Among the participants who reported gambling activity (n = 113), 85.8% were not problematic gamblers, 8.9% were at-risk gamblers, and 5.3% were pathological gamblers. Only 0.2% of all subjects met the criteria for both pathological gambling and pathological video game use. The findings indicate that video gaming and gambling are common leisure times among adolescent students. However, a small but significant minority of these adolescents met the criteria for either severe problem gaming or gambling or both.Adolescence is characterized by emotional instability and risk-taking behaviours that can lead to, among other things, an increased risk of developing pathological video-gaming and gambling habits. The aim of this Study is to assess the prevalence and type of video gaming and gambling habits in adolescent students attending Italian upper-secondary schools. The cross-sectional study was conducted via an online survey using validated questionnaires. The primary outcome measures were the prevalence of past-year video gaming and gambling activities. The sample consisted of 502 adolescent students from first- and second-grade secondary schools. A total of 40.8% of participants were video gamers, 4.8% were gamblers, 17.8% were both video gamers and gamblers, and the remaining 36.6% were not players. Among participants who reported video gaming activity ( Adolescence is characterized by emotional instability and risk-taking behaviours that can lead to, among other things, an increased propensity to develop pathological video-gaming and gambling habits.The eleventh edition of the International Statistical Classification of Diseases and Related Health Problems (ICD-11), which was produced by the World Health Organization, defines gaming disorder as a pattern of recurrent or persistent gaming behaviour manifested by impaired control over gaming, exaggerated priority given to gaming (which takes precedence over daily activities as well as other life interests), and perpetuation or even intensification of gaming despite the occurrence of negative consequences. Furthermore, it defines gambling disorder using the same criteria but referring to gambling instead of gaming behaviours.Growing empirical evidence suggests that each of these two disorders is positively associated with adolescents’ mental health issues , 2, subsTherefore, it is not surprising that pathological use of video gaming and pathological gambling have become an emerging public health problem , 9. HoweThis is a cross-sectional study with convenient sampling. The participants were recruited from five first-grade secondary public schools (‘middle school’) and five second-grade secondary public schools (‘high school’) located in Brescia Province . Data collection was carried out from February 2020 until March 2021 through an online survey. A detailed description of the study protocol was published previously .The Video-Gaming Scale for Adolescents (VGS-A) and the Gambling Behavior Scale for Adolescents (GBS-A) were used to assess video gaming and gambling behaviours, respectively, that occurred during the last year. They classify the respondents as nonproblem, at-risk, or disordered gamer/gambler. Sociodemographic and educational information were also collected. For a detailed description of the measures, see , 12.Descriptive analyses were performed using R version 4.0.2 .n = 294), 68.0% were classified as nonproblem gamers, 24.5% as at-risk gamers, and 7.5% as disordered video gamers. On the other hand, among the respondents who reported gambling activity (n = 113), 85.8% were not problematic gamblers, 8.9% were at-risk gamblers, and 5.3% were pathological gamblers. Only 0.2% of all subjects met the criteria for both pathological gambling and pathological video game use. The demographic information and prevalence rates of video gaming and gambling activities of adolescent responders to the survey are reported in Table A total of 502 adolescent students completed the assessments. The mean age of the participants was 15.9 (SD = 1.93). Most of the participants were female (67.7%), attended high school (79.7%), and had never failed a school year (85.9%). The results indicate that 40.8% of participants were video gamers, 4.8% were gamblers, 17.8% were both video gamers and gamblers, and the remaining 36.6% were not players. More specifically, among participants who reported video gaming activity  of these adolescents met the criteria for either severe problem gaming, severe problem gambling or both. The evidence presented here is consistent with international literature , 8 and wThe major limitations of this study are the cross-sectional design, the use of self-report tools, and a limited sample size that did not allow the use of logistic regression analysis to determine the specific odds for various vulnerability and protective factors.Future studies should expand the sample size and include adolescents who do not attend school and represent different parts of the country. Assessing the prevalence of problematic video gaming use and gambling in adolescents would help to increase awareness about these emergent public health issues and take specific measures for preventing, identifying, managing, and treating these disorders.


Introduction: Several open access platforms are currently available to help facilitate this online learning; however, whether the platforms are generalized or specialized, peers do not evaluate videos, and they may teach unverified techniques. The purpose of this study was to compare the quality of each website’s content using a specific measurement, analyzing the pedagogical quality of Femoroacetabular impingement (FAI) arthroscopic videos on YouTube versus VuMedi. Methods: A prospective study analyzing 20 arthroscopy videos of arthroscopic FAI surgery on two hosting platforms online: 10 on the generalist platform YouTube and 10 on a specialized platform VuMedi. Results: The average length of the YouTube videos was 503 ± 355.02 s (198–1430). The average number of views for the YouTube videos was 56,114.6 ± 116,832.61 . The average length of the VuMedi videos was 797.5 ± 522.5 s (185–1927). The average number of views for the VuMedi video was 10,404.7 ± 10,071.2  The average LAP-VEGaS scores of the YouTube and VuMedi videos were 8.2 ± 3.47 (3–15) and 11.95 ± 2.64 (6–15), respectively (p < 0.0005). Discussion: The use of a specialized website makes it possible to obtain educational videos of better quality. Health professionals need to be aware of this when using it as a resource for learning. Therefore, it should be in the interest of educational institutions to participate in the sharing of videos on this platform or to create their own platform to improve the quality of the information provided and the overall pedagogical experience.Level of evidence: II Femoroacetabular impingement (FAI) is a medical entity that has been historically known since 1936, and its definition was only recently updated by Ganz in 2003 following the development of hip arthroscopy .Continuing medical education (CME) and resident learning are changing with the new rules of the health system modernization . Video phttps://www.youtube.com) is considered the second most popular website in the world, following Google (https://www.google.com) [https://www.VuMedi.com): the first network for video education for doctors. This platform aims at helping doctors optimize patient care decision-making by providing authentic and complete education videos from various institutions and practitioners.In our modern world, several open access platforms are available, with over four billion videos available. YouTube (gle.com) . In addigle.com) , 11. TheHowever, these two platforms are neither peer-reviewed nor professionally verified techniques. This study hypothesized that a specialized platform presented videos of higher educational merit than a generalist platform.This study compares the quality of each website contents using a specific measurement, analyzing the pedagogical quality of FAI arthroscopic videos on YouTube versus VuMedi.A prospective study was conducted between January and March of 2021. Twenty arthroscopy videos of arthroscopic FAI surgery were analyzed on two different online hosting platforms: 10 on the generalist platform YouTube and 10 on a specialized platform, VuMedi. Two surgeons made the initial selection, a junior surgeon and a senior consultant practicing hip arthroscopy. The study was not subject to institutional review by our ethics committee because it did not involve the use of personal data.The selection objective was to choose 10 videos on YouTube and ten on VuMedi. Only one observer made the selection using the following keywords, “FEMOROACETABULAR IMPINGEMENT” or “FAI,” in the search. Before videos were selected, they were sorted by those most viewed. Regarding the YouTube platform, the keyword addition of “HIP ARTHROSCOPY” was required to obtain the ten videos. The following exclusion criteria were used for the videos: incomplete videos, arthroscopies not treating FAI, open surgery, too short, missing video or webinar, FAI rehabilitation, FAI clinic or imaging, patient information included, non-English, and duplicates. The results were deliberately limited to ten videos per platform to simulate a practical search of a junior user, not a systematic search.Two surgeons independently evaluated each video. For each video, the length and number of views at the time of analysis were recorded. The 10 videos in each group were analyzed and evaluated according to two scoring standards.The first LAP-VEGaS-FAI is an adaptation of the LAP-VEGaS score, which judges the character of the pedagogical merit of the endoscopic surgical videos . The LAPThe second score allowed the evaluation of the perceived quality of the video. The reviewers were asked if they would have recommended the video to a peer or resident, and they were also asked if they would have accepted the video for publication and/or a podium presentation, using closed-ended questions and a 5-point Likert scale. © SPSS© Statistics software, version 23.0.0.0. Quantitative variables were presented by mean, standard deviations, minimum, and maximum values. Qualitative variables were matched with their number and percentage. Unmatched quantitative variables were compared using a student test, and the matched variables were compared using a Wilcoxon test. Qualitative variables were compared using a χ2-test. Intra-class correlation coefficients were calculated to define inter- and intra-observer variability for all measured parameters. A difference was considered statistically significant when the degree of significance was less than or equal to 0.05.The statistics were calculated using IBMp < 0.0001) and the second research modality (p = 0.02)  .p = 0.16) and the number of views (p = 0.25).The average length of the YouTube videos was 503 ± 355.02 s (198–1430). The average number of views for the YouTube videos was 56,114.6 ± 116,832.61 . The average length of the VuMedi videos was 797.5 ± 522.5 s (185–1927). The average number of views for the VuMedi video was 10,404.7 ± 10,071.2 . Despite an average of five times more views in the YouTube group and an average duration of 1.5 times longer in the VuMedi group, there was no statistical difference between the two groups regarding the duration . The average LAP-VEGaS scores of the YouTube and VuMedi videos were 8.2 ± 3.47 (3–15) and 11.95 ± 2.64 (6–15), respectively  against a specialized platform (VuMedi). The average score obtained by the LAP-VEGaS-FAI rating was 8.2 for YouTube, compared to VuMedi’s 11.95. Despite a lower viewership , the specialized platform is more beneficial to surgical pedagogy.The advent of information and communication technologies can potentially improve surgical education. We found that video learning is currently a feature of surgical preparation for residents and surgeons. A thriving diffusion presents the opportunity to account for the current flaws in surgical education. However, many ethical entities could be breached while building those surgical videos. Dorfman et al. reported in a systematic review that sharing live videos or patients’ photos in plastic surgery can have significant ethical implications, and particular guidelines should be used . In addiThe LAP-VEGaS-FAI score comes from the LAP-VEGAS score and is conditioned for this study to evaluate videos that treat femoroacetabular impingement . Prior tFinally, teamwork and communication remain essential qualities for safe and effective performance. They have not been explored in this video assessment tool, which focuses on the technical skills of the surgeon . In addiAmong the other limitations, each video was only analyzed twice for simplicity. The study by Celentano et al. reported identical score reliability between two or three assessors . On the This study has a strong potential to be one of the few articles in the field of orthopedic surgery to be interested in surgical educational videos. This can lead to a second comparative study that will assess and compare the quality of training between traditional academic teaching and video teaching.Many surgically treated femoroacetabular impingement videos analyzed in this study did not demonstrate high-quality content when applying the LAP-VEGaS criteria. Using a specialized website makes it possible to obtain better quality and content educational videos. Health professionals need to be aware of this difference when using random surgical videos as a resource for learning. Therefore, it should be in the interest of educational institutions to share optimal and complete surgical videos on those platforms and/ or to create their own platform to improve the quality of the information provided and the overall pedagogical experience.


