and for consistency with past works A = 0.01 is a constant and s is an index that that we approximate to s = −1.5 but depends on the material13. The density of Dimorphos is assumed to be the same as the bulk density of the Didymos system in the calculations, however, we emphasize that the density of Dimorphos alone is not measured. The density of the main body of the DART spacecraft (without the solar panels) was roughly 270 kg m−3 at impact time33. For the sake of this simple approximation, we take the densities of the spacecraft and Dimorphos to be similar enough, because we expect lower densities with decreasing diameter for S-type asteroids34 and assume that the spacecraft was stopped by Dimorphos rather than flying straight through it. We also emphasize that this approximation comes with the caveat that impact physics on small asteroids are still not well-understood, so this impact relation serves as a rough estimate34.In the slower-moving ejecta that makes up the coma, we estimate the particle speed from the fading time of Didymos after impact. This is the time between the moment of impact and the moment the magnitude increased back to Didymos’ original absolute magnitude, m and speeds, v of the ejecta, we can estimate the kinetic energy, KE carried by the initial fast-moving ejecta as well as the coma by MP ≅ 579.4 ± 0.7 kg and velocity on impact is v = UP ≅ 6,144.9 ± 0.3 m s−1. We also compare this to the orbital energy, EO of Dimorphos around Didymos before and after the orbital period change of −33.0 ± 1.0 min (ref. 35) usingr is the semimajor axis of the orbit of Dimorphos around Didymos36. Before the impact, the semimajor axis was measured to be r = 1.206 ± 0.035 km1. We can estimate the original orbital energy of Dimorphos to be EO ≅ −(6.6 ± 0.6) × 107 J. With the orbital period decrease, Kepler’s third law gives a new semimajor axis of r ≅ 1.2 ± 0.1 km resulting in a change of orbital energy of With the estimated masses, Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-023-05852-9.


Fusarium pseudograminearum is a major pathogen for the destructive disease Fusarium crown rot (FCR) of wheat (Triticum aestivum). The cytosolic Acetoacetyl-CoA thiolase II (AACT) is the first catalytic enzyme in the mevalonate pathway that biosynthesizes isoprenoids in plants. However, there has been no investigation of wheat cytosolic AACT genes in defense against pathogens including Fusarium pseudograminearum. Herein, we identified a cytosolic AACT-encoding gene from wheat, named TaAACT1, and demonstrated its positively regulatory role in the wheat defense response to F. pseudograminearum. One haplotype of TaAACT1 in analyzed wheat genotypes was associated with wheat resistance to FCR. The TaAACT1 transcript level was elevated after F. pseudograminearum infection, and was higher in FCR-resistant wheat genotypes than in susceptible wheat genotypes. Functional analysis indicated that knock down of TaAACT1 impaired resistance against F. pseudograminearum and reduced the expression of downstream defense genes in wheat. TaAACT1 protein was verified to localize in the cytosol of wheat cells. TaAACT1 and its modulated defense genes were rapidly responsive to exogenous jasmonate treatment. Collectively, TaAACT1 contributes to resistance to F. pseudograminearum through upregulating the expression of defense genes in wheat. This study sheds new light on the molecular mechanisms underlying wheat defense against FCR. As a result, 50 SNP sites were found among TaAACT1 gene sequences of the different wheat cultivars, and the majority (47 SNPs) distributed among the last 1300 bp sequence of TaAACT1 (nucleotide no. 2028th to 3327th). Interestingly, 13 nucleotide allelic variants seemed to be associated with FCR resistance/susceptibility of wheat and formed at least 2 haplotypes (By means of bulked segregant RNA sequencing (BSR-Seq)  toward tplotypes , of whicF. pseudograminearum infection, the transcript level of TaAACT1 in stems of FCR-resistant wheat cultivar CI12633 was raised, reaching its peak at 4 days post inoculation (dpi) (about 3-fold over non treatment) , then in the wheat roots (about 3.9-fold over noninoculation) of the wheat cultivar CI12633  analysis showed that toward eatment) A. Subseq CI12633 B. In fac indexes  at 4 dpiultivars C. These TaAACT1 were cloned from the partially resistant wheat cultivar CI12633 and had 100% identity with those of CS (TraesCS3D02G005500.1). The TaAACT1 gene contains an open reading frame (ORF) with 1257 bp length. Comparison of the genomic and cDNA sequences showed that the genomic sequence of TaAACT1 was comprised of twelve introns and thirteen exons , belong to peroxisomal thiolases I and can catalyze β-oxidation of fatty acid. Biosynthetic thiolases, also known as Acetoacetyl-CoA thiolases (AACT), belong to cytosolic thiolase II enzymes and catalyze the condensation of two acetyl-CoA to form acetoacetyl-CoA [Aegilops tauschii AACT (XP_0201631811), Triticum dicoccoides AACT (XP_0374108261), Lolium rigidum AACT (XP_0470549061), rice (Oryza sativa) AACT (BAB398721), and corn (Zea mays) AACT (ACG347351) into one branch, while the cytosolic thiolase II (ACAT/AACT) proteins of dicots, namely Arabidopsis thaliana ACAT2 (BAH19918), Medicago sativa AACT1(ACX474701), radish (Raphanus sativus) AACT (CAA550061), and tobacco (Nicotiana tabacum) AACT (AAU956181), were clustered into another branch. AtKAT1 (AEE277361) and AtKAT2 (BAA252481) of Arabidopsis thaliana and rice thiolase I (AAO725881) were categorized to the peroxisomal thiolase I group. Additionally, TaAACT1 is closely related to the corresponding protein of Aegilops tauschii with 100.00% identity at the full-length protein level. These results indicate that TaAACT1 belongs to the cytosolic thiolase II group and that TaAACT1 in wheat might originate from that of Aegilops tauschii.Thiolases in plant cells are divided into biosynthetic thiolases and degradative thiolases according to their functions. Degradative thiolases, also known as 3-ketoacyl-CoA thiolase , the transcript level of TaAACT1 in BSMV:TaAACT1-infected CI12633 was significantly decreased (TaAACT1 gene was successfully silenced in these wheat plants mediated by BSMV.The virus-induced gene silencing (VIGS) mediated by barley stripe mosaic virus (BSMV) was used to investigate the defense function of software A. The oponstruct B. When tt leaves A. These ecreased B, suggesTaAACT1 against F. pseudograminearum infection, TaAACT1-silenced and BSMV:GFP-infected wheat plants were inoculated with F. pseudograminearum WHF220 at the same time. During a period of 28 dpi with F. pseudograminearum, compared with the BSMV:GFP-infected wheat plants, the stems of TaAACT1-silenced CI12633 wheat plants displayed larger lesion sizes of FCR , TaChitinase 3 (TaChit3), TaChitinase 4 (TaChit4), and TaDefensin through RT-qPCR in the TaAACT1-silenced wheat plants and the BSMV:GFP-infected wheat plants inoculated with F. pseudograminearum for 4 d. The results show that the transcript levels of TaChit2, TaChit3, TaChit4, and TaDefensin were significantly decreased in the more susceptibly TaAACT1-silenced wheat plants compared to the BSMV:GFP-infected wheat plants -responsive cis-acting elements (CGTCA motif/TGACG motif) and two abscisic acid (ABA)-responsive cis-acting elements . To explore whether TaAACT1 is responsive to JA and ABA, we tested the transcript level of TaAACT1 in the wheat cultivar CI12633 seedlings sprayed with exogenous MeJA (0.05 mM) and ABA (0.1 mM). As results, upon exogenous MeJA treatment, the expression level of TaAACT1 was significantly increased from 0.5 to 3 h, and reached its peak (~3.40-fold compared with the mock (H2O treatment)) at 0.5 h , the transcript levels of TaChit2, TaChit3, TaChit4, and Defensin were significantly and rapidly increased by exogenous MeJA  were all in response to the stimulation of exogenous MeJA, and that TaAACT1, TaChit2, TaChit3, TaChit4 and TaDefensin should be located in the JA signaling pathway.The promoter sequence of at 0.5 h A. Howeve(TaAACT1 B. Moreovous MeJA C. Taken TaAACT1 without the stop codon was subcloned in fusion to the N-terminus of green fluorescent protein (GFP)-coding gene, generating the p35S::TaAACT1-GFP fusion-expressing vector. The resulting p35S::TaAACT1-GFP and the control p35S::GFP were separately introduced into wheat mesophyll protoplasts with the PEG method and transiently expressed in the wheat cells. These fluorescent proteins expressed were observed via a confocal microscope. The results showed that fluorescence