There are currently limited data comparing the performance of these two radioligands with respect to clinical outcomes or their cost efficacy, which this study aims to address.Amongst others, PSMA-1007 compared to [68Ga]Ga-PSMA-11 , whereas the rate of uncertain findings was significantly greater . The probability of a true positive finding was higher for [68Ga]Ga-PSMA-11  vs. [18F]PSMA-1007 . A significantly (p < 0.0001) higher PPV for [68Ga]Ga-PSMA-11  was found compared to [18F]PSMA-1007 . Intervention efficacy analysis favoured [68Ga]Ga-PSMA-11, where the number needed to image (to achieve a true positive finding) was 10.58 and the number needed to image to harm  was − 8.08. A cost efficacy analysis favours [68Ga]Ga-PSMA-11 in three of the four jurisdictions analysed where health economic data was available  and [18F]PSMA-1007 in one jurisdiction (Denmark).The PET positivity rate was non-significantly higher for [18F]PSMA-1007, but finds significantly greater rates of uncertain findings and false positive findings when compared to [68Ga]Ga-PSMA-11. We find differences in the two tracers in terms of clinical performance and cost efficacy. The method presented herein is generalisable and can be used with clinical or cost data for other countries or tracers.The analysis reveals a non-significantly higher PET positivity rate for [The online version contains supplementary material available at 10.1007/s00259-021-05620-9. We systematically obtain clinical data from patients undergoing PSMA PET/CT with either [68Ga]Ga-PSMA-11 or [18F]PSMA-1007 to inform a Markov chain decision model. In doing so, we obtain data regarding the relative clinical performance and health-care cost efficacy comparing these two radioligands, as well as providing a generalisable methodology, which can be implemented using clinical or cost data from other countries.PC is the most commonly diagnosed cancer in men  and recu imaging –18, the 68Ga]Ga-PSMA-11 was introduced in Switzerland in January 2017, and [18F]PSMA-1007 was introduced at our centre in September 2019 following its temporary approval for use in Switzerland. Cognisant of a learning curve when encountering a new tracer, individuals examined in the first 3 months following the introduction of [18F]PSMA-1007 were excluded from the analysis to eliminate this as a source of bias.The consolidated health economic evaluation reporting standards (CHEERS) statement was followed during the design and execution of this study  which wa18F]PSMA-1007 were identified, of whom 122 underwent imaging for recurrent PC. Likewise, immediately prior to the change to [18F]PSMA-1007 in September 2019, 128 consecutive individuals undergoing [68Ga]Ga-PSMA-11 (May 2019–September 2019) were identified, of whom 122 underwent imaging for recurrent PC and included for analysis, thus yielding a matched pair of cohorts. The study flow chart is given in Fig. From January 2020 to May 2020, 130 consecutive individuals undergoing PSMA-1007, of which 10 were PET-negative . Of the N = 10 men with negative PSMA PET/CT, three were redirected to follow-up PSMA PET/CT. Of the N = 112 men with a positive PET, 21 had uncertain findings (17.2%) which was significantly higher than for [68Ga]Ga-PSMA-11 (p = 0.02). Of these uncertain findings, seven patients underwent further imaging . Of the six patients who were not referred for a follow-up PET/CT, four had PC lesions confirmed and two identified were false positives. Of the 112 patients with positive scans, CSOT was available for 54 patients, with 45 TP and nine FP, yielding a PPV of 0.83 (95% CI 0.65-0.94). Cases where FP lesions were found are outlined in supplementary Table S3. The Markov transition chain is given in Fig. 68Ga]Ga-PSMA-11, the recent meta-analysis of Hope et al. was available and was analysed for quality using the PRISMA statement [p = 0.84).For [tatement . Hope ettatement . The PPV18F]PSMA-1007, no such meta-analytic data for diagnostic accuracy is reported. A systematic literature review performed in July 2021 by the first two authors for the key search terms “PSMA-1007”, “diagnostic accuracy” and “performance” using the PubMed Central archive. This search revealed only three studies reporting data regarding the diagnostic performance of this tracer. Rahbar et al. and Giesel et al. report a 95% rate of positive scans, but do not provide confirmatory follow-up Ga-PSMA-11 was 0.99 (95% CI 0.99–1.0) and was significantly higher (p < 0.0001) than the value for [18F]PSMA-1007 0.86 (95% CI 0.82–1.00). The FDR was lower for [68Ga]Ga-PSMA-11 . The results are shown in Table 68Ga]Ga-PSMA-11 and 0.81 (95% CI 0.66–0.91) for [18F]PSMA-1007 (p = 0.20). The NNI was therefore 10.58, suggesting that for roughly every tenth patient imaged with [68Ga]Ga-PSMA-11 instead of [18F]PSMA-1007, one extra true positive scan would be observed. The lower PPV and higher rate of FP scans  means that the number needed to image to harm (NNTITH) is − 8.08, suggesting that for every eighth patient imaged with [18F]PSMA-1007, potential diagnostic harm may occur as a result of a false positive finding. No significant difference was observed in the likelihood of a negative finding , where the wide confidence intervals seen in Table Mix= 0.9829 and 1.3133 for [18F]PSMA-1007 and [68Ga]Ga-PSMA-11, respectively.In our study, the overall PPV for [https://www.health.gov.il/English/Topics/finance/Pages/default.aspx) and Australia .Costs associated with each examination and associated follow-up imaging are outlined in Table n et al. ). For Ge18F]PSMA-1007 at $3212 vs. $3337 for [68Ga]Ga-PSMA-11. At an outcome level, the cost per TP favours [68Ga]Ga-PSMA-11 at $3697 per TP vs. $3975 per TP for [18F]PSMA-1007. The incremental cost per outcome favours [68Ga]Ga-PSMA-11 at $109, meaning that each TP finding would cost only an additional $109. The incremental cost efficacy ratio was strongly positive at $1153 for [68Ga]Ga-PSMA-11 compared to [18F]PSMA-1007, suggesting that while higher overall costs are associated with the use of [68Ga]Ga-PSMA-11 in Switzerland, these are less than 50% of the cost of a PET/CT and fall under the willingness to pay threshold (cost of a PSMA PET/CT in Switzerland is $2737.88) [68Ga]Ga-PSMA-11 for Switzerland, Israel and Australia but not Denmark, where the lower radiopharmaceutical costs for [18F]PSMA-1007 favoured this tracer, and where the ICER ($5135) was beyond the willingness to pay threshold (cost of a PSMA-PET/CT in Denmark $1387.90 to $1786.11)  . Cost ef18F]PSMA-1007 is marginally, but non-significantly higher when compared to [68Ga]Ga-PSMA-11 . However, we find differences between the two tracers in terms of clinical performance and cost efficacy.In this study we present a Markov chain decision analysis which compares the relative performances of two PSMA radioligands under clinical conditions. Consistent with published retrospective analyses , 34 and Whereas a number of studies reporting the diagnostic performance for PSMA-radioligands report findings on a binary positive/negative scale and without external validation of findings, this dialectic does not reflect clinical reality, where diagnostic uncertainty can and does occur. Furthermore, the patient’s journey does not end with a “positive” or “negative” scan result; any imaging findings inform clinical decision-making and influence subsequent treatment. Although a number of studies investigate the influence of PSMA-PET/CT on treatment decision-making , few ade18F]PSMA-1007 compared to [68Ga]Ga-PSMA-11 [18F]PSMA-1007 PET/CT  had equivocal bone lesions, of which a sizeable minority (11%) were TP findings [18F]PSMA-1007 compared to [68Ga]Ga-PSMA-11 . This translated into a higher rate of additional examinations to clarify uncertain findings, which more often confirmed false positives. Confirmatory follow-up data to a CSOT was available for roughly half of the patients, allowing a direct estimate of the diagnostic accuracy. By comparing the model-derived probabilities for a TP and a FP, the overall patient-level PPV can be calculated. We find that the PPV for [68Ga]Ga-PSMA-11 was significantly higher than for [18F]PSMA-1007 , which corresponded almost perfectly with literature/meta-analysis-derived values for the PPV used in our model (0.99 vs. 0.87), and is taken to be suggestive that there was no significant bias in patient selection in our study or methodology. The lower PPV for [18F]PSMA-1007 might be explained by the higher rate of non-specific uptake, for which further studies are required. As a result, the FDR was lower  and the overall probability for a patient to receive a TP was slightly higher for [68Ga]Ga-PSMA-11 .The choice of radioligand may have both clinical and healthcare economic implications. For example, a number of reports describe increased non-specific radioligand uptake with 1FPSMA-100findings . These s68Ga]Ga-PSMA-11 instead of [18F]PSMA-1007, every tenth patient will benefit through an additional TP (NNI = 10.58). A number needed to image to harm reveals that every eighth patient will suffer an end result of a false positive (NNTITH − 8.08) when scanned with [18F]PSMA-1007. Using clinical data, we are therefore able to quantify, for the first time, the potential clinical impact of choosing one PSMA-radioligand over another.Using the concept of the number needed to treat, we provide an assessment of the effectiveness of PSMA-PET/CT as a healthcare intervention. We