Semiconducting polymer dots (Pdots) are rapidly becoming one of the most studied nanoparticles in fluorescence bioimaging and sensing. Their small size, high brightness, and resistance to photobleaching make them one of the most attractive fluorophores for fluorescence imaging and sensing applications. This paper highlights our recent advances in fluorescence bioimaging and sensing with nanoscale luminescent Pdots, specifically the use of organic dyes as dopant molecules to modify the optical properties of Pdots to enable deep red and near infrared fluorescence bioimaging applications and to impart sensitivity of dye doped Pdots towards selected analytes. Building on our earlier work, we report the formation of secondary antibody-conjugated Pdots and provide Cryo-TEM evidence for their formation. We demonstrate the selective targeting of the antibody-conjugated Pdots to FLAG-tagged FLS2 membrane receptors in genetically engineered plant leaf cells. We also report the formation of a new class of luminescent Pdots with emission wavelengths of around 1000 nm. Finally, we demonstrate the formation and utility of oxygen sensing Pdots in aqueous media. Luminescent semiconducting polymer dots—Semiconductor polymer dots (Pdots) have rapidly garnered interest as an alternative to semiconductor quantum dots and fluorescent dyes in nanoscale fluorescence imaging and sensing applications  (PFBT)  and Poly [2-(5-cyano-methylhexyloxy)-1-4-phenylene] (CNPPP)  were purchased from American Dye Source, Baie-D’Urfe, QC, Canada. Poly  (PSMA) [67:33] (MW 7500) was purchased from Polysciences, Warrington, PA, USA. Palladium catalysts and thionyl chloride were obtained from Sigma-Aldrich, St. Louis, MO, USA. Diamine and boronic acids were obtained from Ambeed, Arlington Heights, IL, USA. Tetrahydrofuran , Phosphate Buffered Saline (PBS), and Bovine Serum Albumin (BSA) were purchased from Fisher Scientific, Hampton, NH, USA. Dulbecco’s Modified Eagle Medium, Fetal Bovine Serum, HEPES buffer and Sodium Pyruvate were purchased from ThermoFisher Scientific, Waltham, MA, USA. Anti-Rabbit IgG (H+L), F(ab) fragment antibody produced in goat, N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide, Tris-HCl, NaCl, Driselase, MES, Poly(ethylene glycol) 3350 MW, Sodium Azide, Tris ruthenium (II) dichloride, skim-milk powder, Tween-20, and Triton x-100 were purchased from Millipore Sigma, St. Louis, MO, USA. GST-tag antibody [HRP] produced in rabbit was purchased from GenScript, Piscataway, NJ, USA. Nitrocellulose membrane and Western blot chemiluminescent substrate kit were purchased from Fisher Scientific, Hampton, NH, USA. Paraformaldehyde (PFA) was purchased from Electron Microscopy Sciences, Hatfield, PA, USA. Murisage and Skoog mixture was purchased from Caisson, Smithfield, UT, USA. DYKDDDDK Tag Recombinant Rabbit monoclonal antibody was purchased from Invitrogen, Waltham, MA, USA. All commercial materials were used as received.Synthesis of Near Infrared Emitting Dyes—The synthesis and characterization of red emitting chlorins P660 bisbis,2,5thiade. Refs. ,45,46,47Pdots Synthesis and Characterization—Near infrared emitting dye-doped Pdots were synthesized via a nanoprecipitation method  produced in rabbit) for 1 h at RT. Afterward, the nitrocellulose membrane was washed three times with TBST buffer (3 × 5 min) and incubated with Western blot chemiluminescent substrate for 1 min, and then was used to expose X-ray films in the dark room.d method . A 1 mL Construction of FLAG-FLS2 transgenic plants—The FLAG sequence-fused FLS2 genomic fragment was generated by a two-fragment polymerase chain reaction (PCR) approach, in which primers including the FLAG sequence were used in two separate PCR reactions. Each fragment of FLS2 was amplified by PCR from genomic DNA of wild-type plants using gene specific primers containing the FLAG sequence. An overlapping PCR reaction was then performed to generate the whole FLS2 genomic fragment with the FLAG sequence inserted behind the signal peptide sequence of FLS2. The PCR products were cloned into pDONR-Zeo ) by BP reaction and subsequently cloned into the binary vector pGWB1 ) by LR reaction. The construct was then transformed into A. thaliana fls2 knockout mutant plants via the Agrobacterium strain GV3101 using the ‘floral-dip’ method bisbis,2,5thiadPhorphyrin Pdots (PPD) show similar absorption spectra a with a Cryo-TEM of antibody coated Pdots—Cryo-TEM measurements, shown here for the first time, reveal the Pdots to be spherical  dyes . The absorbance of Pdots that are doped with BBTD dyes are shown in Oxygen sensitive Pdots—While studies to expand the utility of luminescent Pdots as imaging probes are on-going, there is a growing interest in the development of Pdot-based optochemical nanosensors. Recently, we synthesized oxygen-sensing Pdot based on tris  ruthenium (II) dichloride Ru(dpp)32+. Ru(dpp)32+ features a highly efficient metal to ligand charge transfer (MLCT) transition which results in a long fluorescence lifetime, in the microsecond time scale, andhigh sensitivity to O2 [32+ and is more resistant to water-induced substitution reactions [32+. The structures of the polymer and dye are shown in 32+ shows significant fluorescence enhancement when doped in the Pdot compared to the dye alone. The dye emission shows a strong response to N2 and O2 saturation while the polymer emission remaines constant 32+-Pdots to five cycles of alternating O2 and N2 saturation. 2 was completely reversible, showing that the Pdots are highly stable and effectively measure the level of oxygen under these conditions.ty to O2 . It exhieactions . In formconstant b. To tesPolymer dots as fluorescence probes and sensors have rapidly advanced in recent years. The key development that led to the explosion of Pdot applications came with the discovery of dye dopants. Doping Pdots with organic dyes, either through encapsulation in the polymer matrix or through polymerization, enables combining the superb optical properties of Pdots with imaging and sensing capabilities of their doped organic fluorophores. This paper builds on a previous paper we published where we described the synthesis and characterization of near infrared emitting Pdots in detail . It high


Cdkn1a) was the top hit among genes persistently induced by hyperglycemia and was associated with induction of the p53-p21 pathway. Persistent p21 induction was confirmed in various animal models, human samples and in vitro models. Tubular and urinary p21-levels were associated with DKD severity and remained elevated despite improved blood glucose levels in humans. Mechanistically, sustained tubular p21 expression in DKD is linked to demethylation of its promoter and reduced DNMT1 expression. Two disease resolving agents, protease activated protein C (3K3A-aPC) and parmodulin-2, reversed sustained tubular p21 expression, tubular senescence, and DKD. Thus, p21-dependent tubular senescence is a pathway contributing to the hyperglycemic memory, which can be therapeutically targeted.A major obstacle in diabetes is the metabolic or hyperglycemic memory, which lacks specific therapies. Here we show that glucose-mediated changes in gene expression largely persist in diabetic kidney disease (DKD) despite reversing hyperglycemia. The senescence-associated cyclin-dependent kinase inhibitor p21 ( Persistent diabetic complications despite controlled blood glucose levels, known as hyperglycemic memory, remain a poorly understood phenomenon in diabetic kidney disease. Here the authors identify senescence-associated gene p21 as a regulator of hyperglycemic memory, the suppression of which improves hyperglycemic memory and renal function. Current therapies, particularly blood sugar control and renin angiotensin aldosterone system (RAAS) inhibition, at best delay disease progression2. Sodium-glucose co-transporter-2 inhibitors (SGLT2i) are promising therapeutic adjuvants, but the long-term consequences, the associated risk profile and their ability to halt or even reverse disease progression remain largely unknown3. The persistence of diabetes-associated complications despite improved blood glucose control is known as metabolic or hyperglycemic memory4. Disease reversal, e.g., the improvement of already established DKD, remains an unmet medical need2. Accumulating evidence suggests that epigenetic mechanisms such as DNA methylation or posttranslational histone modifications are mechanistically linked to hyperglycemic memory4, but these insights have not yet led to new diagnostic or therapeutic approaches.Diabetic kidney disease (DKD) is now the leading cause of end-stage renal disease in industrialized countries, affecting approximately 40% of diabetic patients5, yet their potential role in hyperglycemic memory is unknown. Senescence, reflecting permanent proliferative arrest, primarily affects renal tubular cells in chronic kidney disease, including DKD7. The severity of tubular senescence correlates with kidney injury, loss of renal function, and renal fibrosis9. Senescence is associated with inflammation and with mitochondrial reactive oxygen species (ROS), all hallmarks of DKD10.Several pathomechanisms have been linked to DKDShc is epigenetically induced in diabetic patients and mice, and p66Shc deficiency protects against diabetic glomerulopathy in mice12. The cytoprotective and signaling-competent coagulation protease-activated protein C (aPC) reverses epigenetically induced p66Shc expression in experimental diabetes13. These data illustrate that (i) epigenetic changes in diabetic vascular disease are in principle reversible and (ii) that the hyperglycemic memory is linked to endothelial dysfunction, as aPC’s plasma levels reflect endothelial function15.The mitochondrial redox regulator p6616. Tubulointerstitial damage in DKD is characterized by the accumulation of extracellular matrix (fibrosis), inflammation, and senescence of tubular cells17. The contribution of senescence to hyperglycemic memory in DKD remains unknown.Both hypomethylation and hypermethylation have been reported in association with diabetes and glomerular and tubulointerstitial damage in the context of DKDCdkn1a is a top regulator of hyperglycemic memory. Sustained Cdkn1a expression, depends on reduced DNMT1 mediated DNA methylation. Targeting sustained p21 expression by aPC or aPC-based therapies reverses persistent renal injury and improves renal function in DKD.In the current study, we combined hypothesis-free approaches with analyses of human samples, genetic and interventional mouse models and in vitro studies to gain insights into mechanisms of hyperglycemic memory. We show that glucose-mediated changes in gene expression largely persist in DKD despite lowering glucose. The senescence associated gene versus DM + SGLT2i), resulting in lower albuminuria compared to untreated diabetic mice (DM-22), but albuminuria remained increased compared to non-diabetic control mice . Persistent hyperglycemia was induced in mice for 16 weeks , at which stage mice had developed albuminuria Fig. . Subsequ C, Fig. . Thus, wCdkn1a (p21) was among the top persistently induced genes , untreated (DM-22) and SGLT2i-treated (DM + SGLT2i) diabetic mice. Compared to controls (C), 291 genes were induced, and 746 genes were suppressed in diabetic mice  and a similar experimental set up  cell-autonomously induced p21 expression within 24 hours in human kidney  staining. SA-β-gal staining was increased in tubular cells of diabetic mice (T1DM and T2DM models) and remained high despite reducing blood glucose levels using SGLT2i or insulin  in patients with DKD  but not in diabetic patients without DKD (DM-DKD) or non-diabetic controls , but not in healthy controls  and compared these to patients with normal renal function , we obtained renal biopsies from patients with CKD unrelated to diabetes mellitus. Again, we observed p21 expression predominately in tubular cells  the increase of tubular p21 expression in diabetic patients with DKD, but not in diabetic patients without DKD, (ii) the