The potential of obtaining cell cultures with neural crest resemblance  from dental‐related tissues, including human dental pulp cells (hDPCs), has been discussed in the literature. However, most reports include the use of serum‐rich conditions and do not describe the potential for neural differentiation, slowing translation to the clinic. Therefore, we aimed to culture and characterize NCSCs from the human dental pulp in vitro and evaluate their ability to differentiate into neurons; we also investigated the effectiveness of the addition of BMP4 to enhance this potential. Cultures were established from a varied cohort of patient samples and grown, as monolayers, in serum, serum‐free, and also under sphere‐aggregation conditions to induce and identify a NCSC phenotype. hDPC cultures were characterized by immunocytochemistry and reverse transcription quantitative polymerase chain reaction. Monolayer cultures expressed stem cell, neural progenitor and neural crest‐related markers. Culturing hDPCs as neurospheres (hDPC‐NCSCs) resulted in an increased expression of neural crest‐related genes, while the addition of BMP4 appeared to produce better NCSC characteristics and neural differentiation. The neural‐like phenotype was evidenced by the expression of TUJ1, peripherin, NFH, TAU, SYN1, and GAP43. Our results describe the establishment of hDPC cultures from a large variety of patients in serum‐free medium, as NCSC that differentiate into neural‐like cells, as well as an important effect of BMP4 in enhancing the neural crest phenotype and differentiation of hDPCs. Human dental pulp tissue collected from a varied cohort of samples was used to directly establish monolayer cultures in defined serum‐free conditions. In monolayer, the cultures maintained an mesenchymal stem cell (MSC) phenotype regardless of the initial serum‐free conditions. A Neural crest‐derived stem cell phenotype was favored by adding BMP4 before or after transferring the cells to a 3D culture. This work details the acquisition of cells from human dental pulp and describes their establishment and growth in preclinically relevant, serum‐free culture conditions, in comparison to standard serum‐rich cultures. In contrast with the current literature, this article described in greater detail the nature and condition of the samples used from a large group of patients, allowing for a greater understanding in relation to clinical translation. Results provide evidence of neural crest‐related markers in all growing conditions, and the improvement of a neural crest‐like phenotype by growing human dental pulp cells (hDPCs) in 3D neurospheres. Importantly, this article provided evidence that hDPCs that are characterized as neural crest‐derived stem cells can differentiate into neural‐like cells, as well as describing an important novel effect of BMP4 on hDPC neural crest‐like phenotype.1Oral‐derived stem cells (OSCs) have been generally characterized in vitro as mesenchymal stem cells (MSCs). These OSCs include the widely studied dental pulp cells as well as stem cell populations derived from other novel oral sources such as periapical Cyst‐MSCs (PCy‐MSCs). OSCs are regarded as good candidates for autologous cell therapies in regenerative medicine due to their multipotent differentiation, proliferative capacity, and accessibility .However, a factor that can delay translation of OSCs into the clinic is that the isolation, culture, and characterization of these stem cell populations have been commonly performed in medium containing fetal bovine serum  of adult, permanent teeth, and only a small number of key direct applications have been reported.Therefore, we present here a detailed description of the establishment and characterization of hDPCs grown in serum‐free media in vitro, the characterization of their molecular NCSC signature, and the role of BMP4 in enhancing this signature. In addition, we present proof of concept that these NCSCs, generated under serum‐free conditions from hDPCs, can derive neural‐like cells, proposing them as candidates for neural regeneration treatments.22.1The study was conducted in accordance with ethical approval granted by the Leeds East Research Ethics Committee of the UK National Research Ethics Service . Human dental pulp was isolated from samples collected with written informed consent from patients at the Charles Clifford Dental Hospital, Sheffield, UK. Details of the patients' age and the condition and position of the tooth are provided in Table 2.2g. The pellet was resuspended directly into either: (a) standard medium: DMEM, 1× P/S, 1× Glutamax (GIBCO), 10‐4 Ascorbic Acid , 20% FBS (GIBCO); (b) otic stem cell full medium (OSCFM): DFNB (DMEM:F12 (GIBCO), 1% N2 (GIBCO), 2% B27 (GIBCO), 1× P/S (SIGMA), 20 ng/mL bFGF , 50 ng/mL IGF (R&D Systems), 20 ng/mL EGF (R&D Systems); or (c) bone morphogenic protein 4 (BMP4) medium: OSCFM supplemented with 10 ng/mL BMP4 (R&D Systems). OSCFM and BMP4 cultured cells were grown on laminin‐coated six well plates . The time between sample collection and their final deposition in culture conditions was less than 4 hours. The cultures remained undisturbed for the first 4 days. From day 4, half a volume of the respective media was replaced. From day 12 onward, all medium was replaced every 2 to 3 days.Dental pulp was extracted by making a longitudinal groove on the tooth with an electric drill cooled under running water and sectioning the tooth with a mallet and osteotome to expose the pulp. The dental pulp was removed, cut into small pieces and digested with collagenase IV  for 60 minutes, aided by pipetting every 30 minutes. The dissociated tissue was passed through a 100 μm strainer, washed with PBS, and centrifuged for 5 minutes at 4002 using 1:10 Trypsin EDTA  for FBS cultures and 1:80 TE for OSCFM and BMP4 cultures.Primary cultures were passaged at week 4, and cultures able to proliferate and survive at least four passages were described as successful cultures . cumulative cell doubling (CCD) of living cells was calculated using the following formula:2.4hDPC cultures were detached with the corresponding Trypsin EDTA treatment (as described above) and resuspended in sphere medium: DFNB, 1× P/S (Sigma), 20 ng/mL bFGF (R&D Systems), 20 ng/mL EGF (R&D Systems) at 10 000 cells per 100 μL in ultra‐low attachment culture plates . Neurospheres were fed by adding 50 μL of the medium at day 3 and 6. The number of individual spheres and their diameter was measured on day 4. Neurospheres were harvested or used in downstream applications from days 6 to 8. Where indicated, 10 ng/mL BMP4 (R&D Systems) was supplemented to the sphere medium.2.52; Cultrex) coated plates in neuralizing medium consisting of DFNB, 30 ng/mL NT3  and 1 mM dbcAMP . The medium was replaced every 2 to 3 days for up to 2 weeks.Neurospheres were induced as described by Gervois et al2.6hDPC‐FBS cells were transferred to the conditions described for OSCFM and BMP4 cultures for 7 days. Cells maintained in FBS conditions were used as a control. After the 7 days of the transference, hDPC‐NCSC neurospheres were formed. hDPC‐neurospheres were then induced for neural differentiation as described above.2.7After fixation (see below), hDPC neurospheres were subjected to a sucrose gradient of 7.5%, 15%, 22%, and 30% sucrose , and transferred to optimal cutting temperature (OCT) solution for 24 hours (4°C). The tissue was then fast‐frozen on a dry ice/methyl‐butane bath. Cryosectioning was then performed in a Bright OTF5000 cryostat in sections of 5 to 10 μm.2.8Cells and spheres were fixed with 4% Methanol‐free PFA  at room temperature for 10 minutes. For immunocytochemistry, cultures were blocked for 1 hour with 5% normal donkey serum‐PBST . Primary antibodies were added and left overnight at 4°C. Secondary antibodies were added in blocking solution (as above) for 1 hour at room temperature together with DAPI nuclei counterstaining (1:100).Antibodies and dilutions: RBT NFH , RBT PER , MSE TUJ1 , RBT TAU , RBT SOX2 , MSE AP2a , MSE NESTIN , MSE P75 supernatant , MSE SLUG , MSE SNAIL1 , Goat SOX10 , RBT SOX9 , MSE STRO‐1 . DNKa‐MSE Alexa 488 , DNKa‐RBT‐Alexa 568 , DNKa‐GOAT Alexa 568 .The EVOS cell imaging system and InCell Analyzer inverted microscopes were used for fluorescent and brightfield imaging. FIJI image software was used for image processing and analysis. Negative controls were calculated by omitting the primary antibody during the immunocytochemistry.2.9Cultures positively stained for nerve growth factor receptor (P75) and STRO‐1 at passage 0 and between passage 2 and 4 were manually counted and the percentage of positive cells was calculated based on the total cell number defined by DAPI nuclei staining.2.10The Qiagen microkit  was used to extract RNA from the cell cultures according to the manufacturer's instructions and quantified using a Nanodrop spectrophotometer. For cDNA synthesis, at least 200 ng of RNA was used with the Superscript IV retro transcriptase  according to the manufacturer's instructions.2.11hDPC‐FBS transferred into the other culture conditions  and induced to neural differentiation  was performed with Taqman probes using the Thermofisher QuantStudio 12 k