calculate a number needed to image, finding that when imaging with [68Ga]Ga-PSMA-11  and an estimated 25,000 men develop rPC annually in the USA [18F]PSMA-1007, we find that this translates neither into improved clinical performance nor higher cost efficacy PSMA-1007 PET/CT was lower, resulting in improved cost efficacy for this tracer for Denmark. We were unable to assess further the costs of radiopharmaceutical choice on system-level costs; for example, although generator costs may be higher, [68Ga]Ga-PSMA-11 is not under a patent, whereas production of [18F]PSMA-1007 incurs licence fees to the patent holder.One notable finding of this examination was the variable degree of opacity in the reporting of economic data. For two jurisdictions (the UK and Germany), no open-access cost data was available, meaning that a cost efficacy analysis for these two countries was not possible, whereas for Israel and Australia, these data are published on the respective health ministry websites. In the Swiss TARMED system, the amount billed for the radiopharmaceutical is equal irrespective of tracer, and any cost savings incurred by using a different radiopharmaceutical are passed on to neither patient nor insurer. Similarly, no evidence of variation in tracer-specific costs could be found for Israel and Australia. Interestingly, in Denmark, the examination cost for a Ga-PSMA-11 revealed by our analysis, cyclotron produced 68Ga Ga-PSMA-11 is confirmed by a large volume of prospective data and meta-analysis [18F]PSMA-1007, where only two studies reporting data for PPV were identified, and several prospective studies are underway. In mitigation all available data for [18F]PSMA-1007 was systematically reviewed and combined in a meta-analytic model and study quality was formally assessed. In light of the widespread adoption of this tracer, further studies are required to confirm the lower PPV for [18F]PSMA-1007. In two countries (UK and Germany), a cost efficacy analysis was hampered by the lack of publically available health economic data for PSMA-PET scans. Greater transparency in the reporting of these data would be welcome to facilitate future health-economic analyses for nuclear medicine imaging.Although not a study-specific weakness, we draw attention to the fact that the PPV for to [analysis . This wa18F]PSMA-1007 and [68Ga]Ga-PSMA-11. We urge further similar studies from centres with experience of other radioligands, where our model is generalisable and implementable with locally obtained values for other tracers.These retrospective data require further studies to be confirmatory, although we note that strict consecutive inclusion and non-overlapping dates in radioligand availability limit selection bias. Our study is restricted to a single centre analysis, and further multi-centre and ideally international studies should be performed. Finally, our analysis is limited to a pairwise analysis of [68Ga]Ga-PSMA-11 and [18F]PSMA-1007. In keeping with previous studies [18F]PSMA-1007, but find significantly greater rates of uncertain findings and false positives for this tracer when compared to [68Ga]Ga-PSMA-11. The lower PPV for [18F]PSMA-1007 observed in our cohort was in-keeping with literature-derived values and a meta-analytic synthesis of existing data. The higher frequency of false positives, additional imaging or additional intervention to clarify a higher rate of indeterminate findings are indicative of a less favourable clinical performance for [18F]PSMA-1007 compared to [68Ga]Ga-PSMA-11. We find significant differences in terms of clinical performance and cost efficacy for these two radioligands, and the choice of which to implement should be informed by these as well as local conditions, with variation in cost efficacy across the jurisdictions analysed herein.We present a generalisable Markov decision model analysis informed by clinical data obtained from a retrospective analysis of a large single-centre cohort of patients to compare the clinical performance and cost efficacy of the two PMSA-radioligands [Supplementary file1 (DOCX 260 KB)Below is the link to the electronic supplementary material.


Litchi is an important commercial fruit crop widely grown in the world. Graft incompatibility between rootstocks and scions is a major constraint for large-scale cultivation of litchi orchards, popularization of new and excellent litchi varieties, and associated industrial development. Further, the genetic mechanism of graft incompatibility is still unclear in litchi. To reduce the incompatibility problems, this study investigated metabolic and transcriptomic differences between graft compatible and incompatible rootstock-scion combinations of litchi. The result of metabolomics analysis showed that incompatible rootstock-scion interaction modified the profiles of several metabolic substances. However, various compounds of flavonoids, phenolic acids, and lignin predominantly exhibited significantly altered abundance in graft incompatible combinations. Transcriptome analysis identified that graft incompatibility induces dynamic gene differences. The majority of these differentially expressed genes were enriched in biosynthetic pathways of phenylpropanoids. The differential expressions of genes in these pathways could be linked to the differential abundance levels of flavonoids, phenolic acids, and lignin compounds. Integrated metabolomic and transcriptomic analyses revealed a strong relationship between differential genes and differential metabolites identified in this study. In addition, identified hub genes and metabolites were closely associated with graft incompatibility of litchi. This study characterized the abundance of metabolites and genes in graft incompatible combinations and further discussed the genetic mechanism of graft incompatibility in litchi. Our results provide a platform to dissect the molecular mechanisms of graft incompatibility in the litchi fruit. Litchi chinensis, belongs to the Sapindaceae family, commonly known as litchi, a native fruit in Southern China. Litchi is widely cultivated in tropical and sub-tropical parts of China for more than a hundred years  genes had significant coloration with phenolic, flavonoids, and antioxidant enzymes activity in the scion grafted on the incompatible rootstock (indole-3-acetic acid (IAA) along with auxin response factor (ARF) encoding genes mediate various biological changes to promote the vascular connection between the scion and the rootstock  along with Heiye was used as the rootstock and Heli as a scion (YL) . It was The metabolomics analysis is vital to understanding the composition and concentration of metabolic substances for target traits. Thus, ultra-performance liquid chromatography-mass spectrometry was used to investigate the abundance of metabolites in both grafting combinations of litchi. A total of 1,166 metabolites were detected in grafted litchi samples . The majIn addition, all metabolites had shown dynamic variations between LY and YL graft combinations of litchi. In particular, flavonoids compounds such as 5,7,3'-trihydroxy-4'-methoxyflavone, 5,7,4'-trihydroxy-6-methoxyflavone, and 7-methylkaempferol along with other subclasses had significantly higher abundance in graft incompatible combination than compatible. Additionally, phenolic acid compound salicylic acid-2-O-glucoside and coniferin showed similar abundance in this combination . Howeverp ≤ 0.05 and with log2 (fold change) > 1 or < −1. The comparative analysis of LYvsYL showed a total number of 542 DEGs. The distribution analysis determined 314 down-regulated and 228 upregulated genes in YL as compared to LY , cinnamate-4-hydroxylase (C4H), and 4-coumaroyl coenzyme A ligase (4CL). The p-coumaryl CoA then produces basic precursors for the biosynthesis of lateral metabolites , p-coumarate 3-hydroxylase (C3H), caffeoyl-CoA O-methyl transferase (CCoAOMT), cinnamoyl-CoA reductase (CCR), cinnamyl alcohol dehydrogenase (CAD), ferulate 5-hydroxylase (F5H), and caffeic acid O-methyltransferase (COMT)  . A totalon in YL . Their agrafting . This migrafting . So, an via complex chemical reactions regulated by different enzyme encoding genes  and Heiye. In China, Heli is a broad compatibility rootstock variety. In contrast, the Heiye is a weaker rootstock variety as compared to Heli. For mutual grafting, Heiye was utilized as a scion and grafted on the rootstock of Heli (LY). Then, Heli was used as a scion and grafted on Heiye (YL) rootstock. The survival rates of successful grafting were measured after 1 month from 100 plants for each graft combination. The grafted samples were harvested in triplicates from the healing site for graft compatible (LY) and incompatible (YL) combinations of litchi, frozen instantly in liquid nitrogen and stored in an ultra-low temperature refrigerator at −80°C before further analyses. To study the metabolic profiles, the biological samples were converted into powder form with a grinder  at a 30 Hz frequency for 2 minutes. The 100 mg of the powder was mixed into 1.0 ml of 70% methanol containing 0.1 mg/L lidocaine as an internal standard and stored at 4°C overnight to perform the extraction. The overnight stored samples were vortex three times and centrifugation was then performed at 10,000 g for 10 min to produce more sufficient extraction. The supernatant liquid was aspirated, filtered through a microporous membrane (0.22 μm pore size), and finally transferred in injection vials for subsequent metabolomics analysis. In this study, the metabolic datasets were obtained from ultra-performance liquid chromatography (UPLC) (Shimadzu Nexera X2) integrated with tandem mass spectrometry (MS/MS) (Applied Biosystems 4500 QTRAP). The protocol of metabolic detection and their data generation was followed as briefly described in the previous study . The rawf litchi .Total RNA was extracted from graft compatible and incompatible combinations by using the CTAB method . Then, g