increase of urinary p21 in diabetic patients in association with an increasing KDIGO grade of CKD, and (iii) the persistent increase of urinary p21 despite improved blood glucose levels in diabetic patients support a model in which renal tubular p21 expression reflects persistent tubular damage in DKD, contributing to the hyperglycemic memory. These data raise the question of whether reversing p21 induction may “erase” the hyperglycemic memory.Shc expression in diabetic vascular diseases13. To determine whether aPC reverses glucose-induced and sustained p21 expression, we conducted in vitro experiments using HEK-293 and mouse PTCs. HG induced p21 expression in HEK-293 and PTCs, and the expression remained high despite normalization of glucose levels  in PTCs upon exposures to 25 mM glucose for 24 h followed by 24 h of normal glucose levels (5 mM) was normalized upon addition of aPC (20 nM) at the time of returning cells to normal glucose concentrations 24. This point mutation mimics impaired TM-dependent protein C activation due to oxidative damage25. If diabetic, TMPro/Pro mice display increased glomerular damage15, but the consequences for tubular damage and tubular senescence have not been investigated hitherto. In addition to albuminuria and normalized kidney weight, interstitial fibrosis and tubular cell size (reflecting cellular hypertrophy) were enhanced in diabetic TMPro/Pro mice after 22-weeks of persistent hyperglycemia as compared to diabetic wild-type littermates despite comparable blood glucose levels  and KIM-1  expression (mRNA and protein levels) were increased in diabetic TMPro/Pro mice as compared to diabetic wild-type mice -dependent protein C activationels Fig. . Concomiice Fig. . The incice Fig.  and induPro/Pro mice with genetically superimposed p21 deficiency (TMPro/Pro x p21−/−). Despite comparable blood glucose levels among diabetic mice (22 weeks of persistent hyperglycemia), genetically superimposed p21 deficiency markedly reduced albuminuria and normalized kidney weight in hyperglycemic TMPro/Pro mice , we analyzed total DNMT activity. HG reduced DNMT activity in HEK-293 cells in vitro, which remained low despite normalization of glucose levels (HG/NG), but was restored upon concomitant exposure to aPC , Fig. 26. Among these, expression of Tet2 and Kdm6a were persistently induced despite glucose lowering by SGLT2i or insulin , DNMT1 expression was reduced , adding aPC in a subgroup of mice  or (ii) specifically inhibited DNMT1 expression  on top of SGLT2i and aPC treatment in mice with 16 weeks of established hyperglycemia  added the pan DNMT inhibitor 5-aza-2’-deoxycytidine 30, was confirmed in mouse PTCs  and the endothelial protein C receptor (EPCR)12: (i) 3K3A-aPC, an aPC mutant with largely reduced anticoagulant but sustained signaling efficacy and (ii) parmodulin-2, a small peptide mimicking biased aPC signaling via PAR133. Again, after 16 weeks of hyperglycemia we initiated treatment with SGLT2i in combination with phosphate-buffered saline , 3K3A-aPC, or parmodulin-2 , a gene regulating cell proliferation and senescence34, was the top hit among genes that were persistently induced. p21 itself is controlled by DNMT1. Restoring DNMT1 and reducing p21 expression by exploiting aPC signaling reverses already established DKD in mice  the level and duration of p21 induction determine the onset of cellular growth arrest; (ii) p21 expression is sufficient to induce senescence; (iii) p21 expression restricted to tubular cells is sufficient to induce renal fibrosis following acute kidney injury37; and (iv) p21 induces endoreduplication38, which may impair renal tubular cell recovery following acute kidney injury40. While p21-induced cell-cycle arrest and/or endoreduplication are plausible explanations for impaired renal recovery, the exact mechanisms through which p21-induced senescence prevents renal recovery despite glucose normalization remain to be determined. Yet, our data support a function of sustained p21 expression in the tubular compartment given (i) the cell-autonomous, glucose-dependent induction of p21 in tubular, but not in other renal cell types, (ii) the induction of tubular p21 expression before detectable glomerular injury, (iii) the sustained p21 expression in tubular cells despite glucose lowering, and (iv) the tubular, but not glomerular protection in diabetic TMPro/Pro x p21−/− mice. However, given intrarenal crosstalk between the glomerular and tubular compartments we currently cannot exclude that persistent glomerular damage contributes to tubular p21 expression. These data support a model in which prolonged and sustained p21 expression causes tubular senescence and compromises tubular repair capacity in DKD patients41, even after glycemic control has improved.Studies using whole body p21 knock out mice previously established a role of p21 in DKD43, supporting a crucial function of DNMT1 in the renal tubular compartment. However, DNMT1 is not expected to specifically target p21 and its reduced expression in DKD is expected to de-repress (hence induce) other genes. Accordingly, about 93% of genes induced in hyperglycemic mice remained elevated despite lowering blood glucose levels, while only about 36% of genes suppressed in hyperglycemic mice remained repressed , while aPC suppressed p21 expression in vitro and in vivo. Therefore, endothelial dysfunction with loss of endothelial TM expression and associated reduced aPC generation may both trigger (by regulating ROS)12 and maintain  p21 expression. The question of how tubular p21 expression is induced is addressed in ongoing work.While the current results indicate that sustained p21 expression is linked to reduced DNMT1 expression and p21-promoter demethylation, the mechanism initially inducing p21 expression may be unrelated. DNMT1, p21 and γ-H2A.X are associated with DNA damage response (DDR) and, hence, it is possible that increased tubular ROS generation in DKD induces DNA damage and DDR, inducing p21 expressionShc, a mitochondrial protein promoting ROS-generation, in podocytes (glomerular cells) and macrophages13. Furthermore, inhibitors of coagulation factors fIIa (dabigatran) or fXa (rivaroxaban) differentially regulate aPC generation, which is linked to differential gene expression and epigenetic marks49. Epigenetic control of gene-expression by aPC provides a rationale for the long-lasting effects of therapeutic aPC applications despite its short half-life50 and–considering the broad cytoprotective effects of aPC–may be relevant in other disease settings51.The regulation of DNMT1 by aPC raises the intriguing possibility that this coagulation protease may epigenetically control other genes. Indeed, in addition to its effect on p21, aPC reverses glucose-induced expression of p66Shc expression in tubular cells and in podocytes, respectively, is in agreement with the engagement of different receptor complexes and different mechanisms regulated by aPC in the tubular and glomerular compartments. Thus, aPC signals via a heterodimer of PAR3 with PAR2 (human) or PAR1 (mouse) in conjunction with integrin β3 in podocytes, while in tubular cells aPC signals via a heterodimer of PAR1 and EPCR52 (and current results). Mechanistically, aPC targets the unfolded protein response in glomerular cells53 and cellular senescence in tubular cells (this study). These observations support a concept in which different pathomechanisms in the glomerular and tubular compartments promote DKD55. Importantly, aPC ameliorates both glomerular and tubular damage despite targeting different and cell-specific receptor-complexes and pathways, providing a rationale for its high efficacy in animal models of renal injury56 (and current results).The reversal of glucose-induced and sustained p21 or p6650, suggesting that this coagulation protease is a disease-resolving mediator. However, the therapeutic value of aPC itself is limited by its inherent anticoagulant properties. As previously shown in acute disease models57 and here in a chronic disease model, harnessing specifically the signaling-dependent disease-resolving effects of aPC is feasible by mimicking its signaling properties. aPC mutants lacking binding sites for the coagulation factor Va (3K3A-aPC) or small molecules mimicking biased signaling of aPC via the G-protein coupled receptor PAR150 (parmodulins) are devoid of an anticoagulant function, rendering aPC-based therapies feasible not only in acute, but also chronic diseases.aPC has shown beneficial effects in a number of acute and chronic disease models58. Intriguingly, aPC-based therapeutics provided a benefit on top of SGLT2-inhibtion. Therefore, despite recent data suggesting that SGLT2 inhibitors are useful new therapeutic adjuncts in patients with diabetes, additional therapies for full kidney recovery may be required.We used established therapeutic approaches to improve hyperglycemia (SGLT2i and insulin) and observed sustained p21 expression in both cases, supporting the notion that sustained p21 expression reflects the hyperglycemic memory independent of the glucose-lowering intervention and independent of exposure of tubular cells to urinary glucose . Lowering blood glucose levels provided partial protection, as reflected by halted disease progression, which is in agreement with the protective effects of SGLT2 inhibitors in clinical studies59. If confirmed, these patients may not respond to glucose lowering alone, but may profit from a potential therapy targeting tubular senescence.To circumvent the lack of kidney biopsies from diabetic patients before and after improved blood glucose control, we determined urinary p21 levels. p21 was strongly increased in the urine of most diabetic patients and increased with the severity of DKD. Distribution of p21 levels in advanced stages of DKD appeared to be dichotomic, with a small subgroup of patients presenting with low urinary p21 levels despite kidney dysfunction Fig. . Conside60.In summary, we identified a pathway contributing to hyperglycemic memory in the kidney and provided experimental in vivo evidence that targeting hyperglycemic memory in the context of DKD is feasible, potentially by mimicking the cytoprotective effects of aPC The objectives of this study were to identify mechanisms contributing to the hyperglycemic memory in the context of DKD and to test whether this memory could be reversed using suitable therapeutic interventions. We analyzed kidneys from diabetic mice with and without adjusted blood glucose levels using RNAseq, immunostaining, western blotting and quantitative polymerase chain reaction (qPCR). All uncropped blots and gels are provided within the source data. We confirmed our findings in human kidney biopsies and in urine samples from a large cohort of diabetic patients (LIFE-ADULT). Mechanistic studies using gene knockdown and analyses of DNA methylation were conducted in human and mouse renal cells in vitro.CIP1/WAF1 constitutive knock-out (p21−/−) mice were obtained from Jackson Laboratory . Non diabetic C57BLKsJ-db/+ (db/m) and diabetic C57BL/KSJ-db (db/db) male and female mice were obtained from Janvier . TMPro/Pro mice have been previously described24. TMPro/Pro mice were crossed with p21−/− mice, to generate TMPro/Pro x p21−/− double mutant mice. In some mice hyperglycemia was induced in 8 weeks old mice using low dose streptozotocin (STZ) injection following established protocols 52. Age-matched control mice received sodium-citrate. Blood and tissue samples were collected after 22 weeks of persistent hyperglycemia52. Subsets of mice were treated with a sodium-glucose co-transporter 2 inhibitor 53, with insulin glargine , with 5-aza-2’-deoxycytidine 13, an aPC variant lacking specifically anticoagulant function 31, the biased PAR1 agonist parmodulin-2 33, DNMT1 vivo morpholino 62, or DNMT1 mismatch vivo morpholino (Scr.DNMTi). Control mice received PBS every other day . All interventions were conducted for six weeks, starting after 16 weeks of persistent hyperglycemia. Albuminuria indicative