Flex Real‐time PCR system. The reactions were prepared with at least 1 ng/μL of cDNA. The master‐mix, assays, and water volumes were used according to the concentrations specified by the manufacturer's instructions in a final volume of 7.5 μL per reaction in quadruplicate. The relative expression analysis was performed using the 2Taqman probes: RPLP0 (Hs00420895_gH), SOX2 (Hs00602736_s1), OCT4/POUF5F1 (Hs04260367_gH), NANOG (Hs02387400_g1), SOX10 (Hs00366918_m1), P75/NGFR (Hs00609976_m1), NESTIN (Hs04187831_g1), SNAIL1 (Hs00195591_m1), AP2a/TFAP2A (Hs01029413_m1), SYN1 (Hs00199577_m1), NFH/NEFH (Hs00606024_m1), GAP43 (Hs00967138_m1).2.13One‐way ANOVA followed by a Sidak's or Tukey test for pairwise comparison was made to determine differences. A linear regression analysis was used to determine slope differences. The tests were run in GraphPad Prism version 8.3.0 .33.1With the purpose of establishing in vitro hDPC cultures, 48 teeth  were collected with written informed consent from patients at the Charles Clifford Dental Hospital, Sheffield, UK. Patient data collected comprised age, sex, and reason for tooth extraction as described by the clinical staff Table . The ageTo determine whether hDPC primary cultures could be established in serum‐free, neurogenic conditions from the beginning, freshly dissociated hDPCs were seeded in either OSCFM , group 2: 21‐30 years , and group 3: >30 years  was assessed after 4 days in culture Figure . OverallNeurospheres were then analyzed by immunocytochemistry to identify neural crest‐related markers. hDPC‐FBS‐ Figure , hDPC‐OSSNAIL1, P75, SOX10, and AP2a quantitatively by RT‐qPCR. To do this, we analyzed the relative expression levels of each neurosphere sample in comparison to their own monolayer counterpart. Overall, P75 and SNAIL1 expression was significantly higher in the majority of independent neurospheres from all conditions . All the evidence together suggests that the addition of BMP4 to the neurospheres can induce even higher relative expression levels of important neural crest‐related genes.As BMP4 has been shown to drive NCSC identity,4 Figure . HNK‐1 p3.5hDPC‐NCSCs (neurospheres) derived from every initial experimental condition Figure  were ind3.6GAP43, NFH, and SYN1 after neural differentiation , in comparison to the rest of the experimental groups. Furthermore, FBS‐BMP4‐NEU cells also presented a higher significant upregulation of SYN1 when compared to the control group ,We have shown that hDPC can be established in OSCFM directly from the primary tissue with an efficiency of 81%, providing evidence of the suitability of this medium to be used for the establishment and expansion of hDPCs in culture. On the other hand, successfully established hDPC‐BMP4 cultures behaved similarly to hDPCs in FBS and OSCFM media, as judged by marker expression and CCDs. Nevertheless, the efficiency of establishing cultures in BMP4 was lower, with only a third of the attempts being successful. The need to use serum‐free and xeno‐free conditions for dental pulp cell cultures has been recognized, and several authors have described the growth of hDPC in defined media.SOX2, NANOG, and OCT4 were expressed by our cultures, since these have been identified before in hDPCs and other oral‐derived NCSCs, fueling the claim that these populations could be pluripotent.OCT4 and NANOG, together with the lack of SOX2 expression, were not sufficient to suggest a pluripotent state of hDPCs in any of our conditions. Regarding the NCSC phenotype, we observed the expression of a large panel of relevant neural crest‐related markers including SLUG, SNAIL1, SOX9, and NESTIN in our three initial, isolating culture conditions by immunocytochemistry as well as NESTIN by RT‐qPCR. Thus, we provide evidence of neural crest‐like phenotypes already in our monolayer cultures. However, the percentages of P75 and STRO‐1 positive cells varied with passage number. The P75 marker has been proposed as a neural crest marker for hDPC,P75 and that only two hDPC‐FBS cultures were expressing SOX10 by RT‐qPCR in monolayer conditions. The variable behavior observed in all our cultures illustrates the heterogeneous nature of hDPCs when grown as a monolayer, regardless of the serum or growth factors used to supplement the initial cultures. Our findings somehow differ from the study by Al‐Zer et alWe characterized hDPCs grown under OSCFM and BMP4 conditions by RT‐qPCR, and compared them to those derived in the presence of serum. We explored whether markers of pluripotency such as SNAIL1 and P75 mRNA levels, the latter particularly in OSCFM and BMP4 groups. An enhanced NCSC phenotype on BMP4‐derived spheres can be further supported by the expression of SOX2, P75, and SOX10, in contrast to the monolayer cells in which appeared undetected in later passaged cultures by immunocytochemistry and RT‐qPCR. Similarly, cultures from adult palatum, periodontal ligament, dental pulp, or hair follicles grown in defined conditions have been shown to express the neural crest markers Sox10, Sox9, Snail1, Slug, P75, and Ap2a.Our findings led us to hypothesize that heterogeneity in our hDPC cultures could lead to inefficacy in differentiation and that an intermediate population may be required for neural maturation, as shown by observations from the literature on hDPC‐neural differentiation.The addition of BMP4 to the initial growing conditions generally increased the expression of important NCSC markers such as P75 and SOX10 in hDPC‐BMP4 neurospheres, as observed by immunocytochemistry and RT‐qPCR. Additionally, in vitro differentiation into neural‐like cells was qualitatively better from BMP4‐containing cultures, with cells developing neurites and expressing markers of maturing neurons such as NFH, TUJ1, peripherin (PER), TAU, GAP43, and SYN1. However, neuralization into more mature phenotypes would need to be improved further. Cultures from all conditions had undifferentiated, non‐neural cells and in general, cells failed to migrate out of the spheres. We cannot rule out the possibility of obtaining a mature neural phenotype from all our hDPC culture conditions if left for a longer period, as has been described by Gervois et al.However, the observed hDPC‐BMP4 NCSC phenotype, as well as the improved changes observed during their neural differentiation in vitro, support the important role of BMP4 in sensory neuron and cranio‐facial neural crest induction.Notably, the characterization of dental‐related human NCSCs, including those derived from gingival tissue, periodontal tissue, exfoliated deciduous teeth, and adult human dental pulp, has gained increasing interest for their potential use in neurodegenerative disorders. Nevertheless, most reports are limited to describing the molecular resemblance to neural crest cells, while their actual differentiation capacity is usually left untested, masking their actual translational potential for regenerative medicine.Additionally, it would be important to evaluate a combination of strategies to facilitate the application of oral‐derived NCSCs in regenerative medicine, such as their incorporation in advanced manufactured scaffolds or conduits for nerve repair and tissue engineering .Finally, the field of dental pulp stem cells and their applications would benefit from addressing specific diseases or targeted applications from preclinically relevant established cultures if we aim to advance the use of dental pulp cells in regenerative medicine.5We have provided a detailed description of the culture of hDPC from a wide variety of teeth, as well as a robust characterization of the culture conditions. We have also shown that cells can be directly established in serum‐free medium in contrast to the widely used serum‐rich standard conditions. This should prove highly advantageous in a preclinical setting. Also, hDPCs present a basal neural crest‐like phenotype that can be enhanced when induced to form neurospheres (hDPC‐NCSC). Furthermore, BMP4 has a significant role in enhancing the neural crest‐like phenotype in human dental pulp. It is worth noting, however, that there is a substantial level of heterogeneity and patient‐to‐patient variation that should be considered for future clinical applications.Altogether, the present work details the culture and characterization of NCSCs from a broad cohort of human dental pulp samples established in preclinically relevant serum‐free conditions. Our results should facilitate the culturing of these cells under good manufacturing practice‐compliant processes and encourage their use in disease‐specific models to advance their application in regenerative medicine.Although not directly related to the work presented here, Marcelo N. Rivolta is the founder of Rinri Therapeutics. The other authors declared no potential conflicts of interest.O.S.‐C.: collection and assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; F.B.: conception and design, provision of study material, data analysis and interpretation, manuscript writing, final approval of manuscript; M.N.R.: conception and design, financial support, provision of study material, data analysis and interpretation, manuscript writing, final approval of manuscript.