images of 35S::TaAACT1-GFP were distributed in the cytosol, while fluorescence images of the GFP control protein were distributed in both the cytoplasm and nucleus  and two positive regulators, namely TaWAK-6D and TaCRK-7A, were identified to be involved in resistance responses of FCR in wheat [Fusarium pseudograminearum infection. Additionally, a recent study showed that the overexpression of alfalfa thiolase II MsAACT1 in transgenic plants enhanced salinity tolerance and the production of squalene in salt-stress conditions [Wheat is an important staple crop and provides 20% of the food calories consumed by human beings worldwide . Its effin wheat ,26,27. HTaAACT1 associated with FCR resistance of wheat, and provided evidence that TaAACT1, acting as a positive contributor, was required for host defense against F. pseudograminearum infection. By means of analyses in BSR-seq and wheat variant omics, 13 SNPs in TaAACT1, related to FCR resistance, were identified. It is very interesting to develop functional markers based on these SNPs for molecularly breeding wheat with resistance to FCR in future. Transcript analysis results showed that the transcript induction of TaAACT1 by infection of F. pseudograminearum was higher in FCR-resistant wheat cultivars than in susceptible wheat cultivars, and the increase occurred in stems/roots at the greatest extent. Similarly, the expression levels of AtACAT2 and MsAACT1 were higher in roots than in leaves in Arabidopsis thaliana and alfalfa under optimal growth conditions [Arabidopsis mutant of AACT 2 with only one copy is nonviable [TaCYP72A, VIGS was used to conduct functional analyses, and the results showed that TaCYP72A enhances resistance to deoxynivalenol and grain number [TaAACT1, and the results show that the silencing of TaAACT1 decreased resistance to FCR caused by infection of F. pseudograminearum. To our knowledge, this is the first investigation about the role of plant thiolases in plants’ innate immunity. This study deepens the understanding of plant defense mechanisms against pathogens.In the current research, we identified the wheat cytosol thiolase II-encoding gene nditions . Functionditions , and thionviable . Fortunaonviable ,38. For TaAACT1 is required for the expression of TaChitinase 2, TaChitinase 3, TaChitinase 4, and TaDefensin in wheat inoculated by F. pseudograminearum, suggesting that TaChitinase 2, TaChitinase 3, TaChitinase 4 and TaDefensin act downstream of TaAACT1 and are positively modulated by upstream TaAACT1. JA is a primary phytohormone associated with plant resistance responses to necrotrophic pathogens [TaSTT3b-2B in transgenic wheat increased endogenous JA content and the expression of JA synthesis-related genes and enhanced resistance to sharp eyespot and grain weight [TaAACT1 rapidly responded to exogenous MeJA stimulus, and MeJA treatment also significantly induced the expression of TaAACT1-modulated defense genes (Chitinases 2/3/4 and defensin). Thus, JA signaling might play an important role in TaAATC1-mediated immune responses to F. pseudograminearum infection. Taken together, these results suggest that TaAACT1 positively contributes to the resistance of wheat against F. pseudograminearum infection by regulating the expression of TaChitinase 2, TaChitinase 3, TaChitinase 4, and TaDefensin in JA signaling. In plant species, many chitinases and defensin peptides show antifungal activity ,30,35,39athogens . Some kiathogens ,47,52. Fn weight . HoweverArabidopsis thaliana cytosolic Thiolase II , and alfalfa cytosolic Thiolase II/AACT1 , involved in the mevalonate pathway [Arabidopsis thaliana cytosolic Thiolase II/AtACAT2 and MsAACT1, as well as other cytosolic Thiolase II proteins, are classified into the same group, and that TaAACT1 in wheat might originate from that of Aegilops tauschii. The subcellular localization experimental assay indicated that TaAACT1 protein indeed localizes in the cytosol of wheat, like the AtACAT2 subcellular localization result [planta, and that the expression of MsAACT1 increased abiotic stress resistance in bacteria and in planta, strongly suggesting that MsAACT1 positively regulates abiotic stress tolerance by increasing squalene biosynthesis [tobacco mosaic virus, or Pseudomonas syringae pv. tabaci induced accumulation of polyisoprenoid alcohols in the leaves of resistant tobacco plants but did not cause an increase in polyisoprenoid content in susceptible tobacco plants [F. pseudograminearum.Protein sequence analysis showed that the TaAACT1-predicted protein contains two domains: a Thiolase-N domain and a Thiolase-C domain, similar to those of  pathway ,7. Our pn result ,7. In a o plants . In the FCR-resistant cultivars , and susceptible cultivars (Yangmai 6 and Yangmai 158), were used to examine transcript profiles of TaAACT1 with the F. pseudograminearum inoculation. Five wheat cultivars, including F. pseudograminearum were inoculated between the second base sheaths and stems of wheat plants [F. pseudograminearum. The inoculated sheaths of Nivat 14, Chinese spring, CI12633, Yangmai 9, and Yangmai 158 were separately sampled at 4 dpi with F. pseudograminearum for RNA extraction. These samples were quickly frozen in liquid nitrogen and stored at −80 °C prior to extraction of total RNA. The seedlings of the wheat cultivar CI12633 at the two-leaf stage were separately treated with 0.05 mM MeJA and 0.1 mM ABA dissolved in ultrapure water, and ultrapure water solution  for noninoculation and 0.5, 1, 3, 6, and 12 h, as described by Zhang et al. [All of the wheat plants were grown in a 16 h light/8 h dark (22 °C/12 °C) regimen. At the tillering stage, small toothpick fragments harboring the well-developed mycelia of t plants . The inog et al. . The treF. pseudograminearum strain WHF220 was isolated from the FCR-symptomatic wheat sheaths in Shandong by Prof. Jinfeng Yu and Dr. Li Zhang .The pathogenic fungus All primers and their sequences in the study are shown in TaAACT1, TaChit2, TaChit3, TaChit4, and TaDefensin in wheat. RT-qPCR was performed using a SYBR Premix Ex Taq kit  in a volume of 20 µL on an ABI 7500 instrument . Reactions were set up with the following thermal cycling profile: 95 °C for 3 min followed by 40 cycles of 5 s at 95 °C, 15 s at 55 °C, and 32 s at 72 °C, and completed with a melting curve analysis program. All RT-qPCRs were repeated three times. The relative expression of the tested gene was calculated with the 2−ΔΔCT method [Actin gene was used to normalize amounts of cDNA among the samples. Total RNA was extracted from wheat samples tested using Trizol reagent  according to the manufacturer’s instruction. RNA was purified and then checked for integrity. Total RNA was reverse-transcribed to cDNA using the FastQuant R T Kit ; RT-qPCR and specific primers were employed to measure expression levels of T method , where t16) at 20 dpi and used to BSR-seq using the Illumina HiSeq 2500 sequencing technology platform at Beijing Biomarker Technologies. For each pool, three biological replicates were performed. After discarding low-quality raw reads, the generated 6 Gb clean reads from each library were mapped to wheat genomes. RNA-seq data were analyzed as described previously by Wang et al. [TaAACT1 related to FCR resistance of wheat were identified.The purified RNAs were extracted from the resistant and susceptible RILs ) to determine the theoretical pI (isoelectric point) and Mw (molecular weight), The conserved domains and motifs in the deduced protein sequence were predicted by SMART . Sequences were aligned via DNAMAN software. Subcellular localization of TaAACT1 protein was predicted using PSORT Ⅱ online software . A phylogenetic tree was constructed by MEGA 11.0  software. Cis-elements in the TaAACT1 promoter (2000 bp) were analyzed using the PlantCARE . The JA- and ABA-responsive cis-acting elements in promoter of TaAACT1 are shown in One pair of primers (TaAACT1-F1/TaAACT1-R1) for TaAACT1 were designed and used to amplify the full ORF sequence of TaAACT1 from the cDNA of the wheat cultivar CI12633.The predicted protein sequence was analyzed with the Compute pI/Mw tool  derived from the coding sequence of eviously ,45,46,48TaAACT1-silenced and BSMV:GFP-infected (control) wheat plants were inoculated with well-developed F. pseudograminearum mycelia toothpicks and kept at high humidity for 4 d in two batches. After 28 days of fungal inoculation, the average lesion length and width of FCR on the basal sheath of the above wheat plants were measured and recorded, and the FCR disease