of DKD was ensured before initiating treatment. All animal experiments were conducted following standards and procedures approved by the local Animal Care and Use Committee .Wild-type male and female C57BL/6 and p2130. Immortalized Boston University mouse proximal tubular cells (BUMPT) were obtained from Dr. Wilfred Lieberthal (Boston University) and cultured as described previously30. Please refer to supplementary methods for further details.Human embryonic kidney cells (HEK-293) were routinely grown and maintained according to supplier’s instructions . Mouse primary proximal tubular cells (PTC) were isolated and cultured according to established protocols21 were fasted on average for 12.8 ± 1.9 h. LIFE-ADULT participants were compensated with a travel allowance. Urine samples from the FMD study (DRKS00014287) and HEIST-DiC observational study were obtained after ethical approval  and informed consent. For the FMD study, the primary and secondary outcomes are not a part of this study and have been separately published23. The participants did not receive any compensation. Patients receiving de novo a SGLT2 inhibitor were identified in the diabetes outpatient clinic. Please see Supplementary Tables Human renal biopsy samples were provided by the tissue bank of the National Center for Tumor Diseases . Human urine samples obtained from the local outpatient clinic were obtained based on informed consent . All participants of the LIFE-ADULT cohort t-test and ANOVA or non-parametric Mann–Whitney-Test and Kruskal–Wallis test, as appropriate, and post-hoc comparison with the method of Tukey, Bonferroni or Sidak’s multiple comparison. The Kolmogorov–Smirnov (KS) test or D’Agostino–Pearson-Normality-test was used to determine whether the data are consistent with a Gaussian distribution. Prism 9 (www.graphpad.com) software was used for statistical analyses. Statistical significance was accepted at values of p < 0.05.The data are summarized as mean ± SEM (standard error of the mean). Statistical analyses were performed with parametric Student’s Further information on research design is available in the Supplementary Information FileReporting Summary


Aquila fasciata) collected from 2000 to 2022, I show how the inappropriate use of video surveillance cameras could result in negative effects on the reproduction of a threatened species through a before-and-after control-impact study design. Pairs under video surveillance showed lower productivity, lower breeding success and unusual delayed laying dates. The installation of cameras close to the laying date, coinciding with the mating phase of individuals, most of them subadult inexperienced birds; in combination to the reiteration of visits to the nests once the cameras were installed to check the system, particularly during the incubation period and early stages of breeding; and the installation of cameras in a particular area subject to constant human disturbance, might explain these results. Potential management actions to mitigate the effect of the installation of video cameras on birds’ behaviour should include the need to plan the intervention dates, testing the systems beforehand under controlled conditions and adequate post-installation monitoring to avoid unnecessary disturbance to animals. Finally, I urge the scientific community to report the potential negative effects observed in their studies, especially if the target species are threatened with extinction.Video surveillance cameras installed on birds’ nests are a cost-effective tool to study many aspects of ecology and behaviour that would otherwise be practically impossible to obtain. However, although most studies report neutral effects of cameras on birds, very few studies analyse in detail the potential negative effects of their use, particularly on raptors. Here, using a long-term database of a population of Bonelli’s eagle ( The use of camera traps, trail cameras, video cameras, and other remote monitoring devices has made it possible to investigate many aspects of the reproductive ecology of birds that would otherwise be difficult to address through direct observation2. In particular, the use of remote cameras installed in nests allows for getting detailed information on animal behaviour including continuous monitoring throughout the breeding season5, nest predator identification8, prey identification13, behavioural studies of nesting birds19; and even to promote public participation in citizen science projects to study birds’ diet20.The use of video surveillance cameras installed on nests is a cost-effective tool to study many aspects of the breeding ecology and behaviour of birds22 and technological innovations have allowed significant advances in improving both the quantity and the quality of the images received, going from the first prototypes based on 35 mm photographic cameras to video surveillance cameras remotely controlled that allows real-time monitoring and public access through the Internet (review in23). Although most studies report neutral effects of the use of cameras inside nests , very few studies report the possible potential effects that the installation of electronic devices inside nests might cause on behaviour  or direct and indirect effects on breeding performance . For example, among the few studies that exist about this topic, some have reported evidence that the use of camera equipment may reduce nest predation rates28, might bias data collected on predator identity (review in29), or even could cause nest abandonment in extreme cases30. Other studies, in turn, show that, in general, the cameras provide interesting valuable results but alter the behaviour of breeders, at least temporarily13, whereas others report shorter habituation time in those birds with more experience in front of the cameras31. Ultimately, several studies report technological limitations and usual technical failures that make it necessary to check the cameras continuously, with the potential risk of disturbance at nests33.Remote monitoring of nests using electronic devices is a technique with decades of experimentation. The first studies date back to the fifties of the last centuryAquila fasciata) nests in the south of the Castellon province (eastern Spain) to study eagles’ diet. This species is currently highly threatened in Spain, being the only species of a large bird of prey that is experiencing an effective regression of its population in many parts of the country34. Specifically, at the regional level, the species has gone from being considered a “vulnerable” species to “endangered of extinction” in the Valencian Community35. This change in risk category was based on a population viability analysis in which, fundamentally thanks to the combined use of GPS transmitters and long-term field monitoring, it was found that the population trend is compromised in the medium and long term due to a high pre-adult and adult mortality, analogous to that reported in other neighbouring geographic regions38. In particular, since 2000, the Bonelli’s eagle population has suffered a decline of 48.6% in the region, with an observed reduction of the breeding population from 31 territories occupied in 2000 to 18 occupied in 202240. Specifically, in the south of this geographical region, video surveillance cameras were installed by the regional government in the latest two breeding seasons, in an area of high adult mortality41 and where the recreational use of the environment for leisure activities has increased significantly in recent years, which has resulted in a significant increase in disturbance to wildlife in general and the endangered Bonelli’s eagle in particular42.At the end of 2020, upon the request of some forest rangers, the regional government of the Valencian Community  began a project consisting of the installation of video surveillance cameras in Bonelli’s Eagle  methodology45 in territories in which video surveillance cameras were installed and in control territories monitored by field observation throughout the 23-years study period. Once reported the results of this work, it is intended to inform the regional government about the possible causes that could be behind the decrease in breeding performance observed in pairs in which video surveillance cameras were installed. Furthermore, I discuss the advantages and potential drawbacks of the use of video-surveillance cameras to study raptors in a general context.Since the year 2000, I have been collecting an annual monitoring database of the Bonelli’s eagle population in the study area in which information on occupation, breeding performance and demographic parameters is systematically collected thanks to the combination of field monitoring techniques, bird ringing and telemetry information2 and ranges in elevation from the sea level to 1814 m of the highest mountain. The climate is Mediterranean, with annual mean temperatures varying from 17 °C along the coast to 8 °C in the inner highlands. The dominant landscape is composed of Mediterranean scrublands dominated by Pistacia lentiscus, Rosmarinus officinalis, Chamaerops humilis, among others, oak forests  and Mediterranean evergreen forests . Further details on the description of the study area are available at47.The study area is located in eastern Spain and includes the entire Castellón province. The area covers 6612 km47 were visited at least three times: one at the start of the breeding season in January–February to check occupancy, courtship behaviour and egg-laying; a second one at the middle of the breeding season in March–April to check early breeding failure, confirm the onset of reproduction and record the first chicks; and a third visit at the end of the breeding season in May–June to confirm breeding success and nestlings’ age based on feather development48. All observations were made 300 m away from cliff nest sites with 20–60× field spotting scopes (Leica Televid 77 and Swarowski AT80 telescope) during clear days to avoid disturbance to eagles. A territory was considered to be occupied if it was observed nests with green branches, typical pair behaviour, courtship, brood rearing activity or nestlings39. Pair composition was also recorded. To this end, the age of the breeding individuals was recorded according to plumage characteristics. Birds were considered subadults if they were in their third and fourth calendar-year and adults if they were from the fifth calendar-year onwards49. Juveniles in their first and second calendar-year do not breed since sexual maturity is usually reached in their third or fourth calendar-year. Once recorded field data, breeding information was summarized into the following reproductive parameters as usually reported in raptors’ literature: productivity = fledged chicks/pair; breeding success = successful pairs/pairs initiating reproduction  (further details in39). Chicks were considered as fledged once reached the 80% of the normal development .I have been monitoring Bonelli’s eagle population every year from 2000 to 2022. During each breeding season, all known and potential territories based on suitable habitat for the speciesVideo surveillance cameras were installed by regional forest rangers in Bonelli’s eagle’s nests located in the “Sierra de Espadán” Natural Park and its surroundings (southern Castellón province) Table . Accordi50 were used to analyse the variation in productivity in relation to the installation of video surveillance cameras (“camera”), pair composition (“age”) and their interaction. Considering the nature of the dependent variable , the productivity was modelled using a Poisson distribution and log link. The variables “territory” and “year” were included as random terms to account for the non-independence of data51. Five models were fitted: (i) a null model fitted only with random terms; (ii) and (iii) two models including the single effect of each predictor variable (“age” and “camera”), separately; (iv) a model including their additive effect (“age” + “camera”); and (v) a model including