Proton pump inhibitors  commonly are administered concurrently with nonsteroidal anti‐inflammatory drugs  as prophylaxis to decrease the risk of gastrointestinal (GI) injury. However, evidence to support this practice is weak, and it might exacerbate dysbiosis and inflammation.To evaluate the effect of carprofen alone or combined with omeprazole in dogs. We hypothesized that coadministration of omeprazole and carprofen would significantly increase GI permeability and dysbiosis index (DI) compared to no treatment or carprofen alone.Six healthy adult colony beagle dogs.P < .05).Gastrointestinal permeability and inflammation were assessed by serum lipopolysaccharide (LPS) concentration, plasma iohexol concentration, fecal DI, and fecal calprotectin concentration in a prospective, 3‐period design. In the first 7‐day period, dogs received no intervention (baseline). During the 2nd period, dogs received 4 mg/kg of carprofen q24h PO for 7 days. In the 3rd period, dogs received 4 mg/kg of carprofen q24h and 1 mg/kg of omeprazole q12h PO for 7 days. Gastrointestinal permeability testing was performed at the end of each period. Data were analyzed using repeated measures mixed model analysis of variance with Tukey‐Kramer post hoc tests (P = .03). The DI varied over time based on the treatment received (P = .03). Coadministration of omeprazole and carprofen significantly increased fecal calprotectin concentration and DI compared to baseline and carprofen alone.Serum LPS and plasma iohexol concentrations did not differ between treatments. Fecal calprotectin concentrations differed between treatments (Omeprazole prophylaxis induces fecal dysbiosis and increases intestinal inflammatory markers when coadministered with carprofen to otherwise healthy dogs with no other risk factors for GI bleeding. ANOVAanalysis of varianceDIdysbiosis indexGIgastrointestinalHPLChigh‐performance liquid chromatographyLPSlipopolysaccharideNSAIDnonsteroidal anti‐inflammatory drugPPIproton pump inhibitorqPCRquantitative PCRSCFAshort chain fatty acid1Nonsteroidal anti‐inflammatory drugs (NSAIDs) are commonly prescribed to dogs for the treatment of pain and inflammation, often caused by osteoarthritis. The most commonly reported adverse effects of NSAIDs are gastrointestinal (GI) signs such as vomiting and diarrhea.Despite the common practice of prophylactically administering PPIs to otherwise healthy dogs receiving NSAIDs, no published studies have investigated the effects of this combination on intestinal permeability and inflammation in dogs. Our study objective was to evaluate the effect of the NSAID, carprofen, on the development of increased GI permeability, inflammation, and fecal dysbiosis when administered alone or in combination with the PPI, omeprazole, in dogs. We hypothesized that coadministration of omeprazole and carprofen would significantly increase GI permeability and the dysbiosis index (DI) compared to no treatment or carprofen alone.22.1The study was approved by the Institutional Animal Care and Use Committee at North Carolina State University (Protocol # 19‐048‐O). Six healthy adult purpose‐bred beagle dogs from a research colony at North Carolina State University , aged 1.5 to 6 years and weighing 10.0 to 12.0 kg, were used in the study. Dogs were deemed healthy based on lack of a history of GI disease , normal physical examination, and normal laboratory diagnostic test results  obtained within 6 to 15 months of study entry. Dogs were maintained in their original housing throughout the study and their social interaction and feeding schedule were not altered for the study.2.2g for 10 minutes and immediately stored at −80°C. Fecal consistency was graded from 1 to 7 on each day of each treatment period using a standardized fecal scoring system . Diarrhea was defined as a fecal score > 4. Clinical signs, including changes in activity, food consumption, vomiting, and number of defecations, were recorded a minimum of q12h. Fecal and serum samples were shipped on dry ice at study completion to the Gastrointestinal Laboratory at Texas A&M University for measurement of serum lipopolysaccharide (LPS) concentration, fecal DI, and fecal calprotectin concentration.A prospective, sequential study was performed Figure  in which2.3r2) of >0.99, and the back‐calculated concentrations were within 15% of the true concentration of the standard. Plasma samples of 400 μL were loaded onto washed solid‐phase extraction cartridges  and eluted with 1 mL of methanol with 0.25% phosphoric acid. The eluent was evaporated in a water bath at 55°C. The samples were reconstituted with 200 μL of the mobile phase, vortexed, and transferred into the HPLC injection vial. The injection volume was 40 μL.High‐performance liquid chromatography (HPLC) was used to analyze the concentration of iohexol in plasma samples using a method modified from previous studies.The assay met system suitability requirements. Iohexol undergoes endo‐exo isomerism and each peak was resolved as the endo‐ (minor) and exo‐ (major) isomer with the endo‐isomer eluting at approximately 4 minutes and the exo‐isomer eluting at approximately 4.8 minutes. For our results, we added the concentrations of each isomer and reported the total.2.4Serum was collected on day 8 of each treatment period. Endotoxin quantification was performed using Pierce Chromogenic Endotoxin Quant kit (ThermoFisher). Samples were thawed once at room temperature and immediately diluted 1 : 100. Diluted samples were heat‐shocked for 15 minutes at 70°C to inactivate assay inhibitors. The assay was performed according to the manufacturer's instructions with samples, standards, and blanks run in triplicate. The optical density was measured at 405 nm immediately after assay completion, and quantification was calculated using the low standard curve (0.01‐0.1 EU/mL).2.5Faecalibacterium, Turicibacter, Escherichia coli, Streptococcus, Blautia, Fusobacterium, and Clostridium hiranonis as previously described.Fecal samples were collected on days 5, 6, and 7 of each treatment period for analysis of the fecal DI, which was measured on each sample individually. Sample DNA was extracted using a MoBio PowerSoil DNA isolation kit (MoBio Laboratories), following the manufacturer's instructions. To calculate a PCR‐based DI, quantitative polymerase chain reaction (qPCR) assays were performed for total bacteria, 2.6Fecal samples were collected on days 5, 6, and 7 of each treatment period for measurement of fecal calprotectin concentrations. Fecal calprotectin concentrations were measured on each sample individually using an analytically validated radioimmunoassay as previously described.2.7P value adjustment was applied to post hoc tests for all analyses. Statistical significance was defined as P < .05. All statistical analyses were performed using commercial software .A power calculation was not performed before the study. Serum LPS concentration was analyzed for differences between baseline, carprofen alone, and carprofen and omeprazole periods using a single factor repeated measures mixed model analysis of variance (ANOVA). Plasma iohexol concentration, fecal calprotectin concentration, fecal DI, and individual bacterial taxa were analyzed using a 2‐within subject factor repeated measures mixed model ANOVA. Data were tested for effects of treatment, time, and the interaction of treatment and time. Dog, dog*treatment, and dog*time were evaluated as random effects and considered in each model. An unstructured Kronecker product covariance structure was applied to each 2‐within subject factor mixed model ANOVA. A Shapiro‐Wilk test for normality and QQ plots were used to evaluate normality of ANOVA residuals. Levene's equality of variances test was used to evaluate equality of treatment variances. Box plots and Studentized residual diagnostics were performed to evaluate each model for the presence of outliers. Log transformations were required for plasma iohexol and fecal calprotectin concentrations in order to meet underlying statistical assumptions. To control for type I error, Tukey's 33.1P = .32).Serum LPS concentrations for each treatment are graphically depicted in Figure 3.2P < .001). Post hoc tests identified no differences between hours 1 and 2 (P = .72). However, for hours 2 through 6, each subsequent time measurement demonstrated a significant decrease in total plasma iohexol concentration . No significant differences in total plasma iohexol concentration were found between treatments or treatment‐by‐time .Total plasma iohexol concentrations (combined endo‐ and exo‐isomer) over time for each treatment are graphically depicted in Figure 3.3P = .03). However, after adjusting post hoc tests for multiple comparisons, only differences between treatments at each time point were found to be significant. On days 5, 6, and 7, coadministration of carprofen and omeprazole differed from both carprofen and baseline . All dogs had increased DI when receiving both omeprazole and carprofen. No significant differences were observed when carprofen alone was compared to baseline on any day (P > .24). Fecal DI was increased >2 in 5 of 6 dogs on at least 1 treatment day when receiving coadministered omeprazole and carprofen. In the remaining dog, fecal DI was in the equivocal zone of 0 to 2, with a median value of 1.4. In contrast, fecal DI did not increase >0 on any treatment day when dogs received carprofen alone or no treatment. When individual bacterial taxa were considered , Turicibacter, Fusobacterium, and C hiranonis abundances differed between treatments . Post hoc tests determined that coadministration of carprofen and omeprazole resulted in significantly lower average abundances when compared to both carprofen alone and baseline . No differences were observed between carprofen alone and baseline on any day . Faecalibacterium abundances varied over time based on the treatment received (P = .04), but after adjusting post hoc tests for multiple comparisons, only differences between treatments at each time point were found to be significant. On days 5, 6, and 7, coadministration of carprofen and omeprazole resulted in lower average values compared to carprofen alone or baseline . No difference was found between carprofen and baseline on any day . Blautia results varied over time based on the treatment received (P = .002). On day 5, coadministration of carprofen and omeprazole differed from both carprofen alone and baseline . On day 6, after adjusting post hoc tests, no differences were observed between coadministration of carprofen and omeprazole and baseline (P = .05). However, carprofen alone differed between both baseline and coadministration of carprofen and omeprazole . On day 7, all treatments differed  and, as with day 5, coadministration of carprofen and omeprazole resulted in lower average Blautia abundances compared to that for carprofen alone or baseline. No differences were observed over time or between treatments for Streptococcus or E coli .The fecal DI Figure  varied sd Figure , Turici3.4P = .03) but did not vary over time (P = .4), or treatment‐by‐time (P = .35). Post hoc tests determined that coadministration of omeprazole and carprofen resulted in increased fecal calprotectin concentrations when compared to baseline (P = .03) or carprofen alone (P = .04). No differences were observed when carprofen was compared to baseline (P = .46).Fecal calprotectin concentrations Figure  signific3.5All dogs remained active and alert throughout the study. They all maintained strong appetites and consumed 100% of the food offered during each treatment period. One episode of vomiting during baseline treatment, none during the carprofen treatment, and 3 episodes involving 2 dogs occurred during the carprofen and omeprazole treatment period. Fecal scores remained between 2 and 4 during the baseline and carprofen treatment periods, with averages of 2.8 and 2.5, respectively, for days 5 to 7. During the carprofen and omeprazole treatment period, 3 episodes of diarrhea occurred involving 1 dog, and the average fecal score between days 5 and 7 increased to 3.3. This increase was not statistically significant. No blood was observed in the feces at any time during the study based on macroscopic examination.4We evaluated the effect of carprofen on GI permeability, inflammation, and fecal dysbiosis when administered alone or in combination with omeprazole in healthy dogs. Plasma iohexol and serum LPS concentrations were utilized as markers of intestinal permeability. Iohexol is a large osmotic agent that passively diffuses between epithelial cells throughout the entire GI tract and has been used previously for evaluation of intestinal permeability in healthy dogs.The fecal DI and calprotectin concentrations significantly increased during coadministration of carprofen and omeprazole compared to both the baseline and carprofen alone periods. The fecal DI uses a panel of qPCR assays that evaluate the abundance of 7 key bacterial taxa for intestinal health and enumerates them as a single number.C hiranonis, a bacterium with bile acid 7‐alpha‐dehydroxylation ability,C hiranonis. Secondary bile acid production is a key microbiome function known to be impaired after antibiotic administrationClostridioides difficile infections in humans.C difficile is less clear,C hiranonis may confer resistance to infection.When individual bacterial taxa of the DI were analyzed, the combination of carprofen and omeprazole affected 5 of the 7 key taxa evaluated. Short chain fatty acid (SCFA)‐producing bacteria were found to be decreased after coadministration of omeprazole and carprofen. The SCFA, especially butyrate, are essential for intestinal health,Calprotectin is a protein complex found primarily in neutrophils and is used as a noninvasive marker of intestinal inflammation in humans and dogs.Whether carprofen plays a substantial role in the induction of dysbiosis or inflammation when coadministered with omeprazole cannot be determined from our study because we did not include a treatment group in which dogs received omeprazole alone. Additionally, our study had a sequential period rather than a randomized design, in which carprofen was administered first to all dogs separated by a 4‐week washout period and followed by coadministration of omeprazole and carprofen. The half‐life of carprofen is approximately 8 hours and 99.9% of the drug is eliminated within 80 hours after administration.Our study was performed using 6 healthy beagle dogs with a 7‐day treatment period. Despite the relatively short treatment period, coadministration of omeprazole and carprofen induced a fecal dysbiosis and increased intestinal inflammation in these dogs. Future studies with a larger sample size, a group receiving only omeprazole, and a longer treatment period in a population of dogs with additional risk factors for GI injury and bleeding, such as older age and presence of comorbidities, would better represent the clinical population of dogs receiving carprofen and omeprazole long‐term and may allow for detection of additional changes in GI permeability and determine if any positive benefits from omeprazole administration occur that we were unable to detect. Although such additional studies are warranted, our study results lead us to believe that whereas omeprazole is the standard of care for suspected or documented NSAID‐induced gastric or proximal duodenal bleeding, prophylaxis in healthy dogs receiving carprofen provides no benefit to intestinal health and could cause intestinal inflammation and fecal dysbiosis.Authors declare no conflict of interest.Authors declare no off‐label use of antimicrobials.The IACUC at North Caroline State University approved the protocol for this study.Authors declare human ethics approval was not needed for this study.Supplementary Figure 1 Bacterial taxa analyzed by qPCR. Turicibacter, Fusobacterium, and C. hiranonis abundances differed between treatments . The co‐administration of carprofen and omeprazole resulted in significantly lower average abundances of these bacterial taxa when compared to both carprofen alone and baseline . Faecalibacterium abundances varied over time based on the treatment received (P = .04), but after adjusting post‐hoc tests for multiple comparisons, only differences between treatments at each time point were found to be significant. On days 5, 6, and 7, co‐administration of carprofen and omeprazole resulted in lower average values compared to carprofen alone or baseline . Blautia values varied over time based on the treatment received (P = .002). On days 5 and 7, co‐administration of carprofen and omeprazole resulted in lower average Blautia abundances compared to that for carprofen alone or baseline. No significant differences were observed for Streptococcus or E. coli .Click here for additional data file.