index was evaluated. The assessment methods of FCR disease severity were referred to the existing researches [About 21 d post-transfection with BSMV, the searches .BamH I site using the specific primers (TaAACT1-GFP-F/TaAACT1-GFP-R). The amplified fragment was digested with restriction enzyme BamH I and subcloned in-frame into the N-terminus of the GFP coding region in the p35S::GFP vector, resulting in the TaAACT1-GFP fusion construct p35S::TaAACT1-GFP. The p35S::TaAACT1-GFP fusion construct and p35S::GFP control construct were separately individually introduced into wheat mesophyll protoplasts [The coding region of TaAACT1 lacking the stop codon was amplified and added the restriction enzyme toplasts . After iTaAACT1 was identified to be associated with FCR resistance of wheat. One haplotype ofTaAACT1 gene sequences in analyzed wheat genotypes was related to wheat resistance against FCR, and the gene transcript induction by Fusarium pseudograminearum infection was higher in FCR-resistant wheat cultivars than in susceptible wheat cultivars. Moreover, the transcript induction of this gene was the highest in stems then roots, where FCR disease primarily occurs. VIGS-based functional analyses indicated that the expression of TaAACT1 was required for host defense responses against F. pseudograminearum infection, and TaAACT1 positively modulated the expression of the defense genes TaChitinase 2, TaChitinase 3 TaChitinase 4, and TaDefensin in wheat. This study extends knowledge about wheat-F. pseudograminearum interactions and deepens our understanding of the molecular mechanisms underlying wheat plant immunity to fungal pathogens, especially F. pseudograminearum. The TaAACT1 is a candidate gene for highly efficiently improving the resistance of wheat to FCR disease.The wheat cytosol thiolase II-encoding gene


MET exon 14 skipping alterations. The aims of this work were to investigate the potential for drug-drug interactions via cytochrome P450 (CYP) 3A4/5 or P-glycoprotein (P-gp) inhibition. In vitro studies were conducted in human liver microsomes, human hepatocyte cultures and Caco-2 cell monolayers to investigate whether tepotinib or its major metabolite (MSC2571109A) inhibited or induced CYP3A4/5 or inhibited P-gp. Two clinical studies were conducted to investigate the effect of multiple dose tepotinib  on the single dose pharmacokinetics of a sensitive CYP3A4 substrate  and a P-gp substrate  in healthy participants. Tepotinib and MSC2571109A showed little evidence of direct or time-dependent CYP3A4/5 inhibition (IC50 > 15 μM) in vitro, although MSC2571109A did show mechanism-based CYP3A4/5 inhibition. Tepotinib did not induce CYP3A4/5 activity in vitro, although both tepotinib and MSC2571109A increased CYP3A4 mRNA. In clinical studies, tepotinib had no effect on the pharmacokinetics of midazolam or its metabolite 1’-hydroxymidazolam. Tepotinib increased dabigatran maximum concentration and area under the curve extrapolated to infinity by 38% and 51%, respectively. These changes were not considered to be clinically relevant. Tepotinib was considered safe and well tolerated in both studies. The potential of tepotinib to cause clinically relevant DDI with CYP3A4- or P-gp-dependent drugs at the clinical dose is considered low. Study 1 (midazolam): NCT03628339 (registered 14 August 2018). Study 2 (dabigatran): NCT03492437 (registered 10 April 2018).Tepotinib is a highly selective, potent, mesenchymal-epithelial transition factor (MET) inhibitor, approved for the treatment of non-small cell lung cancer harboring The online version contains supplementary material available at 10.1007/s10637-023-01378-z. MET exon 14 (METex14) skipping, tepotinib has been approved by multiple regulatory authorities worldwide /IC50 ratios based on the proposed clinical dose of 500 mg once daily were 8917  and 0.39 (systemic). Both ratios exceeded regulatory cutoff values ), and MSC2571109A showed mechanism-based inhibition of CYP3A4. In addition, both tepotinib and MSC2571109A increased CYP3A4 mRNA levels in vitro. In vitro studies suggested that tepotinib is also a P-gp inhibitor.The potential for tepotinib and its major metabolite to induce or inhibit the major CYPs was investigated in vitro. The results of these studies suggested that tepotinib and MSC2571109A were weak direct inhibitors of CYP3A4/5 , midazolam was administered 4 h after tepotinib.Midazolam absorption is delayed when administered in a nonfasted state, whereas tepotinib is recommended to be administered with food to improve its absorption. To minimize food effects on midazolam and to administer midazolam nearer to tepotinib CBlood samples for midazolam and 1-hydroxymidazolam were collected over 43 h post-dose, which is > 5 times the terminal half-life of midazolam (~ 5 h) and therefore allowed adequate characterization of midazolam pharmacokinetic profile. Similarly, blood samples for dabigatran were collected over 72 h post-dose, which is > 5 times its terminal half-life (~ 8 h).The results from Study 1 demonstrated that tepotinib at a therapeutic dose, did not affect the pharmacokinetics of either midazolam or its primary metabolite, suggesting that the potential for pharmacokinetic interactions with other CYP3A4 substrates is low.max and AUC of dabigatran compared to administration alone by approximately 40% and 50%, respectively. This is generally not considered clinically relevant, but the labeling information advises caution for P-gp substrates for which minimal concentration changes may lead to serious or life-threatening toxicities.The results from Study 2 suggested that tepotinib had a weak effect on the pharmacokinetics of the sensitive P-gp substrate dabigatran. Coadministration with tepotinib increased CIn summary, when tepotinib was administered orally at the therapeutic dose of 500 mg QD, there was no change in exposure to the sensitive CYP3A substrate, midazolam. There was less than a 2-fold exposure increase to dabigatran, indicating that tepotinib is a weak inhibitor of the P-gp-mediated efflux transport of the substrate dabigatran etexilate. Tepotinib was considered safe and well tolerated in these studies involving healthy participants. The potential of tepotinib to cause clinically relevant DDI with CYP3A4- or P-gp-dependent drugs at the intended posology is considered low.Supplementary file1 (PPTX 50 KB)Supplementary file2 (PPTX 213 KB)Below is the link to the electronic supplementary material.


The purpose of this work is to renew the interest and attention for the chain of custody in forensic medicine, its establishment and maintenance, protecting the integrity and validity of evidence as well as to analyze how over time the establishment of the chain of custody and the collection of evidence has evolved also in function of the advent of technology and the use of electronic devices connected to the network. The analysis of the various aspects of the chain of custody demonstrates how necessary it is for the professional figures involved in the phases of the investigation  to know the procedures to follow, trace the movement and the handling of objects subjected to seizure, also for the purposes of toxicological and/or histological investigations. The knowledge of interferences or complications helps to reduce errors and safeguard the validity of the evidence, assuring the proceeding judicial authority that the evidence is authentic and that it is, in other words, the same evidence seized at the scene of the crime. Furthermore, the issue is particularly felt today, with the recent need to guarantee the originality of digital data. Following a careful review and analysis of the literature currently available in this regard, it is worth adding that further efforts are needed to formulate internationally validated guidelines, harmonizing the different reference criteria in forensic science and medical areas, given the current absence of good international practices valid in the field and applicable both in the case of physical evidence and in the case of seizure of digital evidence. Chain of custody is the most important and, at the same time, the most critical process of documenting evidence: in criminal and civil law, the term “chain of custody” refers to the order in which evidence was dealt with during the investigation of a case . When a It is essential to assure the judicial authority that the evidence is authentic and that it is the same that is seized at the scene of the crime. The fundamental point of correct maintenance of the chain of