Surface roughness parameters were estimated using a 3D confocal laser microscope; microstructures were analyzed using a scanning electron microscope. Peak fluence (Fpeak) values of 4 and 8 J/cm2 and irradiation numbers (N) of 20 and 10 shots were selected to create dot patterns in highly translucent zirconia and lithium disilicate glass-ceramics, respectively. Furthermore, Fpeak = 8 J/cm2 and N = 20 shots were chosen to obtain crossed-line patterns in both ceramics. Our results show that lithium disilicate glass-ceramics and highly translucent zirconia exhibit a similar surface morphology under each of the surface treatment conditions. Therefore, FL irradiation of dot or crossed-line patterns (at a distance of 20 and 40 µm) are potential candidates for future investigations.All-ceramic restorations have become increasingly popular in dentistry. Toward ensuring that these restorations adhere to the tooth structure, this study determines the optimal femtosecond laser (FL) treatment parameters for lithium disilicate glass-ceramics and highly translucent zirconia ceramics with respect to surface morphology. For both the ceramics, the following surface conditions were investigated: (1) as-sintered; (2) Al All-ceramic restorations have garnered increasing popularity in dentistry due to their superior aesthetics and biocompatibility compared to porcelain-fused-to-metal restorations. Further, lithium disilicate glass-ceramics and zirconia ceramics are the most commonly used materials for all-ceramic restorations as they satisfy the requirements pertaining to aesthetics as well as mechanical properties .To achieve long-term clinical success with all-ceramic restorations, they must adhere to the tooth structure . PretreaThe current study, thus, proposes femtosecond laser (FL) treatment as an alternative method of surface pretreatment. FL treatment is often referred to as abrasion and can be used to treat metals, silicones, and insulators without resulting in surface damage. However, the optimal FL treatment parameters for lithium disilicate glass-ceramics or highly translucent zirconia ceramics  have not been established to date.Thus, the purpose of this study is to ascertain the optimal FL treatment parameters for lithium disilicate glass-ceramics or highly translucent zirconia ceramics in terms of surface morphology.The following two ceramics were investigated in this study: lithium disilicate glass-ceramics  and highly translucent zirconia ceramics . Fully-sintered square-shaped specimens were prepared (14 × 14 × 2.2 mm) for each ceramic grade.peak = 2, 4, 8 J/cm2) and four different irradiation numbers  were investigated for dot pattern processing; (2) four different peak fluences  and four equivalent irradiation numbers  were investigated for attaining cross-line patterns. The peak fluence is equal to the peak energy irradiated by the Gaussian laser beam, as defined as follows [0 is the radius of the irradiated beam at 1/e2 intensity on the surface of specimens (r0 = 16.1 µm). The equivalent irradiation number for line scan irradiation, defined as the number of pulses irradiated onto a single point, is given as [rep and vscan denote the laser pulse repetition frequency and the laser beam scanning speed, respectively.To determine the optimal laser treatment settings for lithium disilicate glass-ceramics and highly translucent zirconia, an FL  was employed, and the following laser parameters were investigated: (1) Three different peak fluences  (positive control); (3) FL treatment (dot pattern): the specimens were irradiated with three different dot patterns  using an FL. (4) FL treatment (crossed-line pattern): the specimens were irradiated with two different crossed-line patterns (line distances of 20 and 40 µm) using an FL. The details of the FL treatment settings are summarized in Based on the results of the pilot experiment, the following surface conditions were investigated: (1) as-sintered: specimens were preserved untreated on the surface (negative control); (2) AlaS and aR) were calculated using the ProfilmOnline software  with an 80-µm cutoff value.The surface roughness was determined at a magnification of 50× using a 3D confocal laser microscope . Additionally, the surface roughness parameters . Subsequently, the specimen surfaces were examined using a field emission gun-SEM  at 5 kV.peak and the number of irradiated laser pulses. The irradiation parameters listed in 2 with 10 shots resulted in a treated area of approximately 420 µm2 for the lithium disilicate glass-ceramics, whereas a peak fluence of 4 J/cm2 with 20 shots resulted in a treated area of approximately 400 µm2 for the highly translucent zirconia. Additionally, the treated depth was observed to be 6.3 µm harboring a peak fluence of 8 J/cm2 with 10 shots for the lithium disilicate glass-ceramics. On the other hand, the treated depth for the highly translucent zirconia was 7.4 µm with a peak fluence of 4 J/cm2 and 20 shots. The total energy input per unit area (Fpeak × N) was set to the same value (80 J/cm2) for the two ceramics.The relationship between the peak fluence and the treated area or depth is summarized in 2 with the equivalent irradiation of 20 shots was selected for both ceramics, which produced a linewidth of 21.5 µm and a depth of 8.8 µm for the lithium disilicate glass-ceramics, and a linewidth of 22.7 µm and a depth of 7.1 µm for the highly translucent zirconia.For the line pattern processing, the irradiation parameters listed in Using the obtained data, the FL treatment parameters are selected and summarized in 2, 20 shots), the LIPSS period was measured to be 1.14 µm, whereas the IPS e.max CAD HT calculated it to be  1.6 µm.The microstructural analysis conducted using an SEM revealed no microcracks in the lithium disilicate glass-ceramic surfaces irradiated with an FL . On the The effect of FL irradiation on lithium disilicate glass-ceramics and highly translucent zirconia was investigated in this study. Our findings indicate that laser-irradiated surfaces exhibit a higher degree of surface roughness than sandblasted surfaces. Additionally, the SEM images demonstrate the formation of LIPSS and microcracks on femtosecond laser-irradiated ceramic surfaces.rep = 10 kHz, wavelength = 1030 nm, pulse width = 290 fs; for KATANA UTML: Fpeak = 4 J/cm2, N = 20 shots; for IPS e.max CAD HT: Fpeak = 8 J/cm2, N = 10 shots, for dot-pattern processing and Fpeak = 8 J/cm2, Neq = 20 shots for both KATANA UTML and IPS e.max HT for crossed-line pattern processing.Prior to the actual FL irradiation, we conducted a pilot experiment to determine the laser’s actual setting. To obtain structures similar to those of lithium disilicate glass-ceramics and highly translucent zirconia, we used the following FL settings in accordance with a previous study : frep = aR, of laser-irradiated surfaces is significantly greater than that of as-sintered or sandblasted surfaces [2O3 sandblasted zirconia, and FL-irradiated zirconia [aR, of zirconia surfaces irradiated with an FL was significantly greater than that of sintered and the Al2O3 sandblasted surfaces.We investigated the surface roughness in the present study using a 3D confocal laser microscope. Three-dimensional confocal laser microscopy is a non-contact technique applied for determining the roughness of a surface. Both lithium disilicate glass ceramics and highly translucent zirconia exhibit rougher surfaces when irradiated with laser compared to sintering or sandblasting. Our findings corroborate those of Okutan et al., who demonstrated that the surface roughness, surfaces . Garcia-zirconia . Accordi2), which can be partially attributed to the melting of the surface followed by re-solidification. Kakehata et al., described the formation of LIPSS on 3Y-TZP for the first time [2). Even if the LIPSS is formed by the scanning beam’s center, irradiation of the trailing edge of the Gaussian beam associated with the scanning results in LIPSS ablation; this is why the LIPSS observed in dot patterns is not visible in the line scanned samples. Additionally, as demonstrated by the SEM images, the LIPSS contained microcracks, which may reduce the strength of the ceramics [SEM images revealed the presence of clear LIPSS on the FL-irradiated ceramic surfaces with dot patterns. The reason that the vertical and horizontal orientations of LIPSS are different in some images is that the sample orientation was rotated by 90 degrees during observation. LIPSS, on the other hand, was not observed in FL-irradiated ceramics with crossed-line patterns. This type of LIPSS is typically observed in the high fluence region  exhibited similar morphologies in these conditions. Accordingly, FL irradiation of dot or crossed-line patterns at a distance of 20 µm and 40 µm were selected as potential candidates for future investigations. Using these optimal settings, we will further investigate the influence of FL irradiation on the crystallography, mechanical properties, and bond strength of lithium disilicate glass-ceramics and highly translucent zirconia grades. Consequently, the potential clinical application of FL irradiation in surface treatment will be analyzed.In summary, the optimal FL settings were f