The number of insect species and insect abundances decreased severely during the past decades over major parts of Central Europe. Previous studies documented declines of species richness, abundances, shifts in species composition, and decreasing biomass of flying insects. In this study, we present a standardized approach to quantitatively and qualitatively assess insect diversity, biomass, and the abundance of taxa, in parallel. We applied two methods: Malaise traps, and automated and active light trapping. Sampling was conducted from April to October 2018 in southern Germany, at four sites representing conventional and organic farming. Bulk samples obtained from Malaise traps were further analyzed using DNA metabarcoding. Larger moths (Macroheterocera) collected with light trapping were further classified according to their degree of endangerment. Our methods provide valuable quantitative and qualitative data. Our results indicate more biomass and higher species richness, as well as twice the number of Red List lepidopterans in organic farmland than in conventional farmland. This combination of sampling methods with subsequent DNA metabarcoding and assignments of individuals according depending on ecological characteristics and the degree of endangerment allows to evaluate the status of landscapes and represents a suitable setup for large‐scale long‐term insect monitoring across Central Europe, and elsewhere. Malaise trap on the grassland of organic farmland. Insects were collected with Malaise traps, in combination with automated and active light trapping, at identical sites. Subsequently, we weighted the biomass and used DNA metabarcoding to calculate species richness. Catches of the light trapping were further analyzed in respect of species abundances and community composition, considering species‐specific traits. With our data collected during this first year of monitoring, we will highlight the following questions:22.1Our study area is located in southern Germany, 15 km distant to the city Pfaffenhofen. We established four study sites, two in organic, and two in conventional farmland. Malaise traps and light traps were installed at extensively used grasslands. Both grasslands border north–south forest fringes and were mowed twice a year  and without any application of pesticides, however with the use of organic fertilizers. The organic farmland was cultivated without any pesticides since 64 years, but with organic fertilizer (on February 05). Artificial fertilizers were used on the conventional farmland (in 2018 on February 14 and April 05). The following pesticides were applied on the bordering rye and cornfields in conventional farmland: Broadway , Gardo Gold , Callisto , and the shortcut Chlormequat . Distance between organic and conventional grasslands was about 700 m.2.2We installed four standard Malaise traps , with two in each of the two farmland types (organic farmland 48.5073°N/11.5397°E (M1), 48.5072°N/11.5401°E (M2), conventional farmland 48.5090°N/11.5314°E (M3), and 48.5091°N/11.5318°E (M4)). All four Malaise traps were activated from 10 April to 19 October 2018. At each farmland type, one trap was positioned at the edge (forest fringe) and the second at 15 m distance in the open grassland. All traps were simultaneously opened and southwards positioned, with similar exposure to wind. We changed the 600‐ml sampling bottles filled with 80% ethanol simultaneously for all Malaise traps, eleven times throughout the year .2.3Automated light traps  were positioned from sunset to sunrise, 14 times simultaneously in parallel at each of the two grassland sites, from April 10 till October 19 . In addition, we collected nocturnal moths actively at light towers equipped with 12V bulbs, 13 times simultaneously at each of the two grassland sites, from sunset to 01:00 am . As killing agent, we used ethyl acetate. Collected vouchers were identified by A. Hausmann and A. H. Segerer, and, in a few, difficult cases, with DNA barcoding .2.4Dry and wet biomass material was weighted and analyzed separately, according to Ssymanck et al. . Biomass2.5Species identification of organic material in the Malaise traps was performed using DNA metabarcoding. However, macrolepidopterans and orthopterans were a priori sorted out of the Malaise traps and identified by experts at the ZSM. Some macrolepidopterans or fragments of them remained in the bulk sample and were subsequently detected by the metabarcoding approach. All microlepidopterans were identified with the metabarcoding approach in the bulk sample. Each single dried sample  was homogenized in a FastPrep96 machine  using sterile steal beads in order to generate a homogeneous mixture of arthropods and submitted for subsequent metabarcoding (conducted by AIM GmbH). Prior to DNA extraction, 1 mg of each homogenizate was weighed into sample vials and processed using adapted volumes of lysis buffer with the DNeasy 96 Blood & Tissue Kit (Qiagen) following the manufacturer's instructions. For amplification of the CO1‐5P target region and preparation of the MiSeq libraries, a 2‐step PCR was performed. First, a 313 bp long mini‐barcode region was amplified by PCR . Sequences were dereplicated with “derep_fulllength,” first at the sample level, and then concatenated into one FASTA file, which was subsequently dereplicated. Chimeric sequences were filtered out from the FASTA file using the VSEARCH program “uchime_denovo.” The remaining sequences were then clustered into OTUs at 97% identity with “cluster_size,” a greedy centroid‐based clustering program. OTUs were blasted against a custom Animalia database downloaded from BOLD on 28 November 2018, including taxonomy and BIN information, by means of Geneious , and following methods described in Morinière et al. . Data releases have been published for all major arthropod groups, that is, Coleoptera  of an OTU query sequence with its closest counterpart in the database), length of the top BLAST hit sequence, phylum, class, order, family, genus, and species information for each detected OTU was exported from Geneious and combined with the OTU table generated by the bioinformatic pipeline. The combined results table was then filtered by Hit‐%‐ID value and total read numbers per OTU. All entries with identifications below 97% and total read numbers below 0.01% of the summed reads per sample were removed from the analysis. OTUs were then assigned to the respective BIN. Additionally, the API provided by BOLD was used to retrieve BIN species and BIN countries for every OTU, and the Hit‐%‐IDs were aggregated over OTUs that found a hit in the same BIN and shown in the corresponding column as % range Table . To valiSpecies identification in the Malaise trap samples was based on high‐throughput sequencing (HTS) data grouped to genetic clusters (OTUs), blasted, and assigned to barcode index numbers  was assumed as n = l + m − k. This probability is given by  degree of undersampling = 100 (1 − n/nreq).We used two approaches to infer differences in species composition between organic and conventional farming sites. First, we used a random sampling model and calculated for all taxa having at least 15 OTUs the probability of having Sr) between all species, which jointly occur at both farming sites. Significantly, negative Sr values indicate structural differences between the two communities in terms of relative abundances. We used one‐way ANOVA to infer the difference in extinction probabilities between both farming types using average OTU abundance at both sites as the dependent variable.We calculated Spearman's rank‐order correlations . We then assessed for each species whether the peak of emergence was identical in time (within the same sample period) between and within each farming type . Counts of the numbers of these joint emergences in comparison with the total numbers of joint occurrences indicate similar or divergence (habitat specific) phenology.33.1Weight of wet and dry biomass significantly correlated across all samples Table . Malaise3.2The high‐throughput sequencing (HTS) of the samples collected with four Malaise traps yielded a total of 3,117,887 sequences (paired‐end) for Arthropoda and (a few) Euarthropoda , with good sequence quality scores (FastQC). After quality filtering, 2,215,879 sequences were kept, and after dereplication and clustering, 18,531 genetic clusters were obtained, with 6,321 OTUs remaining after chimera detection. Out of these, 6,316 found BLAST hits in the BOLD database. Entries with sequence identity below 97% and total read numbers below 0.01% of the summed reads per sample were removed from further analysis. The remaining OTUs  could be assigned to 3,890 barcode index numbers (BINs) which are considered a good proxy for species numbers (see our discussion). Best represented orders were Diptera with 1,867 BINs, Hymenoptera , Lepidoptera (412), and Coleoptera (254)  yielded 21% more species (BINs) than those of the sites under conventional farming regime (M3–M4). The open grassland trap of the organic farming (M1 versus M3) revealed 10% more species (BINs), the forest fringe trap (M2 versus M4) 30% more. The strongest divergences are found in the holometabolic orders Coleoptera (+43% on the organic farm) and Lepidoptera (+59%), of which the majority of species have ectophagous, phytophagous larvae.Plusia stenochrysis rather than P. chrysitis; Mesapamea secalis rather than M. secalella, Conistra vaccinii rather than C. ligula); in two additional cases (0.5%) , this was not possible because of identity or overlap of nonconspecific sequences. The occurrence of both Scoparia ambigualis and S. basistrigalis in the HTS sequences, however, was ascertained by their phenology . Two false positives (2/412 = 0.5%) were ruled out among the Lepidoptera data, one referring to a Neotropical skipper , which was deposited with an erroneous Wolbachia gene sequence on GenBank, and the other to a Neotropical nymphalid, Agraulis vanillae, which got a 98% and 99% match in BOLD and GenBank blasting, but which had only 9 reads in the Malaise trap M2 most likely due to lab contamination. According to the cleaned data, the Malaise traps on the organic farm (M1–M2) yielded 364 lepidopteran species, 136 more (+59%) than the Malaise traps on the grassland under conventional farming regime (228).The Lepidoptera fraction of the Malaise trap samples was studied in detail  revealing a total of 412 recorded species Table . For a d3.3Species richness and abundance of larger moths (Macroheterocera) from light traps confirm the results and trends from Malaise traps: Light trapping at the two sites between 10 April and 19 October 2018 yielded in total 2,639 macroheteroceran individuals representing 286 species. A detailed list of all Lepidoptera species collected at light is given in Table 3.4F  = 89.01, p < .001).For all sufficiently rich taxa, except for Araneae and Orthoptera, we found significantly lower numbers of joint OTUs than what would be expected from a random sample model, assuming complete sampling of the local species pool Table . However3.5Altogether, 47 species of the Bavarian “Red Book of threatened Lepidoptera species”  were negative Figure . The aveThe species‐specific analysis showed that more than 50% (in most comparisons more than 65%) of