custody consists in the possibility of access to the original exhibit. Consequently, it is appropriate that each person in charge of its custody is fully aware of the responsibility of keeping the evidence intact and of tracing, through suitable documentation, each passage or conservation , which eIn this narrative review, we look for the themes of chain of custody (CC) and forensics (F), considering aspects of important and innovative role in the near future. Three main databases and indexed bibliographic searches were studied. The PubMed database showed 71 articles in English, accessible in an extended version; 45 were considered as subjects of interest, in the years 1990–2022 25 articles corresponding to topics CC and F were considered as review articles ,24,25. NThe chain of custody demonstrates the integrity of an item of evidence ,35,36. AThe chain of custody must contain and document every transmission of the object from person to person since the seizure. The goal is to establish that the evidence is related to the alleged crime, was collected at the scene, and was in its original/unaltered condition rather than having been tampered with or otherwise polluted . To convThe traceability of the registration of the control, of the transfer, and of the analysis of the samples indicates the transparency of the procedure . MaintaiChain of custody documentation has three main purposes: to ask the testing laboratory pertinent questions about the tests, to maintain a chain of custody record, and to document that the sample/test was handled only by authorized personnel and was not accessible for tampering prior to the analyses ,44,45. TThe Investigator or person responsible for collecting evidence must complete the sample container/bag labels and chain of custody forms to enable sample traceability. Each sample container label must bear a unique identification number and other pertinent information such as location, date and time of collection, name and signature of the person who collected the sample, and signature(s) of the witness(es). It is essential that evidence is properly packaged to avoid damage in transit and should preferably be sealed in tamper-evident bags or with tamper-evident tape.-During the investigation, the different chain of custody officers should follow several steps :-document the location of the scene and the time of arrival of the investigator and coroner;-determine evidence repository(s), determine which laboratory(ies) is/are responsible for collecting specific types of evidence, and determine evidence collection priority for fragile evidence;-identify, document, protect and preserve evidence with appropriate containers, labels and preservatives;-document evidence collection by recording its on-site location, time of collection, time and place of disposal, and by whom;-develop personnel rosters, witness lists, and documentation of personnel arrival and departure times.-unique identifier;-name and signature of the person building the sample;-address and telephone number;-details of each sample;-type of analysis required.A separate chain of custody form must accompany the different sets of evidence. The chain of custody form—in order not to be considered invalid for the purpose of demonstrating the correct maintenance of the chain of custody—must contain at least the following information ,47:-uniqHowever, there are no unique forms that document the chain of custody, and lists and registers with different characteristics can also be used .According to an authoritative study by Schum and colleagues, with the development of technology we have worked and will continue to work to increasingly adapt the paper documentation of the chain of custody to digitization, mainly using secure and traceable IT systems . This moWhenever evidence is used for the purposes of the investigation, the signature, date, and time must be entered for the chain of custody form. A sample is in custody if it is actually in the physical possession of the authorized custodian in a secure location without access to unauthorized personnel or exposed to any possibility of tampering.During the trial, if the defense attorney raises concerns about the chain of evidence, the records must show that the chain of custody was never broken. If the inconsistencies persist and the prosecution is unable to prove the chain, it is deemed to have been broken and the defense attorney can seek the annulment of the resulting evidence in court. The same applies to the elements under examination that require toxicological analyses to be performed on them (blood or urine), in these cases the laboratories we rely on will have to comply with certain quality and safety standards that allow the validity of the results deriving from the studies carried out ,50, as w-civil litigation;-doping tests of athletes;-manage the origin chain, for example, to improve the traceability of food products;-the supply of medicines;-seizure of controlled/prohibited substances;-seizure of money/gold ornaments/other valuables by the customs, tax, or revenue offices;-in cases of violence and abuse, with adequate training, also of the health professionals involved in the collection of evidence.In addition to crime scene investigations, other areas that find use of chain of custody include ,52,53:-cThe study of the literature has made it possible to identify multiple critical issues in the chain of custody relating to the seizure of physical and digital evidence. The first and probably biggest problem is the inadequate packaging of evidence: when it is recovered, it must be protected from tampering. If the forensic expert does not personally package the evidence, due to his greater scientific knowledge, he is responsible for clearly explaining to law enforcement which is the optimal packaging for each type of exhibit. Poorly sealed packages are another potential problem: holes in evidence seals or packaging can lead to loss of evidence or the introduction of contamination. Another potential problem is the loss of recovered items or their disappearance. For this reason, it is important that the figures who intervene on the crime scene draw up a list of the stolen and kept objects.Another problem could be the pollution of the crime scene by professionals who intervene before the police and investigative experts, such as rescuers whose sole objective is to protect the patient’s health. By intervening, they can alter, make dirty, or contaminate the crime scene, compromising the validity of the evidence ,55,56,57The technological progress that has affected the field of data production and acquisition, culminating in the digitization and storage of information on electronic devices, has had clear repercussions on the methods of transmission and storage of the same. In fact, in the forensic field, the guarantee of the authenticity and integrity of the digital information, contained in text files or in images acquired with digital photographic equipment, is an essential prerequisite for the correct conduct of the investigations, and for the use of these pieces of information in judicial proceedings ,59.Therefore, nowadays, since the digital medium is one of the main tools used for the acquisition and production of evidence sources, it is necessary to guarantee the integrity of the chain of custody as an indispensable principle to ensure the correctness and the authenticity of the information contained therein.If, on the one hand, the contribution of computer technology has contributed to making many procedures more efficient, rapid, and precise, on the other, it has equally easily led to the vulnerability of the information contained in files and digital media. A photograph or a text file produced with digital media is easily modifiable and, therefore, alterable: this creates the need to protect the authenticity of the computer artifact by relying on a chain of custody . In thisIn the exercise of his or profession, the forensic scientist is often called upon to perform unrepeatable acts, such as the search for “sources of evidence” during a judicial inspection, or to conduct autopsy investigations; in these circumstances, he is called upon to produce photographic documentation aimed at “crystallizing” the crime scene, documenting the existence of artifacts, and drawing up reports whose authenticity and genuineness of content are essential requisites. Likewise, the correctness of the procedures underlying the conservation and transmission of such information, for which a chain of