Staged urethroplasty is performed to treat long-segment obliterating anterior urethral strictures. The technique is particularly challenging when the penobulbar junction is involved, as it requires the transection of the scrotum and the formation of lateral testicular fans. To date, there is no established surgical protocol for this ancillary technique in large volume scrotums with excess skin. We report a case of staged urethroplasty with the necessity of performing T-scrotoplasty due to bulky scrotum. After six months, the T-plasty was successfully resolved, and a new scrotum was formed from the two hemiscrota. In conclusion, scrotoplasty using a bilateral T-plasty is an excellent technique to overcome the necessity of splitting the scrotum by externalizing the urethra to allow for excellent buccal mucosal graft healing over a period of six months. The two separate testicular fans can be reapproximated along the raphe after re-tubularizing the urethra in the 2nd stage, shaping a new scrotum with satisfactory cosmetic results. The treatment of obliterating, long-segment anterior urethral strictures poses a significant challenge to reconstructive urologists. Depending on the history of prior surgeries and the extent of spongiofibrosis, various surgical methods are available, which must be decided on a case-by-case basis. These include free oral mucosal grafts, pedicled penile skin flaps, and flap/graft combinations -3. A staWe report a case of a 66-year-old patient presenting with obstructive low urinary tract symptoms (LUTS) due to a recurrent long-segment penobulbar iatrogenic urethral stricture after two consecutive transurethral bladder tumor resections for low-risk pTa, G2 urothelial carcinoma 10 years prior. To enable regular follow-up cystoscopies, six direct vision internal urethrotomies (DVIU) had been performed in recent years. The patient had no comorbidities and reported satisfactory erectile function with an IIEF-5  score of 23. At the time of initial presentation, urinary diversion via suprapubic catheter was performed following acute urinary retention. The reported IPSS  was 32 with an IPPS-QoL  of 5 (unhappy). A retrograde urethrogram (RUG) showed a long-segment penobulbar stricture with a nearly obliterated urethral lumen Figure . CystoscSonographically, there was marked spongiofibrosis in the entire segment. Due to the extent of the stricture and the multiple prior interventions, we indicated open urethroplasty with oral mucosal augmentation; intraoperatively, a decision should be made regarding a staged procedure if necessary. The surgery was performed by a senior surgeon (LK) with extensive experience in urethroplasty.1st stageUsing the Kulkarni technique, the penile shaft was mobilized and invaginated down through a perineal incision . A ventrThe penile shaft was brought back orthotopically, and the skin was opened longitudinally over the entire stricture. This involved an incision across the raphe from the coronary sulcus, along the scrotum to the existing perineal access. The scrotum was completely divided down to the urethra into two separate hemiscrota. Before graft implantation, the fibrous portions of the urethral plate were resected. Complete resection of the bulbar obliterated corpus spongiosum was necessary.The graft was fixed on the corpora cavernosa with quilting sutures (5-0 polyglactin). The lateral margins of the graft were adapted to the penile skin Figure  using inAt the proximal, perineal end of the neourethra plate, an inverted U-shaped skin flap was prepared and mobilized to the urethra, creating a Blandy perineal urethrostomy using interrupted sutures (4-0 polyglactin) . An intrThe transurethral catheter could be removed on the 10th postoperative day, and spontaneous micturition was possible via the perineal urethrostomy. The suprapubic catheter was therefore also removed. The patient presented again after six months. The urethral plate was found to be adequately healed with a width of 3 cm, and the tissue was well vascularized, soft, and mobile Figure .At this point, the patient reported an unchanged IIEF-5 score of 23, an IPSS score of 2, and an IPPS-QoL of 1 (pleased). Despite his satisfaction with his sexual performance and the relief of his LUTS, he was eager to continue with the second stage of the urethral reconstruction, mainly for aesthetic reasons and his desire to urinate in a standing position. Thus, we proceeded with the second stage repair.2nd stageFirst, the border between the urethral plate and the skin was sharply incised. Lateral dissection of the skin and dartos fascia was performed. Next, the graft was tubularized in two layers using the first layer of interrupted sutures (5.0 polyglactin) followed by a running horizontal mattress suture (5.0 polyglactin) and a 16 French Foley catheter placement. A pedicled tunica vaginalis flap from the left testis was mobilized and fixed to the anastomosis as an interposition layer Figure . SubsequFollow-upFollowing catheter removal, uroflowmetry showed a bell-formed curve with a maximal flow of 25 ml/sec. The patient reported after two years of follow-up a continued satisfactory erection with an IIEF-5 score of 23 without ventral curvature. The reported IPSS was 3 with an IPSS-QoL of 1 (pleased). In addition, there was no reported postoperative incontinence.Staged urethroplasty is an excellent technique to treat long-segment, obliterating urethral strictures, especially when complete resection of spongiofibrosis is required .However, depending on the length and location of the stricture, it is a technically demanding procedure that may present different challenges. Depending on the stricture length, an appropriately sized skin flap or oral graft must be selected. Penile skin flaps are often limited in length and width by the existing anatomy ,9. ScrotFurthermore, the given anatomy must be considered not only when forming the neourethral plate but also during the wound closure. Most publications on staged urethroplasty describe penile strictures, and only a few reports are available on neourethral plate construction and appropriate wound closure at the 1st stage for long-segment strictures extending to the bulbar area , 11. MorIn our case, an ancillary T-shaped scrotoplasty had to be performed in the 1st stage to be able to anastomose the long scrotal skin surface bilaterally with the neourethral plate. Alternatively, part of the excess skin of both hemiscrota could have been trimmed tangentially to allow a simple side-to-side anastomosis with the neourethra. However, this would have resulted in altered scrotum anatomy after reapproximating in the 2nd stage. Considering a possible patient's dissatisfaction with a smaller scrotum due to the surgery, the described T-shaped scrotoplasty offers the possibility of leaving the appearance of the scrotum unchanged in the long term. Furthermore, moving the scrotum away from the midline offers the additional advantage of better graft take since excess scrotal tissue interposition would compromise the effectiveness of the applied compression bolster and the tie-over sutures.The issue of scrotal reconstruction would not be of concern in the case of a single-stage urethroplasty by using a penile invagination into the perineum according to the Kulkarni technique, requiring a much smaller single perineal skin incision . RegardiThe combined technique of Joshi et al. using buccal graft dorsally plus fasciocutaneous penile flap ventrally could be a feasible option, augmenting the urethra up to a 28.6 French caliper, however, requiring an additional circumcising subcoronal incision. He reported in a series of 15 patients a primary success rate of 86.7%, defined as no need for any kind of instrumentation, while the secondary success rate was 73.3%, defined as a Qmax under 10 ml/s or new obstructive LUTS [Limitations of the current technique include the usual complications of every staged urethroplasty, especially following second-stage tubularization including urethrocutaneous fistula, wound dehiscence, and recurrent stricture. Thus, these risks should be addressed by the urologists in the framework of a detailed preoperative written consent process.To the best of our knowledge, the T-shaped scrotoplasty technique has not been described so far and resulted in an excellent functional outcome for our patient and a satisfactory cosmetic outcome following closure.In our case, a successful two-stage urethroplasty was performed for a stricture length of 15 cm. The neourethral plate was constructed with free oral mucosa grafts. The major challenge was a large-volume scrotum. Following cleavage of the scrotal sac and a T-plasty, the two hemiscrota were anastomosed to the neourethral plate without difficulty in the first session. In the second stage, tubularization of the urethra and skin closure with the reconstruction of the scrotum by releasing the T-plasty was performed without problems. The presented T-scrotoplasty technique allows individual scrotal skin placement for anastomosis to the neourethral plate with very good cosmetic and functional results.