species had identical peaks of emergence across the habitat types Table . Importa4Our approach combines quantitative and qualitative assessments and data. Our methods include the calculation of biomass with the use of high‐throughput sequencing of the biomass collected with Malaise traps to calculate species richness. We complement these quantitative data with a qualitative insect assessment, using light traps. We applied automated light traps to collect nocturnal lepidopterans and performed active sampling of nocturnal lepidopterans on light towers. These data provide information on species abundances and community composition. We completed this last step by assigning ecological traits to each single species, such as Red List status. Our data show parallel temporal fluctuations and show higher diversities and abundances, more biomass, and a much larger number of Red List nocturnal lepidopterans in organic farmland if compared with the samples collected in conventional farmland.4.1The combination of a quantitative and qualitative data set provides various advantages, while each single method shows different strengths, but also shortcomings. DNA metabarcoding allows analyses of large arthropod bulk samples of several thousand individuals from hundreds to thousands of species within only few weeks  derive very meaningful conclusions. However, our trends are in accordance with other studies showing a significant loss of biomass and reduced species richness in conventional farmland ; data curation ; investigation ; methodology ; project administration ; supervision ; writing‐original draft ; writing‐review and editing . Andreas Segerer: Conceptualization ; data curation ; investigation ; methodology ; project administration ; writing‐original draft ; writing‐review and editing . Thomas Greifenstein: Conceptualization ; data curation ; methodology ; project administration ; writing‐original draft ; writing‐review and editing . Johannes Knubben: Funding acquisition ; investigation ; project administration ; writing‐original draft ; writing‐review and editing . Jerome Morniere: Conceptualization ; data curation ; formal analysis ; methodology ; software ; visualization ; writing‐original draft ; writing‐review and editing . Vedran Božičević: Data curation ; investigation ; methodology ; writing‐original draft ; writing‐review and editing . Dieter Doczkal: Data curation ; methodology ; writing‐original draft ; writing‐review and editing . Armin Günter: Data curation ; funding acquisition ; methodology ; project administration ; writing‐original draft ; writing‐review and editing . Werner Ulrich: Conceptualization ; investigation ; methodology ; supervision ; validation ; visualization ; writing‐original draft ; writing‐review and editing . Jan Christian Habel: Conceptualization ; methodology ; supervision ; validation ; visualization ; writing‐original draft ; writing‐review and editing .Thomas Greifenstein, Armin Günter, and Dieter Doczkal conducted the field work. Axel Hausmann, Jerome Morniere, Vedran Božičević, and Werner Ulrich performed data analyses. Axel Hausmann, Jan Christian Habel, and Werner Ulrich wrote the manuscript, with further contributions by all other authors. Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.Table S4Click here for additional data file.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Figure S3Click here for additional data file.Figure S4Click here for additional data file.Supplementary MaterialClick here for additional data file.


To assess the prevalence of delirium (DEL) among older patients living at home and periodically visited by their General Practitioners (GPs).Observational study.In Italy, programmed home visits by the GPs are regularly scheduled for their vulnerable and frail patients who are often on poly-drug regimens and suffering from dementia.N = 102 patients among those receiving programmed home visits by n = 6 GP based in the Brianza area (Lombardy).Patients were screened for delirium with the Italian version of the 4AT, with a score ≥ 4 considered as a positive indicator for DEL. The Charlson Comorbidity Index (CCI), the Short Physical Performance Battery (SPPB), the presence of dementia, and benzodiazepine (BZD) use were recorded.p < 0.00001). Analogously, BZD use was over-represented in the DEL+ group with respect to the DEL− one .DEL+ was detected in almost half of the recruited sample (44.1%), and it was clearly associated with increased comorbidity and decreased motor abilities. Pre-existing dementia was documented in most of DEL+ patients (71.1%), while this was the case for only a minority of DEL- , and BZD use. DEL prompt recognition should be one of the goals of GP-programmed home visits, since this treatable and preventable condition is associated to an elevated burden of frailty and risk of death. According to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) criteria , deliriuDelirium is one of the most common acute mental disorders that affect the older people. In acute hospitals, delirium affects nearly one in five patients, but both prevalence and incidence is even higher in surgical wards and in intensive care unit, reaching a prevalence of 80% in mechanical ventilated patients . DeliriuTo date, only a few studies have assessed the prevalence of delirium at home. The East Baltimore Survey  found anIn Italy, General Practitioner (GP)-programmed home visits are regularly performed to check on a special subset of the general population characterized by elevated frailty, moderate to severe disability and chronic conditions that can be managed at home. During the programmed home visits, delirium can be easily missed, if not specifically focusing on it, and especially in the hypoactive form. This information, however, is conceivably of extreme value since delirium is a further important marker of frailty , rather The aim of this observational study was to assess the prevalence of delirium among a cohort of older patients living at home and periodically visited by their GPs due to the elevated burden of their medical conditions.n = 102 consecutive patients were recruited among those receiving programmed home visits by n = 6 GPs based in the Monza-Brianza area . All patients were followed according to this modality because of either, significant logistic/motor difficulties in transporting them to the GP’s office [although truly bedridden patients were n = 11 (10.8%)], or because considered too fragile for moving. Most patients had more than one significant medical condition, but the main reason for being included in this program were: previous stroke (n = 19); severe cardiovascular disease (n = 8); COPD/severe respiratory problems (n = 8); advanced Parkinson’s disease (n = 7); dementia (n = 7); severe osteoarthrosis/orthopedic problems (n = 20); diabetic polyneuropathy (n = 14); age > 95 y.o. (n = 13); age > 85 y.o. and no relatives available (n = 6). These visits were programmed on a monthly basis.Following ethical approval, .). The Charlson Comorbidity Index (CCI) ; all cases were subsequently confirmed by GP clinical impression. The CCI was 4.8 ± 2.3 (1–12) and the SPPB score was 3.1 ± 2.9 (0–12), accordingly to the expected decrease in the functional status of this selected population.The 4AT scale could be administered to all patients [4.6 ± 4.0 (0–12)] and a score suggesting delirium  was obtained in almost half of them , increased comorbidity , and decreased physical performances . Delirium was detected in n = 32 dementia patients out of 35 (91.4%) with respect to n = 13 out of 67 (19.4%) in cognitively spared ones (χ2 48.3 p < 0.00001).Pre-existing dementia was documented in most of DEL + patients (71.1%), while this was the case for only a minority of DEL- . And again, BZD users versus non-users had increased 4AT score , increased comorbidity , and decreased physical abilities [SPPB score: 3.5 ± 2.7 (0–8) vs. 3.5 ± 2.9 (0–12)]. Delirium was detected in n = 33 out of 46 BZD users (71.7%) with respect to n = 12 out of 56 (21.4%) non-users (χ2 25.9 p < 0.00001). Finally, Fig. Analogously, BZD use was over-represented in the DEL + group with respect to the DEL- one . Thus, even when critically evaluating all these issues, the rate of delirium in our home-based patients receiving periodic GP visits appears unexpectedly high.We found a surprisingly elevated prevalence of delirium in these patients (44.1%). Coherently, the 4AT score significantly correlated with both the CCI and the SPPB: as expected, patients with more comorbidities (and drugs) and more impaired physical performance are at higher risk for delirium. The presence of bedridden patients may have led to overestimate delirium prevalence but, plausibly, this is true only for a minority of patients (10.8%). One further limitation consists in the fact that delirium has been ascertained based only on one single 4AT assessment. However, in previous studies, the 4AT has shown good sensitivity and specificity toward DSM criteria, suggesting that is a reliable tool to detect delirium . In addii.e., the more severe is the degree of dementia the higher is the likelihood of developing delirium), GPs need to be formally trained in detecting delirium among their patients cared at home. In fact, delirium is a strong predictor of functional dependence, institutionalization, and mortality in older patients admitted to rehabilitation [The exact relationship between dementia and delirium is a current matter of debate, but dementia is commonly considered the leading risk factor for delirium . In our litation , and to litation . In thishttps://www.sign.ac.uk/, March 2019) recommend BZD deprescription for reduction and management of delirium.The literature indicates a weak relationship between BZD use and increased risk of delirium, in particular, outside of the ICU setting , and theOne major limitation of our study consists in the lack of information on sedative drugs other than BZD. In fact, neuroleptics could be potential confounders, and the global anticholinergic burden of the poly-drug regimens taken by our patients represent a definite source of risk for developing delirium, and, therefore, a potential unaddressed source for bias in our study .As conclusion, our study focuses on delirium from the peculiar prospective of very frail patients in their own home, reporting for the first time a substantial prevalence of the phenomenon. This severe underestimation may have important consequences in terms of public health management since delirium is a harmful condition that can be prevented using multicomponent interventions  and can