custody is invoked, constitutes a guarantee of reliability and truthfulness of these finds, essential characteristics for the correct orientation of judicial investigations and for their use corrected in legal proceedings.In the field of photography, which in forensic medicine plays a central role in the documentation of data and circumstances of forensic interest, a great advantage in guaranteeing data security and quality is given by the use of the RAW image format. The RAW format of an image varies depending on the digital camera used: each manufacturer has its own proprietary RAW format. A RAW image is not yet processed or ready to be printed: it is, in fact, the format that allows you to store more data and information in a photographic shot. Manipulation, understood as tampering, of a RAW image is extremely difficult. The post-production of the RAW image, in fact, determines the conversion of the format and the loss—total or partial—of the EXIF metadata contained in the original file; therefore, a further contribution made by information technology to the implementation of a valid chain of custody of the digital record is the reliable timestamp. Several companies offering digital services provide access to this technology, thanks to which it is possible to stamp a certain date and time on the document, even if it has not been digitally signed. The trusted timestamp ensures certainty and authenticity of date and time for a computer document when it is applied. It consists in the generation, by a time-stamping organism, of a digital signature of the document which can be additional to that of the signatories and, therefore, also affixed to files not previously signed. Reliable timestamping can contribute to the correct maintenance of the IT record chain of custody as it represents documented proof of the existence of a given file at a given instant. The documents to which the timestamp has been affixed are enforceable against third parties in the event of litigation . DigitalThe current state of the art on preserving the authenticity of digital data and which could find a successful use in the chain of custody of digital artifacts is represented by two innovative technologies, the blockchain and the NFT.Blockchain technology is mainly used in the financial sector—it is well known as the technology behind the creation of the Bitcoin cryptocurrency. Blockchain is a shared, immutable record of transactions. Whenever a transaction is performed, all data related to it are stored as a block in the chain. The timing and sequence of transactions are guaranteed; furthermore, the fact that the blocks in the chain are tightly connected to each other prevents any possibility of data tampering. The application of the blockchain in the medical field would allow a very high level of protection, as the data would be reliable and practically impenetrable; however, the blockchain allows data to be updated by those with authorized access .NFTs (Non-Fungible Tokens) are blockchain-based tokens that represent a unique asset, be it digital or physical, thus providing a certificate of authenticity and non-reproducibility for that asset. The use of NFTs is becoming more and more common, from the art world to the automotive industry. The application of blockchain technology and NFTs in forensics could lead to new tools and instruments to ensure the reliability and integrity of digital evidence to the coroner . From thGiven the importance of the establishment of the chain of custody and its correct management in the various phases of evidence analysis, it is essential in the future to properly train the personnel involved in the collection, transport, and analysis of samples . For exaSpecialist doctors who may find themselves involved in the seizure of evidence should also be aware of the need to create and maintain a chain of custody of the evidence. An example could be the surgeon who has to extract a bullet from the body of a patient ,77,78,79


Our understanding of the psychological impact of living with pulmonary hypertension (PH) is growing, particularly for how anxiety and depression present in this group. There is evidence for the use of psychological interventions in other chronic lung conditions; however, trials focusing on adults with PH have yet to be subject to a systematic review.We systematically searched four databases for evidence examining the effectiveness of psychological interventions for adults with PH. A narrative approach has been used to present findings.Overall, 186 unique articles were identified of which four were suitable. Data was gathered from 143 individuals. Cognitive behavioural therapy (CBT), metacognitive therapy (MCT) or counselling were investigated. All three studies reported a significant reduction in anxiety and depression, and some secondary outcomes were also associated with change.The evidence supporting the use of psychological therapies for adults with PH is promising, although limited. There is an urgent need for more research in this area to better understand how we can support this clinical group. Our understanding of the psychological impact of living with pulmonary hypertension (PH) is growing. Evidence exists for the use of psychological interventions in chronic lung conditions; however, trials involving adults with PH have not been subject to a systematic review. We show that although the evidence is limited, the findings are promising and there is a need for more research. The available studies indicate that disease specific factors alone are unable to fully account for the reported impactThere is some evidence for the use of psychological therapies for anxiety and depression in people with other chronic lung conditions.We performed a systematic search in February 2023 of four databases: PsycINFO, MEDLINE, Cochrane library and CINAHL. Search terms were informed by relevant articles known to the authorsTitle and abstract of articles identified were first screened. Most studies were excluded at this stage as they did not investigate PH or were not evaluating a psychological intervention. If relevant, articles were then subject to a full‐text review. Forward and backward searchers of included articles were performed, including searches for articles in previous reviews,Relevant data based on participant, intervention, comparison group and outcome (PICO) were extracted from each article (see Table Four studies were identified as suitable Table . All wer2); suggesting changes in mental health were a secondary aim.Cognitive behavioural therapy (CBT),Two studies included a control group; the other studies were an observational trial or a case study. All studies measured anxiety and depression; other health‐related measures and psychological processes were also assessed. Although for the most part measures were appropriate given the research aims, it is important to recognise that they were typically screening tools and did not aim to diagnose individuals.A significant reduction in anxiety and depression was observed in all samples. There was some evidence to suggest anxiety and depression did not improve in people with PH who did not receive a psychological intervention.Individuals who scored a greater severity of symptoms were more likely to engage in therapyThe importance of integrating treatments for physical and mental health has long been recognised.Advancements in how PH is diagnosed and treated have resulted in people living longer with the condition. There is now a need to extend treatment beyond the physical symptoms, as research has shown this group are significantly impacted by the condition.This is the first systematic review examining psychological interventions for emotional difficulties in people with PH. It is a limitation that a narrative approach was used to synthesise the results; however, at this time, due to the limited data and heterogeneity in methods, samples, interventions and measures, a meta‐analysis would not be appropriate. Articles were screened and data were extracted by one researcher whereas steps could have been taken to protect against bias and human error. Despite this, the evidence is promising for the use of such therapies, but importantly, also highlights the urgent need for more research and better patient involvement and access.Gregg H. Rawlings developed the concept of the review. He developed the protocol, performed the systematic review, data extraction and analysis and write up. He approved the final manuscript for submission. Barbora Novakova was involved in the concept of the review. She provided feedback on an earlier version of the review and approved the final manuscript for submission. Iain Armstrong was involved in the concept of the review. He provided feedback on an earlier version of the review and approved the final manuscript for submission. Andrew R. Thompson was involved in the concept of the review. He provided feedback on an earlier version of the review and approved the final manuscript for submission.This research has received no external funding.Dr Rawlings has received payment from Janssen‐Cilag Ltd for a presentation on depression and pulmonary hypertension (PH).Ethical approval was not required for this article and therefore was not obtained.