To evaluate its potential for clinical application, we compared the e-PTFE graft (n = 5) and our PCL/PDO + 10%DY graft (n = 5) into the carotid arteries of the pigs. No anticoagulant/antiplatelet agent was administered during the follow-up period, and ultrasonography was performed weekly to confirm the patency of the two grafts in vivo. Four weeks later, we explanted and compared the performance of the two grafts by histological analysis and scanning electron microscopy (SEM).A total of 10 pigs (weight: 25–35 kg) were used in this study. We made a new 5-mm graft with PCL/PDO composite nanofiber via the electrospinning technique. We simultaneously implanted a commercially available e-PTFE grafts occluded within the study period. The luminal surface of the PCL/PDO + 10%DY graft in the transitional zone was fully covered with endothelial cells as observed by SEM.No complications, such as sweating on the graft or significant bleeding from the needle hole site, were seen in the PCL/PDO + 10%DY graft immediately after implantation. Serial ultrasonographic examination and immunohistochemical analysis demonstrated that PCL/PDO + 10%DY grafts showed normal physiological blood flow and minimal lumen reduction, and pulsed synchronously with the native artery at 4 weeks after implantation. However, all The PCL/PDO + 10%DY graft was well tolerated, and no adverse tissue reaction was observed in porcine carotid models during the short-term follow-up. Colonization of the graft by host endothelial and smooth muscle cells coupled with substantial extracellular matrix production marked the regenerative capability. Thus, this material may be an ideal substitute for vascular reconstruction and bypass surgeries. Long-term observations will be necessary to determine the anti-thrombotic and remodeling potential of this device.The online version contains supplementary material available at 10.1007/s13770-021-00422-4. Vasculae clinic –5. Howeve clinic .ex vivo [e-PTFE and PET, failed to improve clinical outcomes in small-diameter vessels due to clotting, infection, pseudoaneurysm, thrombosis, occlusion from myointimal hyperplasia, congenital heart failure due to zero somatic growth, kinking, the need for pre-clotting, seroma formation, and venous hypertension [Synthetic vascular grafts have been studied in small-diameter applications for more than three decades. However, most failed or were not satisfactory for clinical use. On the other hand, a cell-based approach has been used to generate a tissue-engineered scaffold with an abundant extracellular matrix (ECM). Regardless of the long-term maturation phase of this graft ex vivo , signifiex vivo –10. Howeex vivo . Similarrtension .l-lactic acid)-co-poly(e-caprolactone) composite, and the results showed enhanced endothelial attachment and spreading in vitro [in vitro data alone cannot be relied upon for better in vivo performance of engineered vascular graft due to the dynamic nature of the blood system. Previous in vivo studies utilizing biodegradable polymers such as poly(glycerol sebacate) [in vivo study, antiproliferative drug such as paclitaxel has been exploited when loaded in polycaprolactone vascular prosthesis to achieve high patency and inhibit smooth muscle proliferation [The introduction of nanotechnology for bioengineering vascular conduits is expected to deliver the “holy grail” of regenerative medicine, i.e., the ability to engineer a scaffold that can mimic the nanostructure of the native vasculature in terms of both mechanical and biological performance. Electrospinning of natural materials and synthetic polymers into nanofibers is a typical application of nanotechnology for tissue engineering –16. Thisin vitro . Howeverebacate) , polylacebacate) , polycapebacate) , 24, andebacate)  as smallebacate) , 27, nitebacate) , 29, andebacate) , 31 in tDipyridamole is an anti-thrombogenic drug is among the most commonly prescribed drugs in the United States  after apin vitro and mechanical characterization. These grafts had several advantages, including excellent tensile properties, good in vitro biocompatibility with human endothelial cells (ECs), and favorable antithrombotic properties. However, immunofluorescence staining and a comparative study with commercially available vascular prosthesis was part of the study limitations. In this study, we compared the in vivo blood compatibility and performance of polycaprolactone and polydioxanone (PCL/PDO) grafts doped with an antithrombotic drug (dipyridamole) with a commercial graft (e-PTFE), for up to 4 weeks in a porcine model. Here, we evaluated the potential of these grafts for clinical application.In our previous study , we fabrMaterials used for this study are poly-ε-caprolactone (PCL)  and PDO  which were purchased from Sigma-Aldrich  and used without modification. 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) and dipyridamole were purchased from Tokyo Chemical Industry Co., Ltd. . Medical grade biodegradable PCL granules were purchased from ROKIT .We utilized a dual electrospinning approach to fabricate a nano-fibrous PCL/PDO vascular graft with an inner diameter of 5 mm similar to our previous report  with minWe evaluated the drug entrapment efficiency of the nanofiber samples after vacuum drying for 48 h at 40 °C. We prepared a nanofiber mat of known area (1X1 cm) and transferred it into 1 ml of HFIP. The amount of drug in the solutions was calculated by UV analysis and compared with the initial amount of drug-loaded during the electrospinning process.n = 3 each). The polymer concentration remained 10% and 8%(w/v) for PCL and PDO respectively. The nanofiber scaffold containing different drug concentrations 5, 7, and 10%(w/w) DY were named PCL/PDO + 5%DY, PCL/PDO + 7%DY, and PCL/PDO + 10%DY respectively. Pre-weighted nanofibers (≈ 0.5 g) were placed in 5 mL of phosphate-buffered saline (PBS) (pH 7.4) at 37 °C with mild shaking. At designated time points, 1 mL of release medium was pipetted out and kept at − 20 °C, and the volume was replaced with an equal amount of fresh PBS. The DY concentration in the release medium was measured by UV–vis spectroscopy  at a wavelength of 405 nm. A standard curve was plotted using a series of known dipyridamole contents in 50% ethanol/PBS.Drug release from PCL/PDO nanofibers loaded with three different concentrations of DY 5%, 7%, and 10% (w/w) with respect to the total polymer weight was analyzed separately . Five castrated adult pigs (weight: 25–35 kg) were purchased from a local pig farm. Each pig was caged separately and maintained at the Animal Center of Chonnam National University Hospital and Medical School, Gwangju, South Korea. The pigs were fed with standard food and the cages were cleaned and disinfected daily.e-PTFE grafts measuring 2 cm in length on the left and right carotid arteries. The pigs were monitored weekly using an ultrasound machine  for up to 30 days.Each pig was implanted with either PCL/PDO + 10%DY or −1), xylazine , and azaperone . They were then weighed and placed in the supine position, and their limbs were restrained on an operating table. The anesthesia was monitored, and when necessary, a muscle relaxant (ATRA®) was administered via the ear vein. An 8-gauge endotracheal tube was carefully inserted through the pig’s mouth to supply oxygen/anesthesia via a mechanical ventilator , while vital parameters  were monitored. While isoflurane was administered , the neck region was shaved and disinfected with povidone and alcohol. A 10-mm incision was made on either side of the neck region to expose the carotid artery. After the carotid artery had been isolated and clamped, 200 U/kg of heparin solution was injected intravenously as an anticoagulant. After 3 min, the proximal and distal ends of the common carotid artery were clamped with non-crushing clamps to stop blood flow. An approximately 1.5-cm segment of the native carotid artery was resected from the mid-section of the clamp. A 20-mm length of ethylene oxide-sterilized e-PTFE or PCL/PDO + 10%DY vascular graft was anastomosed to the proximal end, and then to the distal end by running stitches using 8-0 Prolene sutures . The proximal clamp was released to establish blood flow through the vessel, and continuous hemodynamic monitoring by pulse oximetry (for measurement of CO2 and temperature) was maintained throughout the procedure. Finally, the incision was closed in the standard manner with absorbable 2-0 sutures  for muscle and 5-0 sutures for the skin. After confirmation of blood flow through the graft by Doppler ultrasonography, the animal was woken and transferred to its cage. Aspirin (100 mg/day) and dipyridamole (75 mg/day) were administered for 3 days after surgery. Figure The pigs were administered 200 mg of aspirin, the day before surgery. On the day of surgery, the pigs were anesthetized by intramuscular injection of ketamine  for up to 1 week after the operation and allowed free access to food and water. Movement of the hind limbs was monitored daily for signs of spinal cord ischemia. While graft patency was monitored with an ultrasound machine weekly (weeks 0–4 post-surgery), the peak systolic velocity  was also recorded.In each group, four samples were explanted 4 weeks after surgery. Before graft harvesting, the pigs were anesthetized, and the grafts were retrieved alongside a 2-cm native vessel as an anatomical control. The retrieved grafts were gently rinsed in saline to remove residual blood. The explanted grafts were dissected in cross-section, and the patency and tissue formation on the luminal surface was observed and photographed. The grafts were further divided for histological and scanning electron microscopy (SEM) examination.The