According to the tribological analyses, the adhesion strength of the coated deposition on 316 L was obtained 32.8 N, and the mean coefficient of friction was around 0.3. Biocompatibility studies of coated metallic biomaterials were analyzed on fibroblast cell culture ) in vitro. Hoescht 33258 fluorescent staining was performed to investigate the cellular density and chromosomal abnormalities of the HDFa cell line on the borosilicate glasses coated with germanium–tungsten (W–Ge). Cell viabilities of HDFa cell line on each surface  were analyzed by using -2,5-diphenyltetrazolium bromide) cytotoxicity assay. The antibiofilm activity of W–Ge coated borosilicate glass showed a significant reduction effect on Staphylococcus aureus (ATCC 25923) and Pseudomonas aeruginosa (ATCC 27853) adherence compared to control groups. In the light of findings, tungsten and germanium, which are some of the most common industrial materials, were investigated as biocompatible and antimicrobial surface coatings and recommended as bio-implant materials for the first time.In this study, borosilicate glass and 316 L stainless steel were coated with germanium (Ge) and tungsten (W) metals using the Magnetron Sputtering System. Surface structural, mechanical, and tribological properties of uncoated and coated samples were examined using SEM, X-ray diffraction (XRD), energy-dispersive spectroscopy, and tribometer. The XRD results showed that WGe It is known that heavy metals such as arsenic and cadmium cause zinc to separate from zinc-bound body structures, thereby disrupting the function of certain organs in the body and even causing cancer [Biomaterials have been studied for more than 50 years in the field of medicine and engineering to fulfill the vital function of tissues in the human body with natural or synthetic material types . Biomateg cancer . In thisg cancer . TherefoStaphylococcus aureus and Pseudomonasaeruginosa planktonic [2 intake. Lück et al. [2 or inorganic compounds of Ge are relatively toxic, however, low concentrations of Ge contribute to the physiological functions in the human body. To avoid adverse effects of Ge-132, Ge, and GeO2, the lowest ranges observed are between 42 and 1380 mg/d for average weight (60 kg) adult [Germanium (Ge) is a metalloid, which is known chemically similar to silicon (Si), and mostly used in the electronics industry. This material potentially useful for not only optics, sensors, and catalysis, but also can be used as antimicrobial agents. The potential antimicrobial properties of Ge compounds were proved against the human pathogenic bacteria . Also, tanktonic . Ge is aanktonic . Ge natuanktonic –15. Nagaanktonic  reportedk et al.  also repk et al. . This stk et al. . Moreovek et al. . Thus, tg) adult .Extraordinary physical and mechanical properties of tungsten, especially good wear resistance, high melting point, and hardness made it attractive for using in many areas such as coatings on heat pipes, corrosion-resistant coatings, nuclear power systems, and microelectronic systems –21. PrevIn order to overcome the aforementioned issues such as toxicity, inflammation, bacterial contamination, and reaction of the body, some effective posttreatment of the biomaterials is suggested by coating them biocompatible and corrosion-resistant materials . The mosIn this study, the W–Ge alloy was investigated as a composition material due to the potential biologic properties of Ge and extraordinary physical and mechanical properties of tungsten as mentioned above. Ti was deposited as the first layer on substrates before coating W–Ge due to obtaining good adhesion properties to improve the interfacial strength . This is−6 mTorr and a working pressure of ∼2 × 10−3 mTorr in the argon (Ar) atmosphere. Initially, Ti buffer layer was deposited on the substrate. Afterward, Ge and W were deposited on Ti buffer layer using the co-deposition technique with deposition rates of 50% Ge and 50% W. Deposition rates of samples were determined using a quartz thickness monitor. The deposition temperature during the sample preparation was around room temperature (25–35 C°) and the distance from the target to the substrate was 10 cm. The power, 115 W (RF), 115 W (RF), and 300 V (DC), was applied to Ti, Ge, and W targets, respectively. 200 nm Ti and 1000 nm Ge–W were deposited on the substrates. Physical characterization of all samples was carried out using X-ray diffraction (XRD) (EXPLORER-GNR), scanning electron microscope (SEM) (Quanta FEG 250), energy-dispersive spectroscopy (EDS) (Edax), and tribometer (Bruker UMT).Before the magnetron sputtering process, borosilicate glass  and 316 L stainless steel  substrates were cleaned with isopropanol and distilled water, respectively, using the ultrasonic cleaner for 15 min for each, and cleaning progress ended with air blow-drying. Two inches diameter titanium (Ti), germanium (Ge), and tungsten (W) solid targets with purity (99.99%) were used to prepare samples. Before deposition, to achieve better surface activity and to remove contaminants on the target surface, each target was cleaned using a plasma cleaning process for 10 min. All samples were prepared using a magnetron sputtering system  equipped with two RF, one DC sputter, and two thermal sources. All the depositions were carried out at a base pressure of ∼4 × 10®, USA) included 10% fetal bovine serum  was used to analyze static corrosion behavior of W/Ge coated the borosilicate glass surface. Coated samples were placed in a six-well plastic culture plate with 2 ml DMEM immersion. Culture plates were incubated for 7 days at 37 °C under a humidified atmosphere of 95% air and 5% CO2. After static corrosion was completed, samples were removed from the media and washed with distilled water for scanning electron microscope (SEM) investigations. Surface topology and material components were observed via SEM and EDS analyses [Commercially available Dulbecco’s modified Eagle’s medium  was grown in T25 cell culture flask by using DMEM  included 10% FBS  and 1% Penicillin/streptomycin  at 37 °C, 5% CO2 humidified incubator. After the culture reached the confluency, cells were detached from the flask surface via the use of Trypsin-EDTA  solution. The cell culture was seeded as 1 × 105 number of cells on the tungsten–germanium coated borosilicate glasses in 12-well plate and incubated for 24 h at 37 °C, 5% CO2 incubator to analyze attached cells. Experimental groups were classified as coated materials, uncoated borosilicate glasses, and negative control (cell culture plate surface), and each group was analyzed in triplicate samples.Human fibroblast (HDFa) cell culture . Coated and uncoated materials were placed in a fresh culture plate with the aid of sterile tweezers. The cell cultures were fixed by using 4% glutaraldehyde solution at +4 °C for 30 min. Fixed cells were washed with 1x PBS and 1 µM Hoescht 33258 fluorescent dye added to each well and incubated for 5 min at room temperature. The stained sample surface was monitored with a fluorescent microscope at ×10, ×20, and ×40 magnifications.Hoescht 33258 staining was performed to detect the cellular density and chromosomal abnormalities of the HDFa cell line on the borosilicate glasses coated with tungsten–germanium. After 24 h of incubation, cell cultures in 12-well plates were washed with phosphate-buffered saline  solution was used according to the manufacturer’s instructions. Briefly, coated and uncoated materials were rinsed with DMEM media and 0.5 ml of fresh DMEM + 10% MTT solution was applied to the samples and incubated for 3 h at 37 °C, 5% CO2 humidified incubator. Formazan crystals were dissolved by using dimethyl sulfoxide (Sigma-Aldrich®), and each well was analyzed using a plate reader  at 570 nm wavelength.3--2,5-diphenyltetrazolium bromide  and P. aeruginosa (ATCC 27853) were kindly provided by Dr. İskender Karaltı . These bacteria were cultured in Mueller Hilton broth  medium at 37 °C overnight. The bacterial suspension was adjusted to 0.5 McFarland standard in MHB to evaluate antimicrobial and antibiofilm properties.S. aureus and P. aeruginosa. Overnight cultures were adjusted to 0.5 McFarland and seeded on the MHA plate. Inhibition zones were checked.Antimicrobial activity was performed using halo inhibition zone tests . BrieflyBorosilicate glasses coated with tungsten–germanium were sterilized for 24 h with UV after 10 min 70% EtOH incubation. Borosilicate glasses coated with tungsten–germanium were placed in a 12-well plate and then 0.5 McFarland bacterial cells were added. After incubation for 48 h at 37 °C, they were washed with a 0.25% Trypsin solution to collect the biofilm from the sample. The solution was evaluated by the 600 nm the OD measurement. W–Ge coated borosilicate glass samples were sterilized in 70% Ethanol at 30 min and UV at 48 h. Then, samples and bacterial cultures seed in six-well plate in MHB medium. Borosilicate glasses are used for baseline control. Incubated samples were washed twice with PBS and fixed 2.5% glutaraldehyde at room temperature for 30 min. After fixation, samples dehydrated in ethanol series  for 10 min. Finally, samples were observed via scanning electron microscope (SEM-Quanta FEG 250) .p < 0.05.Statistical analyses of the numerical data obtained from the studies were performed with GraphPad Prism 7; ANOVA Dunnett’s multiple comparison test was used following the two-way ANOVA analysis for statistical evaluation and the statistical significance level was accepted as S. aureus (ATCC 25923) and P. aeruginosa (ATCC 27853) bacteria strains.Materials used in biomedical applications were applied in different forms and used for specific properties to accomplish a particular task. Materials such as metals, which were shown to enhance certain functions in the human or animal body, were often used as implants . In prevFigure As shown, Fig. Figure 3O (JCPDS: 96-152-8030), which was coming from a passive oxide layer on the 316 L substrate. As mentioned above and showed in Fig. 2, which is shown in Fig. 2 is the dominant peak in W–Ge coating.Figure Lc), which were obtained from the scratch test. Initially, the friction coefficient has the values around to 0.24, and slightly increased to around 0.32. Because of the smooth morphology of the sample surface, the friction coefficient was found quite stable, and the mean friction coefficient was investigated to be around the value of 0.3. With increasing normal force from 0 to 50 N, as expected Ff also increased to around 20 N. It is known that, in order to investigate the critical adhesion load value, normal force increased gradually and when the scratch tip reached to the substrate, which causes to the adhesive failures in the coating sample and this leads to dramatic changes on the friction coefficient and Ff at the data. From this point of view, the critical load value of the coating deposited on 316 L substrate obtained from the data is 32.8 N. Also, adhesive failure occurred as a result of the radial crack in the sample is shown inset picture in Fig. 2 composition, Ge2O and Cr3O metallic oxides were also seen in the coating deposition due to either the oxide layer on the substrate or oxidization after removing the deposition chamber. As a result of these oxidations of metals and crystallized form of WGe2 in the film, wear properties and the friction coefficient of the film became lower. In the literature, friction coefficient and hardness values were shown inversely proportional to each other according to Archard’s model [Scratch test is a well-known technique when determining the adhesion properties of materials . The con’s model . The low’s model –48. GeneTo investigate the corrosion behavior of W–Ge coating, static corrosion analysis was performed by using DMEM in place of the SBF (simulated body fluid) . After 7Studies claimed that germanium functionalized graphene could be integrated into biomedical devices because of its antimicrobial and hemocompatibility properties. If a graphene material