Experiments on choice-predictive brain signals have played an important role in the debate on free will. In a seminal study, Benjamin Libet and colleagues found that a negative-going EEG signal, the readiness potential (RP), can be observed over motor-related brain regions even hundreds of ms before the time of the conscious decision to move. If the early onset of the readiness potential is taken as an indicator of the “brain’s decision to move” this could mean that this decision is made early, by unconscious brain activity, rather than later, at the time when the subject believes to have decided. However, an alternative kind of interpretation, involving ongoing stochastic fluctuations, has recently been brought to light. This stochastic decision model (SDM) takes its inspiration from leaky accumulator models of perceptual decision making. It suggests that the RP originates from an accumulation of ongoing stochastic fluctuations. In this view, the decision happens only at a much later stage when an accumulated noisy signal (plus imperative) reaches a threshold. Here, we clarify a number of confusions regarding both the evidence for the stochastic decision model as well as the interpretation that it offers. We will explore several points that we feel are in need of clarification: (a) the empirical evidence for the role of stochastic fluctuations is so far only indirect; (b) the interpretation of animal studies is unclear; (c) a model that is deterministic during the accumulation stage can explain the data in a similar way; (d) the primary focus in the literature has been on the role of random fluctuations whereas the deterministic aspects of the model have been largely ignored; (e) contrary to the original interpretation, the deterministic component of the model is quantitatively the dominant input into the accumulator; and finally (f) there is confusion regarding the role of “imperative” in the SDM and its link to “evidence” in perceptual decision making. Our aim is not to rehabilitate the role of the RP in the free will debate. Rather we aim to address some confusions regarding the evidence for accumulators playing a role in these preparatory brain processes. For example, when we see a car crossing our lane on the highway, we step on the break to avoid a collision. An important factor is the level or quality of sensory information. For example, when driving in broad daylight we instantly see the dangerous car. But when it is foggy, we might be uncertain about whether it is a car or just a random pattern in the mist. In that case, we might need to look at the pattern for a bit longer and gather evidence across time. A popular approach for explaining perceptual decision making (PDM) under such varying levels of sensory evidence is the accumulator model  was proposed by In the original version of the SDM , two difIn our model this solution amounts to simply shifting premotor activation up closer to the threshold for initiation of the instructed movement and waiting for a random threshold-crossing event”  in their implementation. On every time step, an increment or decrementIn this section, we will go into more detail about how the model works and which evidence is provided in support of it. We will focus on two papers, xi , left, oAt each time step, This means that the increment/decrement that is added to The first term,c are fixed scaling constants).The second term,The third term,When the accumulated signal The next two rows of the figure show the consequences of removing the imperative  term vs. the noise term. If the noise term is removed, the signal changes in evidence. Note that the trial-by-trial variability of the response time is only affected by the noise term. When the constant term is zero, the behavior of a perceptual accumulator is governed by the noise term  is based on the perceptual decision-making model, but with zero constant evidence, with only the noise active, thus making it similar to perceptual guessing. It has indeed been shown previously that perceptual guesses  and spontaneous decisions indeed elicit similar activation patterns in posterior parietal cortex . Thus, idata see .The roles of imperative and noise in the model have been implied to reflect a two-stage sequential process:In our model this solution amounts to simply shifting premotor activation up closer to the threshold for initiation of the instructed movement and waiting for a random threshold-crossing event.” . In the model, there are also no two discrete stages, in the sense that first there is a pre-stationary stage that moves the system toward threshold alone and then a stationary stage where the system waits for a threshold crossing. Half of the decisions happen during the early stage that is dominated by the imperative moving the signal closer to the threshold . This is not the case. Instead, the fluctuation time series are latent and hypothetical variables of the model that are not directly measured (as is the case in many other neuroimaging models). The fluctuating time courses were not measured at the single-trial level.Next we would like to address a number of confusions that seem to have originated in the literature on the SDM. For example, summaries provided in various papers give the impression that the empirical analysis of the EEG data and of behavioral waiting times provides Furthermore, in recent years, there have been substantial challenges to the ubiquity and nature of a core feature of the SDM, that is the accumulation process: Especially in situations where sensory evidence is brief rather than distributed across time, accumulation might not always take place , in otheIn order to provide more direct evidence, the authors point to converging studies on animals. Potential evidence for the neural implementation of a SDM for endogenous tasks was reported by majority of predictive time periods are around the start of the trial or even before the onset of the trial, which is the opposite of what would be expected in the case of an accumulator model. We will return to this later. Furthermore, the predictive signal is transient. Thus, this specific signal does not seem to map on to any variable of the SDM model, neither to a continuous stochastic or constant input, nor to an accumulator continuously integrating input across the trial. Instead, it could simply reflect a cognitively interpretable bias signal, such as an expectation on that trial of when the delayed reward will occur. If anything, a different subpopulation of neurons exhibits a ramping-like behavior, but mostly toward the end of the trial. Importantly, there is a considerable temporal dissociation between the time where most of the time windows are informative, and the time when the putative accumulator in their data ramps toward threshold  or differences in motivation or impulsivity (in the case of spontaneous movements). Interestingly, even out-of-the-box this fluctuation-free model makes very similar predictions for features of perceptual decisions to the accumulator. Importantly, it predicts the typical heavy-tailed reaction time/waiting time distribution. When used in a similar way to predict readiness potentials, the ballistic accumulator model also provides a good fit to the RP, despite its simplicity and the absence of random fluctuations during the trial . When the sensory information level is absent, I is set to 0 and the behavior is driven purely by the noise. In the SDM with the published parameters, the situation is not like decision making without sensory information. I is not set to 0, so there is a constant driving input. However, as we have seen most of the interpretations in the literature focuses on the role of the noise fluctuations.If we look back to the perceptual decision-making case, the roles are quite