grafts were rinsed with normal saline after explanation and subsequently fixed in 2.5% glutaraldehyde for 4 h. The samples were then transferred into PBS and allowed to stand overnight, after which they were rinsed five times with PBS. Samples were dehydrated with a series of ethanol solutions of increasing concentration . For critical point drying, we transferred the samples into a fresh solution of 1:2 hexamethylenediamine (HMDS):ethanol 100% for 20 min. The samples were transferred to a fresh solution of 2:1 HMDS: ethanol for a further 20 min. Finally, the samples were transferred into 100% HMDS for 20 min; this step was repeated, after which the samples were allowed to stand overnight in a fume hood with the cover loosely open. The samples were then sputter-coated with gold and examined by SEM . Similar to a previous study , we perf2O). Hematoxylin and eosin (H&E), Masson’s trichrome (MT), safranin O (SO), and Verhoeff-Van Gieson (VG) staining were performed according to the manufacturer protocols. Subsequently, the samples were dehydrated through an ascending ethanol series  and mounted on slides for observation under an optical microscope.Cryosections (8-µm-thick) were deparaffinized by placing them in xylene three times, for 5 min each time, followed by hydration through a descending alcohol series  and double-distilled water  for 10 min at 97 °C. The samples were blocked with 1% bovine serum albumin (BSA) in PBS for 1 h and washed with PBS three times, for 5 min each time. Immunohistochemical staining was performed using mouse anti-human α-smooth muscle actin (SMA) antibody  or anti-cardiac myosin heavy chain (MHC) monoclonal antibody  as primary antibodies. Labeling was visualized using Alexa Fluor 597-conjugated goat anti-mouse secondary antibody (Invitrogen). ECs were stained using FITC-conjugated anti-Von Willebrand factor (vWF) primary antibody  and Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (Invitrogen). Collagen I/III was stained using rabbit anti-pig collagen I/III (Bio-Rad) polyclonal antibody. Newly recruited macrophages were visualized using a monoclonal antibody to CD68 as the primary antibody (Invitrogen) and Alexa Fluor 597-conjugated goat anti-mouse secondary antibody (Invitrogen). The nuclei were stained by incubation in 4′,6-diamidino-2-phenylindole (DAPI) < . All antibodies were used according to the manufacturer’s protocols. The samples were washed with PBS three times for 5 min each time and mounted onto slides using a medium. Fluorescence images were captured using a laser scanning confocal microscope; ZEISS LSM 800. Native pig carotid artery was used as a positive control.Briefly, paraffin was removed from the 8-µm-thick cryosections of explants by placing in xylene three times, for 5 min each time, followed by three different alcohol concentrations  and subsequent washing with ddHe-PTFE grafts in the left and right carotid arteries. All pigs were monitored at four experimental time points  by ultrasonography. All pigs survived for 4 weeks. Surgery duration and vessel clamp time were similar between the two grafts, as summarized in Table e-PTFE grafts (Supplementary Video 1).Two groups of five pigs each were implanted with either PCL/PDO + 10%DY or in vivo. The grafts were stored at room temperature after sterilization with ethylene oxide. The inner diameter of the graft (5 mm) could be measured easily using a Vernier caliper or ruler . At the end of the follow-up period (4 weeks), Doppler ultrasound revealed that, for the PCL/PDO + 10%DY grafts, patency of up to 96% (4/4) was maintained, in contrast to 0% (0/4) for e-PTFE grafts. PCL/PDO + 10%DY grafts showed excellent patency and strong synchronous pulsation with the host artery (Supplementary Video 2), and there was no evidence of stenosis or aneurysm. The regular and clear pulsation seen at 4 weeks indicated excellent integration with the host tissue. Before occlusions were observed in the e-PTFE grafts, retrograde flow signals were often seen at the distal arteries, suggesting thrombus formation.Graft patency was monitored using a combination of ultrasonography and manual palpation of the carotid artery pulsation, as shown in Fig. e-PTFE grafts were all occluded from 2 weeks after surgery. The peak systolic blood flow through the PCL/PDO + 10%DY grafts fluctuated over the study period, but the values were within the normal physiological range  were positively stained, as shown by the yellow circles distributed throughout the graft, especially within the vicinity of polymer remnants  are essential for good vessel function. Our immunofluorescent staining data at 4 weeks indicated the presence of smooth muscle cells and a more organized medial layer for the PCL/PDO + 10%DY graft Fig. . Macrophnts Fig. .Fig. 8ReThe amount of ECM deposition on the graft indicates the impact of host cell infiltration. The graft wall contained significant amounts of collagens I and III Fig. , and thePositive staining for MHC in the neoartery indicated a contractile smooth muscle phenotype Fig. . This mo2 from tissue to blood. However, PCL/PDO + 10%DY graft surfaces were smooth and clean, with ECs fully covering the luminal surface  performance of a new reinforced antithrombotic acellular graft in a porcine carotid artery model . In thatmobility . TherefoIntimal hyperplasia and thrombosis remain the greatest challenge in vascular surgery, especially in small-diameter vessels. Despite numerous investigations, graft failure due to intimal hyperplasia and thrombosis has yet to be eliminated. Furthermore, for clinical translation, synthetic vascular grafts require certain characteristics, such as cost-effectiveness, cell-free status, good mechanical properties, biodegradability, a highly porous structure for cell infiltration, easily scalable fabrication, and off-the-shelf availability. Vascular substitutes should also be anti-thrombogenic, have the structure of VSMCs/ECs , and be e-PTFE grafts failed due to thrombosis at 2 weeks, similar to previous reports [e-PTFE grafts show remarkable plasma leakage after anastomosis which was not observed on PCL/PDO + 10%%DY grafts. Previous studies have explored the potential of PCL and PDO to fabricate vascular grafts [The most interesting findings of the present study regarding small-diameter (< 6 mm) PCL/PDO + 10%DY grafts at 4 weeks are that the grafts were patent and showed synchronous pulsation with the native vessel, whereas all  reports , 52. Addr grafts . Althougr grafts , 54. We in vivo, the graph showed a high patency rate with complete endothelial coverage and no thrombi on the luminal surface, coupled with the formation of medial and neoadventitial layers; in contrast, the e-PTFE graft occluded at 2 weeks. A comprehensive assessment of remodeling cell types, such as inflammatory cells, and ECM production further confirmed positive remodeling of PCL/PDO + 10%DY grafts during the observation period in this study.Here, we used an electrospinning approach to combine a rapidly degradable polymer (PDO) as the luminal layer and a combination of PCL and PDO, spun simultaneously, as the abluminal layer. employing this strategy, the luminal layer will be replaced with endothelium as it degrades faster, while the thin abluminal layer will degrade slowly over time. The polymer solutions were all doped with an antithrombotic drug (dipyridamole) intended to inhibit clot formation. Finally, the graft was reinforced by 3D printing, using biodegradable PCL to increase the mechanical strength for ease of surgical handling. After 4 weeks Rapid reendothelialization is known to have antithrombogenic effects , 56. Henin vivo disparity between PCL/PDO + 10%DY and e-PTFE grafts may have been due to differences in the physical and mechanical properties of the grafts, such as the reinforcement that enhanced kink resistance in PCL/PDO + 10%DY grafts, and to the compliance of the grafts, which can significantly alter the hemodynamic flow [The results of ultrasonography demonstrated a slight reduction in diameter of the PCL/PDO + 10%DY grafts which subsequently stabilized at 4 weeks. The reduction of lumen size was apparently due to tissue formation on the luminal and abluminal sides, as confirmed by H&E staining. During this period, normal blood flow  was observed. However, at 2 weeks, all e-PTFE grafts were occluded. The mic flow .in vivo implantation of reinforced electrospun biodegradable grafts showing synchronous pulsation with the native vessel as early as 4 weeks after implantation in a porcine model. This device is novel in that it combines nanofibers using electrospinning and 3D printing to generate an arterial substitute with compliance similar to that of the native vessel.This is the first preclinical study of in vivo performance of electrospun PCL/PDO + 10%DY graft and commercially available e-PTFE grafts at 4 weeks in a porcine model. The ability of the PCL/PDO + 10%DY graft to promote the attachment/growth of ECs and inhibit clot formation within the experimental window supports the use of this device as a vascular substitute. Histological analysis showed that the PCL/PDO + 10%DY graft was well tolerated, and no adverse tissue reactions were observed. Furthermore, the favorable production of VSMCs and ECM proteins , coupled with synchronous pulsation with the native vessel, suggested that this device has potential for clinical use. These positive results of our short-term study in a large animal model indicate the potential for future clinical application, especially in patients with cardiovascular disease.In conclusion, we compared the Supplementary file1 (MP4 5837 kb)Supplementary file2 (MP4 27910 kb)Below is the link to the electronic supplementary material.