is combined with germanium, there would be a greater antimicrobial and biocompatible property compared to the bare graphene molecule . FurtherS. aureus and P. aeruginosa. This result shows that the coating material is not released into the petri dish. The media may not have been suitable for W–Ge release [P. aeruginosa and S. aureus solution obtained from borosilicate glasses coated with tungsten–germanium with trypsin treatment were 0.025 and 0.018 nm, respectively. In contrast, the OD values of baseline for P. aeruginosa were 0.0534 and for S. aureus 0.020 nm as shown in Fig. S. aureus and P. aeruginosa. In the current study, glass without coating is used as a baseline control [In the holo-inhibition test used to measure antimicrobial activity, the area without reproduction was evaluated. However, no inhibition zone was observed in either the baseline control group or samples Fig. . It was  release . However release . OD valu control . It is c2 chemical compound was observed with a dense structure in (110) crystalline phase. The mean fraction coefficient of the W–Ge film on Ti buffer layer was obtained ~0.3, which was quite lower than the uncoated 316 L substrate. The adhesion strength value of the coated film was obtained 32.8 N. The static corrosion analysis showed that there was no significant change in surface morphology and the atomic ratio of W–Ge coatings after 7 days of DMEM immersion. Also, biocompatibility analysis showed that HDFa cell cultures were favorably attached to the sample surfaces, and cells were spread with favorable homogeneity. Cell viability assay puts forth that there was no significant difference between W–Ge coated surfaces and the cell culture plate. Finally, the antibiofilm activity of coatings was shown by using two different pathogenic bacteria strains (S. aureus and P. aeruginosa).W–Ge composition films were deposited using the reactive magnetron sputtering technique first time in the literature. The WGe


The total postoperative opioid requirements in G1 were significantly higher than those in G0 and G2 ). A multivariate regression analysis revealed that G1 was independently associated with increased postoperative opioid requirements . Consequently, male patients who had ceased smoking within 1 month of undergoing a laparoscopic distal gastrectomy with gastroduodenostomy had higher postoperative opioid use than patients who had ceased smoking for more than 1 month and nonsmokers.Smoking is clinically associated with high postoperative pain scores and increased perioperative analgesic requirements. However, the association between the duration of smoking cessation and postoperative opioid requirements remains unclear. Therefore, this study aimed to evaluate the association between the duration of smoking cessation and postoperative opioid requirements. We retrospectively analyzed the data of 144 male patients who received intravenous patient-controlled analgesia (IV PCA) after laparoscopic distal gastrectomy with gastroduodenostomy. All patients were divided into three groups: G0, nonsmoker; G1, smoker who quit smoking within 1 month preoperatively; G2, smoker who quit smoking over 1 month preoperatively. Analgesic use, pain intensity, and IV PCA side effects were assessed up to postoperative day 2. As the duration of smoking cessation increased, the amount of postoperative opioid consumption decreased ( Postoperative pain is one of the major causes of distress in patients during the postoperative period . Given tNicotine is considered to have an analgesic effect and smoking supplies nicotine to the central nervous system –8. This One possible explanation for this could be the pathophysiologic changes that occur during smoking cessation, including nicotine withdrawal and altered nicotinic receptor availability –16. ThesThis retrospective study was approved by our institutional review board and registered at the University of Ulsan, Seoul, Republic of Korea (2020-0573). First, we searched our institution's Information Technology of Service Management (ITSM) system between September 2013 and February 2014 using the terms “early malignant neoplasm of the stomach,” “distal gastrectomy,” and “gastroduodenostomy.” Data on a total of 549 patients were retrieved after the first ITSM search. Further, patients undergoing laparoscopic surgery, patients receiving total intravenous anesthesia, male patients, and patients receiving intravenous patient-controlled analgesia (IV PCA) with oxycodone were included in the study. The rate of female smokers was less than 5% in this cohort study. Considering that the disproportionate smoking rates between sexes could be a confounding bias, women were excluded from our study. A total of 148 patients were thus eligible for inclusion after the second ITSM search was performed. A total of four patients who discontinued IV PCA because of dizziness, nausea, vomiting, or postoperative bleeding before the postoperative day (POD) 2 were also excluded. Finally, 144 patients (110 smokers and 34 nonsmokers) were included and analyzed .Smoking status was defined based on a previous study's definition . Nonsmokµg/kg) or oxycodone (0.1 mg/kg) was injected 30 min before the end of the surgery. Before initiating IV PCA with oxycodone, 0.3 mg of IV ramosetron was administered to prevent postoperative nausea and vomiting. The IV PCA device  was set up with the following conditions: basal infusion rate, 1 mL/h; bolus dose, 1 mL; lockout interval, 15 min; and maximum infusion volume, 5 mL/h. The IV PCA regimen comprised oxycodone in 0.9% saline . The amount of oxycodone in the IV PCA was adjusted based on the patients' age and total body weight. Those who were aged <65 years received 200 mg if their total body weight was ≥50 kg and 160 mg if their body weight was <50 kg. Those who were aged ≥65 years and had a body weight ≥50 kg received 140 mg and received 100 mg if their body weight was <50 kg. After the surgery, when postoperative analgesia was considered insufficient , the patients received additional doses of fentanyl (1 µg/kg) or oxycodone (0.1 mg/kg) in the postanesthesia care unit. For patients who experienced shivering, meperidine 25 mg was injected. In the general ward, rescue analgesics, including ketorolac 30 mg, tramadol 50 mg, and meperidine 25 mg, were administered to reduce postoperative pain. Ketorolac was initially administered when patients complained of significant postoperative pain . If the pain did not subside and the patients required additional analgesics, meperidine was administered. In patients with renal insufficiency, tramadol was initially administered instead of ketorolac to reduce postoperative pain.Total intravenous anesthesia was used for the induction and maintenance of anesthesia. To prevent severe postoperative pain after emergence, fentanyl (1 μg = meperidine 100 mg = oxycodone 10 mg) , P < 0.001; G1: β = 16.22; 95% CI: [9.31–23.13]; P < 0.001)  .β = −0.46, 95% CI [−0.74 to −0.17], P=0.001; G1: β = 12.80, 95% CI [5.81–19.80], P < 0.001). The side effects recorded up to POD 2 were not different between the three groups (These relevant factors associated with postoperative opioid requirements after the univariate linear regression analysis were included in the multivariate linear regression analysis. As a result, age and G1 were independently associated with postoperative opioid requirements (age: e groups .The current study presented three main findings. First, postoperative opioid requirements decreased in accordance with an increased duration of smoking cessation in patients who were smokers. Second, male patients who ceased smoking within 1 month preoperatively had more postoperative opioid requirements and showed higher pain intensity than nonsmokers and patients who ceased smoking more than 1 month preoperatively. Third, younger age and smoking cessation within 1 month of surgery were independently associated with increased postoperative opioid requirements.Several previous studies have demonstrated that opioid requirements and pain intensity increased in smoker patients during the postoperative period , 14, 19.Altered pathophysiologic changes as a result of smoking cessation have been well established. For example, significant improvements in blood pressure and heart rate are observed within 24 hours of smoking cessation, lung function improves within 3 months, and the risk of coronary heart disease decreases to half after 1 year of smoking cessation , 22. Altβ2 nicotinic acetylcholine receptor availability is increased, which is associated with increased pain sensitivity [These findings may be explained, at least in part, by nicotine withdrawal and altered nicotinic receptor availability –16. Smoksitivity . These psitivity . Therefositivity . In the sitivity , 23.Younger age is well known to be a risk factor for increased pain intensity and opioid requirements postoperatively , 24. TheThis present study has some limitations. First, this was a retrospective study comprising a small sample size, which could limit and weaken the results. However, we performed various statistical analyses, including multivariate linear regression analysis. Hence, we believe that these methods strengthened the statistical significance of our results. Second, we included not only current smokers but also ex-smokers. In this study, current smokers had ceased smoking to achieve better surgical outcomes, at least 1 week or a maximum of 6 weeks before the surgery, and they were almost included in G1, except for one patient who was classified as G2 because the duration of smoking cessation was 6 weeks. However, ex-smokers ceased smoking to reduce their risk for smoking-related disease or healthcare costs, at least 6 months or a maximum of 45 years before the surgery, and they were all included in G2. Thus, the duration of smoking cessation for the ex-smokers was longer than that for the current smokers. These differences may have affected the pathophysiologic changes observed as a result of smoking cessation subsequently. Consequently, further evaluation of current or ex-smokers only is warranted. Third, although we used 1 month of smoking cessation as the criterion when dividing smokers into two groups, further study with various durations of smoking cessation to reduce postoperative pain is necessary. Fourth, although subjects with chronic pain or those who were taking pain medication were not considered for exclusion when designing the present study, through the strict review of medical records, we confirmed no patients who had chronic pain or used any pain medication for chronic pain in our cohort.In conclusion, male patients who had ceased smoking within 1 month of undergoing a laparoscopic distal gastrectomy with gastroduodenostomy had higher opioid requirements than male patients who had ceased smoking over 1 month before surgery and nonsmokers. Postoperative opioid use appeared to be reduced according to an increased duration of smoking cessation in smokers. After adjusting for other clinical variables, smoking cessation within 1 month was considered an independent factor associated with increased postoperative opioid use. Therefore, more postoperative analgesics may be required to provide sufficient analgesia in patients who cease smoking within 1 month of surgery.