the combination of the additive effects and the potential multiplicative effects of the combination of variables of interest (e.g. “camera” * “age”). All models were computed using the package “glmmTMB” for R52. The GLMMs were compared and ranked through an information-theoretical approach based on Akaike’s Information Criterion (AIC). The conditional and marginal R2 of all GLMMs were calculated using the lognormal approximation following53 in order to assess the relative contribution of random and fixed effects through the R package “MuMin”54. All computations and figures were done in R version 4.1.2. (https://www.r-project.org) with the “ggplot2” package. Statistical significance was set at α < 0.05.Descriptive statistics were computed as mean and standard deviation for all variables. Differences in productivity and breeding success before and after the installation of video surveillance cameras were tested through non-parametric Wilcoxon rank-sum tests with continuity correction. Generalized Linear Mixed Models (GLMM)All field methods were carried out in accordance with relevant guidelines and local regulations to avoid disturbance to birds. This is an observational study. Therefore, there is no need for the ethical approval of any licensing committee.1,21 = 358.7, p < 0.0001, R2 = 0.945).Overall, 496 breeding attempts were recorded in 33 different territories in which 515 chicks fledged during the period 2000–2022. Population size decreased significantly from 31 occupied territories at the start of the study period to 18 occupied territories at the end  in comparison to the average laying date of the overall population . Furthermore, these laying dates were also delayed in comparison to the average laying dates of these particular territories all over the study period . On the other hand, the two pairs under video surveillance that bred successfully in 2022 showed laying dates one within the average values of the population (7th February) and the other one unusually advanced (22th January) in comparison with the average values of the population. Nonetheless, these values were within the average values of the same territories all over the study period .Average productivity of the whole population was 1.04 ± 0.84 chicks/pair . Considering all territories, overall average productivity did not vary in the study area before  and after  the installation of video surveillance cameras . Considering the two breeding seasons in which surveillance cameras were operating (2021 and 2022), there were no general differences in productivity between years . However, in the same period, there were significant differences in the productivity between territories under video surveillance  and those without video surveillance   , respectively; and 87.70% and 88.23% of the variance was explained by the entire model, including both fixed and random effects , respectively. Remarkably, those nests under video surveillance showed the lowest values of productivity across the long-term (23-years) temporal dataset  included the age and camera as predictors. The two top-ranked models showed one differences in productivity among territories in relation to pair composition ; and the other one the additive effect of pair composition and the installation of video surveillance cameras  Table . Fixed eAverage breeding success was 66.80% ± 12.92 successful pairs all over the study period (2000–2022). Considering the period in which video surveillance cameras were installed (2021–2022), pairs under video surveillance showed lower average breeding success  than pairs without cameras .1. Today, thanks to technological advances, they constitute a cost-effective tool and their application and usefulness have been demonstrated over several decades of use, as reported in the literature55. Although most studies report neutral effects of cameras on birds27, or directly assume no effects without a formal test for it, very few studies analyse in detail the potential negative effects of their use, particularly on raptors. The advantages of using video cameras in nests are evident. However, as with all technologies, they have their limitations and drawbacks. Among them, it should be noted that the analysis of the images obtained through video surveillance is time-consuming since it requires long hours of viewing the recordings to obtain the data. In addition, the fact that they have to be installed outdoors inevitably exposes the technology to environmental conditions that are sometimes adverse  and for which the technology has not been originally designed, particularly in most cases where the equipment is readapted to operate outdoors. To this fact must be added the difficulty of camera installation, especially in the case of working at height such as on cliffs or trees where many species of birds nest (e.g. raptors). This entails the need to have to fine-tune all the parameters before installation, since any repair or readjustment that has to be done afterwards implies access to nests in the middle of the breeding season, coinciding with the most sensitive period for bird reproduction56. In this paper, thanks to the availability of a database obtained through long-term monitoring of a population of Bonelli’s eagle, I show how the inappropriate use of video surveillance cameras could result in negative effects on the reproduction of a threatened species. Far from being a critique of technology, this paper analyses the effect of video surveillance camera installation on reproduction and then discusses what factors might be behind the statistically significant reduction in breeding performance observed.Video surveillance cameras are a useful and practical tool to study many aspects of ecology and animal behaviour that would otherwise be practically impossible to obtain58 and other eagles60.According to the results of this work, the installation of cameras in Bonelli’s eagle nests has caused a reduction in the productivity and breeding success of the territories under video surveillance. My results show no differences in breeding performance between the years 2021–2022, when cameras were operating, but significant differences in breeding parameters between territories under video surveillance and those without video surveillance in the same period. Considering the overall dataset (period: 2000–2022), there were no differences in breeding performance before and after the installation of video cameras. When all variables were taken into account, the results of the GLMM showed significant differences in productivity among territories in relation to the installation of video surveillance cameras and pair composition. Pairs composed of two subadult birds or by one subadult and one adult bird were less likely to be successful in reproduction, which is in agreement with the general pattern reported in this31. Furthermore, in the case of the four pairs that were subjected to video surveillance in consecutive years, one of them changed the nest in the first breeding season to another cliff located 4.35 km from the usual breeding site; the second pair failed to breed in 2022 when it had two chicks in the nest, possibly due to a heavy rainstorm; the third pair, the one that unusually delayed reproduction in 2021, did not lay eggs in 2022; and the fourth pair did not lay eggs in either of the 2 years.Interestingly, the installation of video surveillance cameras might affect the laying date. The two pairs that bred successfully of the four monitored in the first year showed an unusual delay in the laying date. Subsequently, the recovery on laying dates within the normal regime in the next breeding season might be attributed to the effect of habituation by the eagles42; (iv) the installation of cameras in nests of unexperienced pairs composed by at least one subadult bird, with the consequent lower breeding experience58; and (v) a potential lack of experience in work at height of the team of forest rangers in charge of installing the cameras, which resulted in long operating times and the need to repeat new visits once the equipment was installed.The potential causes of the observed reduction in breeding performance remain unknown. Based on the legal rights to access to environmental information in Spain, I requested the regional administration  detailed information on the Bonelli’s eagle video surveillance project. Unfortunately, the technical specifications of the whole monitoring system  and, specifically, about the number of interventions done in each eagle’s territory once cameras were installed were not provided. Nevertheless, according to the specific information included in the forests rangers’ report and my field experience with the species all over the 23-years study period, I would take into account the following facts: (i) the installation of video cameras by forest rangers took place at the end of the year Table , hence cIn some cases, breeding failure took place after the pair arranged the nest, even though no egg laying was observed. In other cases, video-surveilled pairs failed reproduction after incubation and egg hatching, when chicks were in the firsts week of development. Technical failures including the failure of the SIM card, power cable failure, lack of phone signal and even the deterioration of the antennas by herbivores made necessary reparation after camera installation. This implied repetitive visits the top of the cliff where the antennas and the transmission system were installed or even the nest. This reiteration of visits, along with the occurrence of natural events such as heavy rainstorm that affected the entire population during critical stages of the breeding season , in combination to the pair composition  could potentially explain the decrease in breeding performance of video-surveilled pairs in comparison to non-surveilled pairs.72, among many others). Therefore, the timeliness and novelty of the intervention, if other goals are not considered, could also be questioned.This paper is not intended to critique video camera technology for nest monitoring. On the contrary, the technology used by experienced researchers with clear scientific objectives constitutes an ideal complement to fieldwork. Therefore, this study is aimed at warning of the risks that the inadequate use of technology could have on endangered species if the necessary cautions are not taken into account. This includes the requirement of establishing clear scientific objectives, which were absent in this case. For example, the study of Bonelli’s eagle diet, which was the main justification for the installation of cameras in Bonelli’s eagle nests by the regional government, has been a topic deeply studied for decades (e.g.Aquila chrysaetos), cameras must be used judiciously because camera installation creates a persistent manipulation at the nest13. In addition, the use of video cameras should only be used as part of well-planned scientific studies, and researchers should follow protocols that minimize disturbance to eagles during installation and post-operational functioning.In order to avoid unnecessary adverse effects, potential management actions to mitigate the effect of the installation of video cameras in nests on birds’ behaviour would include the need to plan the intervention dates in advance, separating them as much as possible from the mating period of the eagles. It would be also convenient to test the systems beforehand under controlled conditions or with non-threatened species in which a reduction in reproduction could not have significant effects on population dynamics. Ultimately, the post-installation monitoring is critical in order to avoid unnecessary disturbance to eagles and to ensure positive outcomes. In agreement with a similar study on the golden eagle (Finally, I urge the scientific community to report the potential negative effects observed in their studies of the use of video surveillance cameras in bird nests, especially if the target species are threatened with extinction.Supplementary Figure S1.