International graduates have been shown to experience comparatively poor outcomes in their transition to the host labour market after course completion. In Australia, international graduates typically fare worse than domestic graduates in the labour market, with new evidence pointing to a deterioration in their relative labour market position over the years. The contributing factors for the deterioration, however, remain unclear. In this article, we analysed unique large-scale survey data from the Australian Graduate Survey to identify the factors underlying the deteriorating labour market outcomes of international graduates from 2000. Our findings indicate that the deteriorating labour market outcomes of international graduates can be largely linked to the declining share of these graduates who are a citizen or permanent resident of Australia. The rising percentage of international graduates who are non-native English-speakers also played a role, albeit a modest one. These findings call attention to the persistent labour market disadvantage of international graduates and highlight the inadequacies of existing policies aimed at temporary retention and stronger English language skills in promoting labour market integration. We first provide a review of existing literature on the retention of international graduates in Australia, with an emphasis on factors thought to influence their transition to the host labour market after course completion. We then describe the data and methods employed to examine their unfavourable labour market position, followed by a discussion of the results. In the last section, we discuss the significance of the present study and its policy implications.In the early years of opening up its education services to the international market, Australia had no clear post-study migration and employment pathways for its international graduates. In view of rising global competition for high-skilled migrants, Australia introduced several changes to its immigration policies at the turn of the 21st century to retain international graduates following the completion of their studies. Specifically, exemptions were put in place to waive international graduates from the strict requirements of permanent residency application on discipline-related work experience and English language testing, along with bonus points for their Australian education credentials , and those who were participating in paid work . The latter are further broken into part- and full-time employed. We follow the Organisation for Economic Co-operation and Development (OECD)  and examTo identify qualification mismatch, we employ the job analysis method using the Australian and New Zealand Standard Classification of Occupations (ANZSCO), First Edition, Revision 1 ABS  as the aWe further employ the job analysis method to capture field-of-study mismatch. Specifically, we follow the OECD  and utilFor skill mismatch, we use the direct self-assessment method and analyse graduate perceptions of the importance of non-technical skills acquired during the recent course to their employment. In this case, respondents are considered mismatched if they indicated that these skills were not important to their employment. In contrast, those who responded formal requirements were important or somewhat important, are categorised as matched.characteristics effect captures differences in the composition of the two groups. For instance, higher wages received by white men, compared to black men, in the United States could be attributed to their higher level of educational attainment . This large effect is likely to reflect the influence of citizenship and residency status given its importance in accessing discipline-related work in the Australian labour market (Blackmore et al., Despite their alleged desirability, international graduates appear to experience relatively poor outcomes in the Australian labour market after the completion of their studies, with new evidence pointing to deteriorating outcomes since the initiation of post-study migration and employment pathways in 1999 (Tang et al., The Blinder-Oaxaca decompositions revealed that a considerable part of the deterioration was linked to an influx of non-native English-speaking international graduates. The over-representation of non-native speakers amongst international graduates was also responsible for their disadvantageous labour market position relative to domestic graduates. These results came as no surprise, given poor English language skills amongst recent waves of international graduates (Birrell et al., Instead, the decreasing percentage of international graduates who were a citizen or permanent resident of Australia was identified as the key factor contributing to their deteriorating labour market outcomes. While citizenship and permanent residency were found to provide a diminishing protective effect over time, the characteristic remained critical to gain a foothold in the Australian labour market. In particular, the results demonstrated that citizenship and permanent residency were increasingly relevant to securing discipline-related jobs over the period of analysis. In other words, international graduates who remained in Australia with a temporary visa were progressively less likely to obtain work experience in their area of expertise. This finding ironically runs counter to one of the main objectives underpinning the recent policy shift to promote and prioritise temporary retention of international graduates; that is, to provide them an opportunity to accumulate discipline-related work experience in the Australian labour market Spinks, .The smaller share of international graduates who took up paid work during the final year of study was also an important contributing factor for their worsening labour market position. The lack of paid work experience amongst recent cohorts of international graduates was shown to account for a large portion of the rise in economic inactivity and unemployment, as was the case for their below-par outcomes compared to local graduates in these domains. However, the dearth in local work experience cushioned the deterioration and widening gaps with respect to part-time employment and education-job mismatch. As pointed out earlier, these contradictions possibly reflect the nature of the jobs typically available to international graduates: low-skill, casual work with limited relevance to their study. The results also underline the tendency of international graduates to remain in these jobs following course completion, an issue that has attracted critical scholarly attention over the years (Jackling, Taken together, the present study provides novel quantitative empirical evidence which enhances our understanding of the key forces shaping the labour market outcomes of international graduates in Australia. Drawing on a large sample of international graduates who stayed on since 2000, the study presents additional, robust evidence in relation to the relevance of Australian citizenship and permanent residency, as well as local discipline-related work experience. It demonstrated that language background and English language skills play a less prominent role than suggested by the existing body of qualitative research. The study also offers some insights into the relevance of field-of-study choices, especially in the areas of Management and Commerce, Education and Health.It is important to acknowledge two limitations in this study. First, the present study was not able to take into account the role of some elements of work readiness, including soft skills and cultural fit - owing to a lack of information on these factors in the AGS. The links between these personal qualities and the deteriorating labour market outcomes of international graduates remain to be elucidated and should be the focus of future research. As pointed out earlier, these largely subjective characteristics might have been used as a subtle exclusion criterion to discriminate against international graduates in the Australian labour market (Blackmore et al., Despite these limitations, the present study offers important lessons to policy and practice by challenging existing Australian approaches to international graduate retention. Specifically, our findings reinforced recent findings of the persistent labour market disadvantage experienced by international graduates in Australia. The results further highlighted the inadequacies of some of the existing policies and practices designed to improve the integration of international graduates into the Australian labour market. Amongst other things, the current policies on temporary retention seem to have done little to promote local discipline-related work experience amongst international graduates. These findings call for better interventions to help international graduates integrate into the Australian labour market, including recognising and addressing the structural barriers they face in their transition to work. One of the key structural barriers that warrants immediate policy attention is the differential treatment and stereotypes international graduates have been reported to experience in a labour market that still favours the employment of permanent migrants (Robertson, The COVID-19 pandemic has drawn further attention to the vulnerability of international graduates - as well as other temporary migrant workers - who were expected to return to their home country when they could no longer financially support themselves against the backdrop of a weak labour market and a lack of government income support (Gibson & Moran,


The mitochondrial genomes (mitogenomes) of bilaterian animals are highly conserved structures that usually consist of a single circular chromosome. However, several species of parasitic lice (Insecta: Phthiraptera) possess fragmented mitogenomes, where the mitochondrial genes are present on separate, circular chromosomes. Nevertheless, the extent, causes, and consequences of this structural variation remain poorly understood. Here, we combined new and existing data to better understand the evolution of mitogenome fragmentation in major groups of parasitic lice. We found strong evidence that fragmented mitogenomes evolved many times within parasitic lice and that the level of fragmentation is highly variable, including examples of heteroplasmic arrangements. We also found a significant association between mitochondrial fragmentation and signatures of relaxed selection. Mitochondrial fragmentation was also associated with changes to a lower AT%, possibly due to differences in mutation biases. Together, our results provide a significant advance in understanding the process of mitogenome fragmentation and provide an important perspective on mitochondrial evolution in eukaryotes. Multiple independent origins of fragmented mitochondrial genome evolution in parasitic lice provide insights into eukaryotic mitochondrial evolution. However, there are a few notable exceptions scattered across the animal kingdom. The most pronounced examples of variant mitogenome structure are in organisms outside of Bilateria, including in some hydrozoa and jellyfish8, which have mitogenomes that consist of multiple (in some cases hundreds), fragmented linear chromosomes. Within Bilateria, fragmented mitogenome chromosomes are only known from a genus of nematode (Globodera)9 and three groups of insects: thrips10, book lice11, and parasitic lice12. In all four of these cases, the mitogenomes consist of multiple smaller, circular chromosomes. Of these, parasitic lice have the most extreme variation in mitogenome structure14. However, the extent and causes of mitogenome fragmentation in lice remain largely unknown.Most animals have mitochondrial genomes (mitogenomes) that consist of a single, circular chromosome that is between 12,000–18,000 base pairs (bp) long. This conserved structure is likely a result of the crucial and nearly universal mitochondrial function in eukaryotic cells, as deviations from this structure are usually associated with cell death and/or diseasePediculus humanus corporis), which have 20 circular fragments (“minicircles”) each containing 1–3 genes12. Subsequent studies indicated that fragmented mitogenomes in lice were ancestral in a clade of “mammal” lice 19 including the human louse, but more recent studies suggest mitogenome fragmentation is more widespread, although perhaps sporadic, across lice13. In agreement with these findings, full or partial mitogenomes with multiple chromosomes have been reported from a genus in Ischnocera (Columbicola)20 and from several genera in Amblycera21, indicating that mitogenome fragmentation evolved multiple times within