P < .00001). Higher complication rates were observed in TS with global ballooning pattern compared to apical ballooning pattern . Higher complication rates were observed in patients with age < 50 years than patients >50 years . Recurrence occurred exclusively in patients with PPGL‐induced TS . PPGL‐induced TS was characterized by more global ballooning's pattern , and lower left ventricular ejection fraction  compared to exogenous catecholamine‐induced TS. In conclusion, catecholamine‐induced TS was characterized by a dramatic clinical presentation with extensive left ventricular dysfunction, and high complication rate.Innumerable physical stress factors including externally administered catecholamines, and pheochromocytomas and paragangliomas (PPGLs) have been reported to trigger Takotsubo syndrome (TS). A systematic search of PubMed/MEDLINE identified 156 patients with catecholamine‐induced TS up to December 2017. Data were compared within the catecholamine‐induced TS cohort, but some comparisons were also done to a previously published large all‐TS cohort (n = 1750). The mean age was 46.4 ± 16.4 years (72.3% women). The clinical presentation was dramatic with high complication rates in . The most common TS ballooning pattern was apical or mid‐apical , followed by basal pattern , global pattern , mid‐ventricular , focal , and unidentified pattern . There was an increase in the prevalence of apical sparing ballooning pattern compared to all‐TS population (37.7% vs 18.3%,  Thus, in this systematic review and meta‐analysis, the clinical features, complications and outcomes of 156 published cases of externally administered epinephrine‐ and norepinephrine‐, and PPGL‐induced TS are described. Furthermore, a comparison was made between the whole group and a study including all‐TS population. The externally administered catecholamine‐induced TS is also compared to the PPGL‐induced TS.2All cases of epinephrine‐induced TS, norepinephrine‐induced TS, and PPGL‐induced TS from 1990, the year where the Japanese term Takotsubo was introduced, to December 2017 were critically reviewed. The cases were retrieved by systematical searches in PubMed/Medline using the search terms “Takotsubo,” “apical ballooning,” “stress cardiomyopathy,” and “broken heart syndrome,” and linking them with the terms “pheochromocytoma,” “paraganglioma,” “catecholamines,” “epinephrine,” “adrenaline,” “norepinephrine,” and “noradrenaline.” Cases with PPGL‐induced transient left ventricular dysfunction where the clinical features and course were consistent with TS were also included. Articles were initially screened by title for relevance and then by abstract, with full‐text articles of potentially relevant reports reviewed. The reference lists of the retrieved full‐text studies were scanned to identify additional relevant reports. Only case‐reports or reports on a series of cases where enough information was available on every case were included  was considered inappropriate because these instruments had not been developed to study case reports or series. The PRISMA  guidelines were followed.The following information was extracted from the publications: the age and gender of the patients, the clinical presentation, the type of ECG changes and the cardiac biomarkers in all patients. The TS localization was deemed by the description in the text or the available figures in the manuscripts. The results were compared with 1750 patients from the study by Templin et al2.1t test or Chi‐square test was used as appropriate to compare categorical data, and two‐tailed unpaired student's t test was used for continuous variables. A P < .05 was considered significant.Continuous variables are presented as means ± standard deviations and categorical data as absolute values and percentages. Fisher's exac3The systematic search identified 391 potentially relevant records, with an additional 10 records (including one case of PPGL‐induced TS from 19873.1The mean age was 46.4 ± 16.4 years (range 16‐86 years). Women constituted 72.3% of the cohort. In all the patients, either external epinephrine (n = 42) or norepinephrine (n = 7) administration or PPGLs (n = 107) was documented as the most probable physical trigger factor for TS. However, in 75 of 156 patients (48.1%) additional potential trigger factors were identified . In most of the patients with external epinephrine‐ or norepinephrine‐induced TS, the disease conditions, which indicated catecholamine administration, could have been a potential trigger factor for TS.The most common clinical presentation was chest pain, which occurred alone in 22.4% (n = 35) of the patients. Other presenting symptoms were syncope , dyspnea in , abdominal pain , and headache . Signs and symptoms suggestive of PPGLs Information on electrocardiographic (ECG) changes was available in 141 patients . In contrast, ST‐depression and peaked T waves were significantly more prevalent in basal ballooning pattern compared to that of apical ballooning's pattern .STEMI‐like ECG changes and T‐wave inversions were significantly more prevalent in apical ballooning pattern compared to basal ballooning pattern . It was increased in 97.7% (n = 126). In 121 patients (77.5%), information on coronary angiography was available. In 98.3% (n = 119), the coronary angiography was normal. Only 1.7% (n = 2) had signs of obstructive coronary artery disease.3.2P = .0022). The complications occurred in 71.1% (32/45) of the basal ballooning pattern of TS compared to 58.5% (38/65) of the apical ballooning pattern (P = .227). There was no difference in the rate of complications between men and women . In contrast, the complication rates were higher in patients aged <50 years than patients aged >50 years . Inotropic medications were used in 33.1% (50/151). The most common used inotropic medication alone or in combinations were dobutamine, norepinephrine, epinephrine, amrinone, or milrinone. Mechanical ventilation was used in 25.8% (34/132). Extracorporeal life support (ECLS), veno‐arterial extra‐corporeal membrane oxygenation (ECMO), percutaneous cardiopulmonary support, left ventricular assist device, and intra‐aortic balloon pump were used alone or in combination in 21.5% (31/144). Recurrence occurred exclusively in patients with PPGL‐induced TS .Information on complications was available in 151 of 156 patients (96.8%). Complications occurred in 68.2% (n = 103) (Table P = .35). Death occurred in 3.7% (n = 4) of the patients with PPGL‐, 4% (n = 2) with exogenous catecholamine‐induced TS. Mortality during recurrence of PPGL‐induced TS was high . Different inotropic medications alone or in combinations were used in most patients who died ; this may have contributed to the deaths.In total 3.8% (n = 6) died; 7% in the male group (n = 3), and 2.7% (n = 3) in the female group . The disease severity was reflected in significantly higher complication rate (68.2% vs 21.8%), more cardiogenic shock (37.7% vs 9.9%) and higher use of inotropic medications (32.7% vs 12.2%). Significantly higher recurrence rate was reported in catecholamine‐induced TS  than all‐TS population.Compared to all‐TS population,3.4The disease in PPGL‐induced TS was more severe with significantly increased prevalence of global TS‐pattern and lower left ventricular ejection fraction Table . Complic4This systematic review and meta‐analysis reports hitherto on the largest number of patients (n = 156) with exogenous (epinephrine and norepinephrine) catecholamine‐ and endogenous (PPGLs) catecholamine‐induced TS. The main findings were that a substantial number of patients presented with severe hemodynamic compromise such as pulmonary edema, cardiogenic shock, circulatory failure, and arrhythmias. Considerable numbers of patients had global left ventricular dysfunction and marked depression of left ventricular ejection fraction. This was reflected in high complication rates during admission where two‐thirds of the patients suffered complications and one third had multiple complications. Worth mentioning is that the description of LVWMA was not always accurate especially in the cases described before the TS‐era. Five out of nine PPGL‐induced TS published before 2000 were described to have myocardial depression or severe left ventricular dysfunction with a clinical course consistent with TS.Higher complication rates were observed in patients aged <50 years than those >50 years. Mortality was higher in men (7%) than women (2.6%). These findings are in line with what other investigators have reported in TS in general.There was an increased prevalence of apical sparing ballooning pattern . In PPGL‐induced TS, high recurrence rate (16.8%) was reported. The high recurrence rate of TS in PPGL‐induced TS population was most likely attributed to the delay in the PPGL diagnosis where episodes of massive catecholamine elevation had acted as a trigger factor.The more severe disease in PPGL‐induced TS than in exogenous catecholamine‐induced TS may be attributed to higher catecholamine surge in the former.4.1One hypothesis in the pathogenesis of TS is epinephrine‐induced switch in signal trafficking in the apical left ventricular myocardium causing apical ballooning.4.2The analysis of catecholamine‐induced TS was based on retrospective studies of case reports or series with the inherent limitations of all retrospective studies, in particularly that of ascertainment bias. The absolute levels of the cardiac biomarkers and catecholamine levels could not be utilized for the estimation of comparable mean values because of lack of standardization and uniformity across the case reports. The TS localization pattern was not always accurate, and this point has been discussed previously.5The clinical features, complications and outcome of the hitherto largest number of patients with exogenous and endogenous catecholamine‐induced TS are described in this systematic review and meta‐analysis. Catecholamine‐induced TS was characterized by a dramatic clinical presentation with extensive left ventricular dysfunction, and high complication rates.The authors declare they have no conflict of interest.