This study aimed to examine the association between Machiavellianism and gift-giving in live video streaming, as well as the mediating role of desire for control and the moderating role of materialism in this relation. A sample of 212 undergraduate students  with experience of gift-giving in live video streaming was recruited to complete questionnaires on Machiavellianism, desire for control, materialism, and the frequency of gift-giving in live video streaming. The results showed that Machiavellianism was positively associated with gift-giving in live video streaming through the mediating role of desire for control; and the mediating effect of desire for control was moderated by materialism, with this relation being stronger for individuals with a higher level of materialism. Though with several limitations , this study could deepen our understanding of the influencing mechanism of gift-giving in live video streaming, which could also provide practical implications for the sustainable development of the live video streaming industry. With the development of society, especially information technology, the manners of social interaction and entertainment have profoundly changed. In recent years, live streaming platforms , where anyone can deliver real-time broadcasts or watch and interact with the streamers , have beA notable feature of live streaming is that users could interact with the streamer by sending messages and giving paid gifts. Gift-giving is a common feature of most live streaming platforms with specific interpersonal purposes—viewers send gifts to express their encouragement and “like” for the streamer, with the aim of getting special feedback from the streamer (such as streamers’ warm and immediate response), as well as superior social status in the live streaming channel of the streamer ,5; in adHowever, previous studies mainly focused on the factors underlying viewing live video streaming . A numbeThough no research examines this issue directly, there are several forms of empirical and theoretical evidence that could provide the basis and foundation for this study and the corresponding hypothesis.The attempts to examine the influencing factors of gift-giving in live video streaming are limited. Previous studies mainly focused on the social motivational factors of gift-giving in real life, defining gift-giving as a process that serves communication, social exchange, economic exchange, and socialization functions ,10. It hIndividuals’ social interactions may be greatly influenced by their personal traits, insofar as people with different personal traits may have different motivations and tendencies in daily social interaction and relationships . As one The process of gift-giving could be seen as a special social interaction between viewers and streamers in live video streaming. When viewers send gifts, the information about the viewer and gifts will be presented in the information bar which all viewers could see, and the streamers will also express their gratitude to the gift-givers in public; afterward, the streamers would also respond warmly to the gift-givers, for example, by answering their questions immediately or conducting specific behaviors  at their request. Thus, it is assumed that this process could satisfy the need to seek social status, and individuals would tend to engage in more gift-giving activities. Relevant studies also revealed that the motivations for social status, social competition, social recognition, and the special attention from streamers were closely associated with gift-giving in live video streaming ,6,7. TakThe desire for control refers to the extent to which individuals are motivated to control their environment or exercise dominance over interpersonal situations . The desTo some extent, live video streaming provides a perfect platform for viewers to satisfy the psychological needs motivated by the desire for control by easily sending gifts. First, to attract more viewers and get more paid-gifts, streamers would adopt various strategies  to make the viewers feel comfortable, influential, and dominant, which could satisfy the psychological needs driven by the desire for control ,8. Then,In addition, research also indicated that the desire for control was closely associated with Machiavellianism. On the one hand, manipulating or exploiting others in social interaction is a defining element of Machiavellianism ,15. PartMaterialism is defined as the importance attached to the possessions and the acquisition of material goods in achieving major life goals or desired states ,27. It iAs discussed above, the nature of paid gift-giving in live video streaming is an exchange for social relationships, attention, power, and status with money, which fits well into the mindset of materialism . Thus, iTo sum up, the present study aimed to examine the association between Machiavellianism and gift-giving in live video streaming, as well as the underlying mechanism—the potential mediating role of desire for control and the moderating role of materialism. In addition, undergraduate students are active in various online activities, and they are also fairly active in live video streaming ,6,38. AtConvenience sampling was adopted in this study to recruit undergraduate students  to participate in this study. Two hundred and twelve undergraduate students , who have the experience of gift-giving in live video streaming, were recruited from a university in central China to participate in this study. This study was approved by the Ethical Committee for Scientific Research at the researchers’ affiliated institution, and complied with the ethical guidelines protecting human participants. In the study, they were asked to complete a set of scales on the main variables and report relevant information—age, gender, and the frequency of viewing streaming in their daily lives.The Mach-IV scale , a widelχ2/df = 2.74, RMSEA = 0.05, NFI = 0.95, CFI = 0.97, and Cronbach’s alpha was 0.85.The scale developed by Burger and Cooper was adopted to assess individuals’ desire for control . It contThe Chinese version of the Material Value Scale  was usedAn item (“How often do you give paid gifts to the streamers when you are viewing the live video streaming”) was created to measure the frequency of gift-giving in live video streaming, and participants were asked to assess this item on a five-point Likert scale ranging from 1 “rarely” to 5 “always”, and a high score indicates more frequent gift-giving when viewing the live video streaming.All the statistical analyses were conducted with the SPSS 23.0 . First, the descriptive statistics were computed for the main study variables, and then Pearson’s correlation analysis was also conducted to examine the relationships among the variables. Then, the PROCESS macro , which wThen, the hypothesized moderated mediation model was subsequently tested using Hayes’ the SPSS macro PROCESS with 5000 bootstrapping sampling . Gender,The main results of the moderated mediation model analysis consist of two parts: the regression analysis model and the conditional indirect effect analysis, and they are presented, respectively, in Then, as can be seen from This study examined the psychological mechanisms contributing to gift-giving in live video streaming. First, the results found that Machiavellianism was positively associated with gift-giving in live video streaming, consistent with the findings on Machiavellianism. As one of the “offensive yet non-pathological personalities” , MachiavFurthermore, the results also examined the mechanism underlying this relationship and found that desire for control could mediate the association between Machiavellianism and gift-giving. The desire for control is a common and prominent social motivation driving one to seek control over the environment and events in life . TheoretIn addition, the results also found that materialism could moderate the mediating effect of the desire for control (moderating the association between the desire for control and gift-giving). Especially, the mediating effect of desire for control was stronger for individuals with a higher level of materialism. Materialism refers to the cognitive tendency to how important a person values possessions and acquisition of material goods ,27. IndiIn the current information era, this study examined the psychological motivations of gift-giving in live video streaming by investigating the association between Machiavellianism and gift-giving in live video streaming and its underlying mechanism. First, this study deepens our understanding of live video streaming by focusing on gift-giving and individuals’ personalities (Machiavellianism). Second, by exploring virtual platforms, these findings extend previous research on gift-giving and provide new insight into the social motivations that underpin it. Thirdly, these results also provide practical implications for the sustainable development of the live video streaming industry. For example, the functions that could satisfy viewers’ psychological needs should be offered by the live video streaming platforms to attract viewers; at the same time, individuals with certain characteristics  should be paid special attention to, and some measures should also be taken to avoid getting too much involved in paid gift-giving . Finally, this study also extended studies on Machiavellianism and trait activation theory, suggesting that they could also influence and explain individuals’ behavior in online space.Several limitations also should be acknowledged. First, due to the cross-sectional method and the small sample , causal inference and good generalizability cannot be achieved . Future