clear. When the sensory information level is high then the accumulator primarily integrates this evidence throughout the trial  and the noise. Thus, movement toward the threshold is achieved by a combination of the continuous “push” of the constant and the variable (zero-mean) “rattle” of the random input. The combined and accumulated effect of these variables is additionally subject to a leak. One way to compare the contribution of the stochastic and the non-stochastic component to crossing of the threshold is to assess how much of the distance the accumulator travels between zero and the threshold can be attributed to each component. So we will sum up all the stepwise contributions of the noise component .Third, we can assess the A simple illustration of the net input of both sources is shown in backwards from the time of threshold crossing. The histograms show the cumulative input between trial onset and threshold crossing separately for the constant imperative and for the noise (and separately for the two published variants of the SDM). For the first SDM version . The idea there would be that the constant signal initially drives a “stochastic exponential transition period”  decisions being made. Second, from this plateau the system waits for a random threshold-crossing event.Let us consider the time point 5 s into the trial where the accumulator has on average reached around 90% of its asymptote see , middle.Please note, in the extended version of the SDM with pink instead of white noise as input , the logI = 0.1, k = 0.6 and threshold = 0.1256) the SDM would converge to 0.1/0.6 = 0.167 based on imperative alone and without noise. The threshold in this model is at 0.1256 so the threshold would be crossed without any noise in the model. Taken together, as mentioned above, it is not accurate to think of the SDM as a two-stage model, with phase 1 being “climb closer to threshold (but do not cross it yet),” and phase 2 being “OK, now you may cross the threshold at any time.”With the reported parameters .In perceptual decision making, “evidence” refers to one variable having information ic times . In lineic times , say thaor noise) might reflect evidence in the SDM after all. Already an early review paper interpreted the original study asThere seems to be some confusion in the literature about whether one of the signals .“And then:accumulation of evidence might operate primarily on stochastic neural fluctuations in the motor systemThese models assume that decision time in the Libet task is based on a process of accumulation of evidence to a threshold, just like in other decision-tasks. Because the decision is not based on perceptual or other external evidence, this ” .“And finally:gathering of evidenceThis means that the RP and the LRP do not reflect a ballistic process that necessarily leads to action but rather a ” .“Thus, it appears that some authors consider that the noise plays a role of evidence, and that there is some additional signal involved, here termed “internal information.” Also in other papers, there is some ambiguity as to the respective roles of the variable factor  and the constant factor (evidence/imperative):Schurger et al. propose that the motor system constantly undergoes random fluctuations of RPs and that this random premotor activity is used as a substitute for actual evidence” . One possibility could be that there is a single overall mechanism, but with two different types of input, one being sensory evidence and the other being imperative.When considering one variable having evidence about another one would want it to fulfill some additional requirements. For example, the evidence should be able to “stand in” as a proxy of what it is representing . In the context of the SDM, the constant (or mean) component of the “evidence” in perceptual decision-making is replaced by a different constant factor, an imperative to move given by the demand characteristics of the task. To avoid confusion, we recommend using the term “imperative” in the context of the SDM rather than “evidence.” Sixth, we remind the reader that a key aim of the SDM, to provide a pre-decisional account of the RP, cannot be fully addressed by the model because this hinges critically on the nature of the noise fluctuations as being “objectively random” versus “epistemically random.” This of course is true of any scientific model that incorporates randomness, and may be very difficult to decide empirically, but at least the case is far from closed.The aim of this paper was to assess the level of evidence for the SDM and to address certain confusions that have arisen in the literature. First, we highlighted that there is no direct evidence based on neuronal-level measurements for the role of stochastic fluctuations in the RP and movement initiation and that the analyses are based on macroscopic signals averaged across many trials. This does not rule out the SDM as a model, but it clarifies what kind of data will be required to definitively validate the model. Second, we found that the purported evidence for the SDM from animal studies is limited and may not favor the SDM over other models. Third, we showed that a quasi-deterministic model where the parameters are fixed at the beginning of the trial (the LBA) makes very similar predictions to the SDM, including for the interrupted version of the Libet task, and fits well with a population of neurons found in an invasive monkey study. Fourth, we found that the literature has tended to ignore the deterministic component of the SDM, the imperative signal that accounts for an important part of signal input toward the threshold. Both the stochastic fluctuations and the imperative are necessary for reaching the threshold in realistic time periods with the published parameters of the SDM. Fifth, there is a confusion regarding the link between perceptual decision making and spontaneous movements. We have argued that the SDM is not just a special case of perceptual decision making, but without the evidence. Although mathematically identical to a leaky stochastic accumulator used to model perceptual decision making, the SDM does not incorporate any “evidence” There are some other questions that need to be addressed: Where is the ramp? The strong contribution of the constant (imperative) predicts a large initial rise of the accumulator signal at the beginning of the trial. This would predict a very stereotypical ramp at the beginning of the trial in the corresponding brain signals. In fact, invasive recordings from human single neurons have revealed a slow gradual ramping up of the signal prior to the time of decision .Could the ramp not be the buildup of the intention? The fact that the imperative signal plays such an important role in bringing about the decision could point to a re-interpretation of the components of the SDM model. One way would be to interpret the imperative signal as the largely deterministic and gradual buildup of the intention to move  and the experiments. These lab experiments impose constraints that are not present in real-world free-ranging actions. For example, there is an explicit or implicit affordance to move within a reasonable time-frame  and at the same time avoid being predictable or rhythmic, which The participant was required to perform the movement not rhythmically, but in irregular intervals” ([…] try not to decide or plan in advance when to press the button, but to make the event as spontaneous and capricious as possible.” (let the urge to act appear on its own at any time without any preplanning or concentration on when to act” (Sorry, but I never felt an urge to move”? As has been pointed out previously (It could also be useful to extend the scope from thinking about spontaneous movements in general to what happens specifically in spontaneous movement  to act” , p. 625.eviously  the key eviously , inhibiteviously , or geneeviously . StickinThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical approval was not required for the study involving humans in accordance with the local legislation and institutional requirements. Written informed consent to participate in this study was not required from the participants or the participants' legal guardians/next of kin in accordance with the national legislation and the institutional requirements.CB: Conceptualization, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing. BG: Conceptualization, Investigation, Writing – review & editing. J-DH: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing.