Dermestes maculatus  resemble caterpillars but actually feed on dry tissues of dead animals, and sometimes on human corpses. To hide and metamorphose into adult beetles, these larvae dig deep tunnels called pupation chambers. These holes and pits are usually observed in skins, horns, furs, or surrounding materials such as wood. They have also been reported on archaeological bones and experimentally observed on fresh pig bones, but never on humans. In this context, we investigated whether larder beetle larvae could also dig pupation chambers in human bones, and under which conditions this could occur. For this purpose, we placed medieval dry human bones as well as fresh calf and beef bones (control) with Dermestes maculatus larvae. After 1 month, we observed tunnels corresponding to pupation chambers only on dry human bones, and under conditions of high larval density. Despite these results being preliminary, they are, nonetheless, of particular interest in a forensic context, as they could help to understand taphonomic bone modifications or even the chronology of mass grave deposals.Larvae of the larder beetle Dermestes maculatus  larvae were placed in a dermestarium with different types of bones varying in species (Bos taurus or human), age (adult or immature), and preservation method (fresh or dry). Our results show that dermestid larvae caused multiple lesions, including larval mandible traces on cortical bone, cortical perforations, drilling of pupation chambers, destruction of the trabecular network, and the perforation of cartilage. Bone destruction was mainly observed on aged dry bones, while fresh bones only exhibited soft tissue and superficial cartilage lesions. According to these results, pupation chambers could indicate the simultaneous presence of several corpses at different decomposition stages, or the addition of new corpses while others were already skeletonized. These conclusions are particularly important in the case of mass graves, where chronology is sometimes difficult to establish.Dermestid beetles (Coleoptera: Dermestidae) are necrophagous insects feeding on mummified carcasses. After six to seven molts, the larvae stop feeding and dig pupation chambers to hide and safely evolve into adults. Such pupation chambers have already been observed on archaeological mammals’ bones, but the attribution and interpretation of these osteological lesions lack experimental evidence in a forensic context. To observe whether dermestid larvae dig pupation chambers in human bones, 20 or 40  D. frischii, D. undulatus, D. peruvianus, D. lardarius, D. haemorrhoidalis, D. maculatus, D. bicolor, and D. ater [Dermestid beetles (Coleoptera: Dermestidae), also known as larder beetles, are necrophagous insects feeding on dry organic material, including human corpses. These insects are fairly common, and were initially known as pests of stored products and for their use in natural history museums to clean skeletal remains. In a forensic context, they are often observed on human corpses during late decomposition stages, but have also been sampled as early as 10 days after death ,3. DurinLarder beetles are holometabolous insects, which means they have four main stages during their life cycle: egg, larval, nymph, and adult. After mating, the females lay tens to hundreds of eggs in cracks or cavities of the cadavers. The larvae feed on dry flesh: they bore holes of 2–5 mm in diameter into desiccated muscle tissue, thus progressively creating an irregular network . During Due to their ability to remove the remains of flesh from bones, taxidermists and forensic anthropologists have widely used dermestid larvae to deflesh skeletons ,13,14. HIndeed, just before nymphosis, dermestid beetle larvae dig tunnels called pupal chambers to hide and reduce the risk of predation as well as cannibalism by other larvae ,21,22. TAlong with an a posteriori analysis, only a handful of experimental studies have been performed so far: Huchet et al. (2013) found pupation chambers on polystyrene and wood, while Zanetti et al.  reported them on pig bones ,23,24. HDermestes maculatus (Coleoptera: Dermestidae)  is one of the most synanthropic dermestid species, and is commonly found in forensic cases in Europe [De Geer, 774 is onn Europe ,7,24,25.n Europe .To observe whether dermestid larvae dig pupation chambers in bones, 20 or 40 larvae were placed in a dermestarium with food and different types of bones. The different conditions detailed below were designed to observe the effect of bone freshness as well as the link between larval density  and the digging of pupal chambers ,26,27.2) while 40 larvae experiments were placed in 14 × 9 × 9.5 cm boxes (ground density of 0.31 larvae/cm2). This second condition artificially increased the average density of larvae, reproducing overcrowding situations.The experiments took place in polycarbonate boxes supplied with dry food ad libitum  and damped cotton ,28. BoxeBos taurus or human), age (adult or immature), and preservation method (fresh or dry)  destined to be re-buried: one proximal extremity of a left adult femur, one right adult calcaneus, two adult vertebrae , two fragments of an immature radius, and a proximal epiphysis of a right immature ulna (included in the same box).Eight experiments were conducted simultaneously with three different types of bones varying in species ( or dry) . Each bo®, Noisy-le-Grand, France), with a 15 × 10 cm field of view and a spatial resolution of 90 microns. Multi-planar reconstruction (3D-MPR) was performed using CS-3D Imaging software. The 3D analysis and post-treatment were performed using Avizo 9 . Manual segmentation tools were used to calculate 3D volumes and surfaces. All bones were photographed before and after the experiments with a digital camera mounted on a 3D stand monitored with the Helicon Remote 3.8.6w software. Picture compilation was achieved with the Helicon focus 7 software and Adobe Photoshop CS4. Microscopic analysis was performed on a Nikon SMZ18 microscope (software NIS-Element BR) and a Leica M80 stereomicroscope (software LAS Core). After experiments, bone artifacts were scanned using Cone Beam Computerized Tomography . We also observed nymphs still present inside some of these cavities  placed at low larval density did not show any deep nor visible superficial lesions after experiments .D. maculatus larvae to create deep osteological lesions on human bones. Such entomological alterations are especially important in a forensic context, as they provide information on the history of the bone remains and the taphonomic processes involved in their degradation. However, it must be kept in mind that the results of this study are based on a limited set of experiments and need to be replicated. Thus, the following interpretations must be considered with precaution and regarded as hypothesis still needing more experimental confirmation.This study demonstrates, for the first time, the ability of The deep osteological lesions observed in the present study on human bones comply with the characteristics of pupal chambers previously established by Martin and West on other substrates (1995) . Their dFrom the outside, the passage of dermestids can be misinterpreted as accidental injuries drilling into cortical bone. However, the scalloped margins on the edge of entry orifices, the thin scratches observed on cortical surface, and the pupation chambers drilled in the trabecular bone, proved to be the action of the mandibles of dermestid larvae . In the Pupal chambers were observed only on dry human bones exposed to high densities of larvae: none of the fresh bones nor those exposed to low larval density had such osteological lesions. However, many perforations were observed on dry flesh, some still containing nymphs in situ. According to these observations, it can be concluded that dermestid larvae preferentially dig pupation chambers into the soft desiccated flesh remaining on the surface. Larvae used dry bones as sites for pupation only if the predation pressure from conspecifics was high and soft mediums were not available in the surroundings.These behavioral observations could seem anecdotal but may have important consequences in a forensic context . HoweverFirst, our results indicate that pupation chambers were excavated only in dry bones, not fresh ones. This finding implies that bones with pupation chambers were already dry when pupation chambers were dug. However, larvae cannot feed on skeletonized remains and need food (flesh) to develop and reach the pupal stage: the existence of pupation chambers cannot be explained without the close proximity of flesh. Accordingly, the digging of pupation chambers directly into bones should occur on cadavers with asymmetric decomposition (some parts skeletonized while flesh is still presents on others). In this case, the topological distribution of the marks should be considered: chambers only dug on the limb could suggest a delayed conservation of the trunk, while chambers dug on the axial skeleton illustrate complete skeletonization of the corpse when chambers were dug. In this latter case, the presence of another corpse, not yet skeletonized, should therefore be necessary to explain the successful development of the larvae.In the case of pupation chambers observed on an extensive set of bone remains or a whole skeleton, our results suggest the presence of several corpses at different decomposition stages, or the addition of new corpses while others were already skeletonized . In suchFinally, the occurrence of bone pupation chambers only under high larval density/overcrowded conditions could help to explain why these alterations are uncommon. Indeed, according to our results, the digging of pupation chambers directly into bones should occur only under certain conditions allowing the development of massive larval populations. Larder beetles and their larvae are most active during the hot and dry summer months, when rapid desiccation of carrion is more likely and larval development is fast ,3. ConseD. maculatus larvae bred at low density with food ad libitum do not alter fresh bones [Larder beetles are used by natural history museums to clean skeletal remains. Their ability to quickly remove flesh from bones is also of interest to forensic anthropology laboratories. Indeed, the use of mechanical methods to remove soft tissues can damage the surface of the bones, and simmering with detergent or bleach is not DNA-safe. However, it is necessary to ensure that dermestid larvae do not create superficial or deep lesions during the cleaning process. Together with former studies, our results indicate that sh bones . We do, The interpretation of osteological lesions is of primary importance in forensic taphonomy to determine the cause of the death as well as the peri-mortem and post-mortem events. Information provided by insect activity has been underestimated so far. On the contrary, archaeological literature has long been interested in the impact of environmental factors, including insect artifacts, on bones. This study expands across these two fields to provide the first experimental evidence of larder beetle pupation chambers on human bones. While preliminary, these results could bring decisive information in the interpretation of mass graves.