This guideline will provide a practical roadmap for management of SSc that builds upon the previous treatment guideline to incorporate advances in evidence-based treatment and increased knowledge about assessment, classification and management. General approaches to management as well as treatment of specific complications will be covered, including lung, cardiac, renal and gastrointestinal tract disease, as well as RP, digital vasculopathy, skin manifestations, calcinosis and impact on quality of life. It will include guidance related to emerging approved therapies for interstitial lung disease and account for National Health Service England prescribing policies and national guidance relevant to SSc. The guideline will be developed using the methods and processes outlined in Creating Clinical Guidelines: Our Protocol. This development process to produce guidance, advice and recommendations for practice has National Institute for Health and Care Excellence accreditation. The term began on 27 February 2012 and the current renewed accreditation is valid until 31 December 2023. More information on accreditation can be viewed at The current British Society for Rheumatology (BSR) guideline for SSc was published in 2016 , EUThe working group has identified the following key issues and draft questions related to them. The key issues and draft questions will be used to develop more detailed review questions, which will guide the systematic review of the literature.The working group recognizes the importance of timely diagnosis of SSc and that there have been advances in understanding early identification of the disease. Delays in diagnosis should be minimized. As previously, it is appropriate that early dcSSc is a particularly important diagnosis because of the early risk of severe internal organ complications and the need for specialist referral and initiation of disease-modifying treatment with immunosuppression.What is the best approach to timely diagnosis and specialist referral?How can people with SSc be classified for stratified medicine and management?What are the best treatments for early dcSSc?When and how should people with SSc be screened for concurrent malignant disease?What non-pharmacological treatments are supported by evidence, including vitamins, supplements, massage  and psychological support for early new diagnosis and established disease?This section will update and expand the management and treatment recommendations included in the previous BSR guidelines. Trials for several new treatments have been published for complications such as SSc-ILD and PAH. In addition, new approaches to treatment with established therapies are likely to be considered, including autologous haematopoietic stem cell transplantation. These topics will be considered in the following questions:Which people with SSc should be considered for autologous stem cell transplantation, taking into account the risks as well as potential short- and long-term benefits?How should ILD in SSc be managed and treated, taking into account new approved drugs such as nintedanib with emerging evidence supporting the use of targeted biological agents?What is the best evidence-based management and treatment for pulmonary hypertension, including PAH?What is the best evidence-based management and treatment for cardiac involvement?What is the best evidence-based management and treatment for Raynaud’s phenomenon (RP)?What is the best evidence-based management and treatment for digital ulceration?What is the best evidence-based management and treatment for critical digital ischaemia?What is the best evidence-based management and treatment for gastrointestinal complications, including nutrition, oropharyngeal and dental aspects (sicca syndrome)?What is the best evidence-based management and treatment for scleroderma renal crisis?What is the best evidence-based management and treatment for skin manifestations in addition to skin thickening and fibrosis such as telangiectasis and pruritis?What is the best evidence-based management and treatment for calcinosis in SSc?What is the best way to manage and treat neurological complications of SSc, including peripheral neuropathy, allodynia, cranial neuropathy and neuralgia?What is the best way to manage and treat musculoskeletal manifestations of SSc, including soft tissue loss, contractures, arthritis and bone health?What is the best evidence-based management and treatment for pregnancy and reproductive health problems, including male and female sexual dysfunction?What are the best interventions for general impact of SSc on health status and quality of life, including fatigue?The working group recognizes that there are challenges delivering high-quality equitable care for SSc across England and the devolved nations. This reflects the infrequency of SSc in primary and secondary care and its clinical diversity, as well as the need for comprehensive multispecialist and interdisciplinary clinical care. In addition, while treatment options are increasing, it is recognized that clinical impact can be limited, and even with optimal management there is a significant unmet medical need for people with SSc.The group will provide feasible and appropriate ways of benchmarking delivery of care and assessing standards so that audits of service delivery as well as patient-specific evaluations can be undertaken in a robust and standardized way. It is likely that SSc will provide a template that may be relevant to other uncommon multisystem autoimmune rheumatic diseases managed across the NHS.How can an audit of the guideline be best performed to assess service delivery and to undertake a patient-specific audit?The guideline is expected to be published in 2024.Chris Denton (chair), Voon Ong (rheumatologist), Ariane Herrick (rheumatologist), Maresa Carulli (rheumatologist), Francesco del Galdo (rheumatologist and EUSTAR guideline liaison), Elizabeth Renzoni (respiratory physician), John Pauling (rheumatologist), Emma Derrett-Smith (rheumatologist), Jeremy Royle (rheumatologist), Muditha Samaranayaka (rheumatologist), Nuala O’Donoghue (dermatologist), Michael Hughes (rheumatologist), Athiveeraramapandian Prabu (rheumatologist), Karen Douglas (rheumatologist), Bridget Griffiths (rheumatologist), Maya Buch (rheumatologist), Clare Pain (paediatric rheumatologist), Julia Spierings (autoimmune disease stem cell transplant expert), Luke Gompels (rheumatologist), Caroline Cotton , Aoife Tynan (pharmacist), Kim Fligelstone (patient representative), Georgina Pantano (patient representative), Begonya Alcacer-Pitarch , Louise Warburton , Emma Blamont and Sue Farrington (Scleroderma & Raynaud’s UK patient organization), Nina Goldman (respiratory medicine fellow), Enrico De Lorenzi (rheumatology fellow) and Elen Roblin (rheumatology fellow).