Malnutrition is a prevalent problem occurring in different diseases. Hemorrhagic transformation (HT) is a severe complication of acute ischemic stroke (AIS). Few studies have evaluated the association between malnutrition risk and hemorrhagic transformation in patients with acute stroke. We aim to investigate the influence of malnutrition risk on the risk of hemorrhagic transformation in patients with AIS.A total of 182 consecutive adults with HT and 182 age- and sex-matched patients with stroke were enrolled in this study. The controlling nutritional status (CONUT) score was calculated to evaluate the malnutrition risk. HT was detected by follow-up imaging assessment and was radiologically classified as hemorrhagic infarction type 1 or 2 or parenchymal hematoma type 1 or 2. Logistic regression models were conducted when participants were divided into different malnutrition risk groups according to the objective nutritional score to assess the risk for HT.P = 0.027; odds ratio, 3.960 , P = 0.048, respectively].The prevalence of moderate to severe malnutrition risk in patients with AIS was 12.5%, according to the CONUT score. Univariate analysis showed that the CONUT score is significantly higher in patients with HT than those without HT. After adjusting for potential covariables, the patients with mild risk and moderate to severe malnutrition risk were associated with a higher risk of HT compared to the patients in the normal nutritional status group [odds ratio, 3.180 , Malnutrition risk, measured by CONUT score, was significantly associated with an increased risk of hemorrhagic transformation in patients with AIS. Malnutrition has emerged as a common condition in older patients with chronic diseases that can lead to poor prognoses –3. It isThe reported prevalence of malnutrition at admission after stroke has varied from 9 to 20% , 8. Due Hemorrhagic transformation (HT), the bleeding of the ischemic brain area after stroke, is a severe and notable complication occurring in patients with AIS , 19. It We enrolled consecutive participants from the Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University clinical database of HT between January 2009 and April 2022 in this retrospective study. This study was approved by the Institutional Review Board and Research Ethics Committee of the Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University. No requirement for informed consent because this study was retrospective, and all included data were anonymous. The human data collection and the article writing were in accordance with the ethical guidelines of the Declaration of Helsinki.Patients were included if they (a) were aged between 18 and 90 years, (b) were admitted within 7 days of the onset of symptoms, and (c) were diagnosed as AIS by a brain computed tomography (CT) or magnetic resonance imaging (MRI) scan during hospitalization or right before it. Patients were excluded if they (a) were identified as having a transient ischemic attack (TIA) or hemorrhagic stroke, (b) were unable to check for repeatable CT or MRI, or (c) lacked laboratory data.Demographic characteristics, vascular risk factors, and radiological images were obtained by trained research coordinators. The baseline vascular risk factors included hypertension, diabetes mellitus, dyslipidemia, atrial fibrillation, previous history of stroke, smoking status, and current alcohol consumption. Laboratory data in the blood samples  and blood pressure measurements were collected within 24 h after admission. They were measured at the clinical laboratory in our hospital using routine laboratory methods. All the laboratory measurements were conducted by experienced laboratory technicians who were blinded to the baseline characteristics and clinical outcomes of the study participants. Treatment information on all the patients with AIS, including thrombolytic therapy, anticoagulant, and antiplatelet therapies before admission, was gathered. The size of the infarction area involving more than one-half of the middle cerebral artery territory was defined as large , 29. StrPatients were screened for malnutrition risk using the CONUT score . The CONIn this study, we combined moderate and severe malnutrition status as a moderate-severe malnutrition risk for analysis.Patients were diagnosed with HT by a follow-up CT or MRI performed within 24 h and 7 days (±2) from the onset of stroke. In addition, image examinations were conducted whenever the patients' clinical condition appeared to worsen for quick detection of the presence of HT. Two experienced neurologists, who were blinded to the clinical characteristics of each patient, retrospectively confirmed the results of the official neuroradiology report to agree on the presence and subtypes of HT. Hemorrhagic events were further radiologically divided into hemorrhagic infarction (HI) (types 1 and 2) and parenchymal hematoma (PH) (types 1 and 2) according to the recommendations of the European Cooperative Acute Stroke Study .t-test and analysis of variance (ANOVA), while the non-normally distributed variables were compared using the Mann–Whitney U-test and the Kruskal–Wallis H-test. The CONUT score was used to evaluate the prevalence of malnutrition risk at admission. Identified variables related to HT in the univariable analysis and main baseline confounders were selectively entered into the multivariable analyses with a conditional logistic regression model to explore significant predictors of HT. According to the malnutrition risk assessed by the CONUT score, the normal group was selected as the reference to compare the risk for HT, HI, and PH between the three malnutrition risk groups. All statistical analyses were conducted with SPSS for Windows, version 22.0  and GraphPad Prism, version 8.0.2. Additionally, a two-sided P-value < 0.05 was judged to be statistically significant.For descriptive analysis, continuous parameters were presented as means with standard deviation or median with interquartile range, while categorical variables were reported as frequencies (percentages). Statistic comparisons were performed with different methods on the basis of the nature of the data. The chi-square test, or Fisher's exact test, was used for categorical variables. The normally distributed variables were compared using the student's At baseline, a total of 182 consecutive participants with HT and 182 age- and sex-matched ischemic stroke patients without HT were enrolled in this study, among whom 345 (94.8%) patients had information on all the lymphocyte count, total cholesterol, and albumin data. The median age of these included patients was 74.0  years, ranging from 28 to 90. Most of these patients were men (56.3%). The median National Institute of Health Stroke Scale score (NIHSS) was 6.0 . Among the patients diagnosed with hemorrhagic transformation, hemorrhagic infarct type 1 (HI-1) occurred in 30 (16.5%) patients, hemorrhagic infarct type 2 (HI-2) in 80 (44.0%), parenchymal hematoma type 1 (PH-1) in 49 (26.9%), and parenchymal hematoma type 2 (PH-2) in 23 (12.6%). The subcategorization was based on imaging features.The median CONUT score was 2.0  in the total population. By CONUT score, 167 (48.4%) patients had mild malnutrition, 40 (11.6%) patients had moderate malnutrition, and three (0.9%) patients had severe malnutrition. The baseline demography and disease characteristics of the patients with different malnutrition risks based on CONUT scores are displayed in 2 = 21.243, P < 0.001; P = 0.001; 72.1 vs. 34.8%, chi-square test P < 0.001, respectively, P = 0.026). The prevalence of different malnutrition risks across three subcategorized groups of HT was significantly different  vs 2.0 (1.0–3.0), P < 0.001, respectively, P = 0.329].The proportion of patients diagnosed with HT was significantly different among three malnutrition risk groups using the chi-square test , P < 0.001, respectively, P = 0.016; P = 0.079; P = 0.004; OR = 2.276, (95% CI = 0.859–6.029), P = 0.098, respectively]. The risk for HT was also related to atrial fibrillation, the large size of the infarction, elevated NIHSS score on admission, and anticoagulant therapy before admission.According to the CONUT scores calculated by the equation as the categorical variable grouped previously mentioned, we performed the conditional logistic regression models. Similarly, univariable analyses indicated that mild malnutrition risk and moderate to severe malnutrition risk showed significant associations with an increased risk of HT without adjustment , while no apparent difference was found between the mild malnutrition risk and the normal group . In multivariate analysis, the association between varying degrees of malnutrition risk and HT remained significant. The OR for HT was higher in patients with a mild malnutrition risk and moderate to severe malnutrition risk . However, the risk of HI was not significantly higher in the moderate to severe malnutrition risk group . However, there was no significant association between moderate to severe malnutrition risk and increased risk of HI or PH based on the adjusted multivariate regression analysis. Interestingly, we found patients with mild malnutrition risk were more likely to undergo HI . We supposed that higher malnutrition risk patients could be more likely to experience PH, representing a more severe radiological category and poor clinical outcomes in patients with stroke. However, the association between varying degrees of malnutrition risk and PH was insignificant after full adjustments. Due to the limited number of patients in our study, we hope more studies can provide further evidence to confirm these results.Previous studies have explored the association between objective malnutrition scores and the occurrence of adverse outcomes. A higher CONUT score could predict cardiovascular death and all-cause mortality in the general population, according to a prospective study in the United States . López ESeveral factors constituting the nutritional status score may account for the observed association between malnutrition index and HT. First, serum albumin functions as a major plasma antioxidant defense and constitutes the primary oxygen radical trapping –51. As aThere are several limitations to our study. First, this study was a single-centered and retrospective analysis, which could not prove cause and effect. Second, due to the unavailability of necessary variables, we did not make a comparison between different nutritional scores and were unable to assess malnutrition status by gold standards, including the European Society for Clinical Nutrition and Metabolism diagnostic criteria for malnutrition. However, the CONUT score used in this study was reported to be relatively accurate in evaluating the malnutrition risk and was of great importance for the guidelines of clinical therapy. We hope more studies can further investigate the variation among different evaluation tools and verify the findings of our study. Third, we take the infarct size as a categorical variable in our analysis owing to the limitations of the experimental condition. It is more precise to calculate the infarct size using the Alberta Stroke Program Early CT Score (ASPECTS) system by trained radiologists. Additionally, the sample size of this study was limited. More studies need to be conducted to further confirm the relationship between malnutrition risk and HT. Finally, we evaluated malnutrition risk only at admission but not at dynamic observation. It is unclear whether malnutrition status changes over time could influence the risk of hemorrhagic transformation. Further study is warranted to fully understand the influence of nutrition interventions on malnourished patients.In conclusion, this study demonstrated that malnutrition risk, measured by the CONUT score, was significantly associated with an increased risk of hemorrhagic transformation in patients with AIS.The datasets presented in this article are not readily available because the research data is confidential. Requests to access the datasets should be directed to the corresponding author.The studies involving human participants were reviewed and approved by the Institutional Review Board and Research Ethics Committee of the Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University. Written informed consent was not provided because this study was retrospective and all included data were anonymous.C-XY and B-LH designed the study. C-XY and X-YC interpreted the data. C-XY wrote the manuscript. Y-NZ prepared the figures. Y-NZ and X-YC performed the statistical analyses. B-LH supervised the study. All authors have made an intellectual contribution to the manuscript and approved the submission.This study was supported by grants from the projects of the National Natural Science Foundation of China  and the Clinical Scientific Research Foundation of the Second Affiliated Hospital of Wenzhou Medical University (SAHoWMU-CR2017-01-212).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.