lice. However, despite these recent developments, it is not known how common fragmented mitogenomes are in parasitic lice, nor how frequently fragmentation has evolved in the group. In addition, levels of mitochondrial fragmentation and gene arrangements on the fragments can even be variable within a genus of louse20. However, it is unknown how stable the mitogenome organization is among individuals (or populations) within a species of louse. Heteroplasmy can also be present in some mitogenomes of lice, either in the form of divergence between homologous genes or multiple chromosomal arrangements within a single individual22. These reported cases suggest heteroplasmy could be much more prevalent in parasitic lice, particularly in lice with fragmented mitogenomes.Fragmented mitogenomes in lice were first described in the human body louse  nucleotide composition, whereas lice with single mitogenomes have base compositions more similar to other insects25. This difference could be related to mutation biases. Specifically, a lower AT% in fragmented mitogenomes could be a result of fewer deamination mutations occurring during replication26, because the chromosome fragments are smaller and replication can occur in a shorter time period. However, these hypotheses have not been explored in detail beyond observations of general patterns.In conjunction with high substitution rates and nonadaptive processes, fragmentation could also lead to a shift in nucleotide composition of the mitogenomes. Most insects  have highly AT-biased  mitogenomesHere, we assemble, annotate, and compare mitochondrial genomes across a broad diversity of lice to understand the extent, causes, and consequences of mitogenome fragmentation in parasitic lice. Specifically, we address the following questions: (1) how prevalent are fragmented mitogenomes across parasitic lice? (2) how often has mitogenome fragmentation evolved in lice, (3) is there evidence that mitogenome fragmentation is a non-adaptive process? and (4) does fragmentation result in lower AT% of mitogenomes? Altogether, this study considerably advances our understanding of the process of mitogenome fragmentation in parasitic lice, the implications of which can provide new insight into basic cellular and metabolic function across eukaryotes.27, these newly assembled mitogenomes have highly rearranged gene orders. Of 25 genera of Ischnocera (including new and previously published mitogenomes) 15 showed strong evidence for having more than one mitochondrial chromosome, 13 of which have (to the best of our knowledge) never been reported before. Most taxa with a fragmented mitogenome  had at least one fragment with strong evidence for circularity based on read-mapping coverage and continuity (Supplementary Data Pessoaiella) to 16 (Columbicola)20. The lengths of different fragments in Ischnocera also vary depending on the genus, with complete fragments ranging from 1130 to 11430 bp  contains the arrangement cox3-nad4-trnM-rrnS and a shorter fragment  is missing nad4 with the arrangement cox3-trnM-rrnS (a 1269 bp deletion)   across heteroplasmic regions and paired-end reads with pairs that spanned the 5′ and 3′ ends of the contigs.We also found evidence of heteroplasmy in mitogenome organization, defined here as multiple chromosome arrangements in an individual. on) Fig. . Falcolion) Fig. . Both he28. This phylogeny represents the most current and well-supported hypothesis of evolutionary relationships among parasitic lice. We combined information from mitogenomes assembled in the current study with previously sequenced mitogenomes to match this prior sampling, for a total of 36 louse taxa. Using an Equal-Rates (ER) model (AICc = 52.6), phylogenetic reconstruction favored fragmented mitogenomes as the ancestral state in lice, with at least eight transitions to single-chromosome mitogenomes  had a fragmented mitogenome, a scenario that is slightly favored in the ER model. Second, there are conserved blocks of gene arrangements within louse clades that were likely broken up by fragmentation. For example, Megaginus, Pessoaiella, and Campanulotes share a long block of 16 genes from trnI to nad4 . However, these three taxa do not form a clade, and the sister taxon of each has a highly fragmented or rearranged mitogenome  is a sister to a clade consisting mostly of lice with fragmented mitogenomes. Finally, there are diverse clades of lice, for example, mammal lice, that all have highly fragmented genomes and there is no evidence of reversal to single chromosomes within these clades, suggesting that mechanistic reversal from a fragmented genome to a single chromosome could be extremely difficult.To investigate the evolutionary history of mitogenome fragmentation in lice, we tested for phylogenetic signal of fragmented vs. single mitogenomes in lice using a published phylogenomic treeome Fig. . Third, C. passerinae 2 (the number reflects likely cryptic species diversity in C. passerinae31). These individuals were collected from four different host species and several geographic regions23. Mitogenome organization was mostly consistent across all 11 individuals of C. passerinae 2. We did initially find, however, that there were some differences in gene arrangements among the 11 individuals of C. passerinae 2  and atp8 (four unique arrangements) . In contrast, comparisons between species indicate consistent differences in gene order and sequence variation  Fig. . The seqrsa Fig. . Howeverite Fig. . In othe25, we found that lice with fragmented mitogenomes have a significantly lower AT% (mean 65.8%) than lice with single mitogenomes (73.8%)   had a much higher rate of C → T mutations (49.1%) than Columbicola (fragmented mitogenomes) (24.8%), and a higher proportion of deamination mutations overall    or fourfold degenerate sites   Fig. . The nucAT% mean 5.8% than05) Fig. . HoweverAT% mean 5.8% thandN/dS ratios towards 1.0, indicating less stringent purifying selection) along the test (transitions from single to fragmented mitogenomes) branches relative to the background branches using both nuclear  and mitochondrial  phylogenies. In both analyses, most codon sites increased towards 1 along test branches, indicating most sites are under stronger purifying selection in background branches   revealed that fragmented mitogenomes have evolved from single chromosome mitogenomes many times in the group Fig. . This pa26. During replication, the sense strand can be left unbound for up to two hours, leaving the strand particularly exposed to deamination mutations32. In fragmented mitogenomes, replication would theoretically take less time, thus limiting the exposure time for deamination mutations to occur relative to larger, single chromosome mitogenomes. Consistent with this idea, some of our results showed considerably more C → T mutations in a group of lice with single chromosome mitogenomes (Physconelloides) compared to a similar group with fragmented mitogenomes (Columbicola)  is thought to be mainly caused through G → A mutations or the occurrence of deamination mutations, especially C → T mutations33 and large mitogenomes of certain plants34. However, relaxed selection in mitochondrial PCGs alone does not explain why some lice have fragmented mitochondrial genomes and others do not. It may also be the case that the nuclear genome in lice overall is subject to relaxed selection, as suggested by their simplified morphology , and this could lead to relaxed selection for mutation repair. Given that many mitochondrially targeted genes, including those involved in mitochondrial replication, are encoded by the nuclear genome, some of these genes may have experienced knockout mutations 13 resulting in increased mitochondrial mutation rates. Under this scenario, whether a lineage or clade of lice has fragmented mitogenomes is determined by the random loss of particular nuclear genes.Relaxed selection (or reduction in the efficiency of selection) in conjunction with high rates of mutation suggests that fragmentation could be a nonadaptive process . This would be consistent with other systems, including the highly fragmented genomes of endosymbiotic bacteria of cicadas22), and can result from a lack of sorting of mitochondrial mutations within the germ line or paternal leakage of mitochondrial DNA36. The majority of these cases are differences at single sites or in gene lengths, and although there are instances of deleterious heteroplasmic alleles22, it is seemingly rare for heteroplasmy to result in changes in gene order.Despite considerable variation in mitogenome organization among different louse taxa, we found stable organization within a single species Fig. –5, and aCaenorhabditis briggsae some strains have a heteroplasmic copy of a partial mitochondrial genome that has a large deletion (including the nad5 gene) compared to the full chromosome37, similar to the situation in some lice. The frequency of the heteroplasmic strain can increase in experimental populations, even though possessing the heteroplasmic chromosome is deleterious. This increase occurs through the biased transmission of the heteroplasmic chromosome in which it acts as a selfish genetic element37. A similar process could be occurring in lice. If a heteroplasmic fragment containing a large deletion arises in a lineage , this fragment could initially persist through biased transmission. If the original full chromosome then experiences a knock out mutation of an essential gene, but one which was still intact on the heteroplasmic fragment, this would then maintain the fragment through strong selection, because without the fragment the knock out mutation would be lethal. In this scenario, the original chromosome is also retained, because it contains other essential genes that do not occur on the heteroplasmic fragment. Further knock out and deletion mutations on either chromosome might even be selected for, as long as they do not knock out a gene contained on only one of the two chromosomes. This process could continue until both chromosomes in aggregate contain only a single copy of each gene, resulting in two chromosomes both maintained by strong selection. This cycle could repeat if new heteroplasmic fragments of the two smaller chromosomes arise. This process could also continue until each chromosome contains only a single or very few genes, similar to the situation seen in mammal lice and some bird lice. Such a process could also explain why relaxed selection (and in particular an upward shift in dN/dS) is associated with transitions to fragmented mitogenomes in lice, but under a selective model . When both chromosomes are in the stage where they each contain a copy of the same functional gene, both copies might accrue amino acid replacements more readily than if only a single copy of the gene existed. Once a knock out mutation occurred, the lineage would be “stuck with” the only functional copy, but one that has now accrued many amino acid replacement mutations. Further evidence for this possible mechanistic pathway could be obtained by examining in more detail clades of lice that differ in mitogenome fragmentation by uncovering whether there is a stepwise process consistent with a heteroplasmic fragment as an intermediate step.Heteroplasmy in mitogenome organization may also provide a clue for a potential mechanism for fragmentation to initiate and become more extreme. Within the nematode Our results also highlight the need for future work on different taxonomic and evolutionary scales. For example, it could be that fragmentation has an underlying genetic component conserved as far back as the common ancestor of Acercaria . On a smaller evolutionary scale, our results suggest that investigations within specific clades of lice will likely be fruitful for further understanding mitogenome fragmentation. For example, comparisons among many louse taxa with variable mitogenome structures in a clade could allow for a more detailed reconstruction of the process and direction of fragmentation. Working with more closely related taxa could also help clarify the nature of relaxed selection. Although we found a significant signal of relaxed selection associated with fragmentation, the signal was weak overall, likely because the taxa in our data set are highly divergent. Despite these limitations, our findings are a crucial development in our understanding of mitogenome fragmentation in lice. Our results help establish the broader patterns of mitogenome structure across parasitic lice, provide evidence for a selective explanation for fragmentation, demonstrate a shift in nucleotide composition as a likely consequence of fragmentation, and build a foundation for future research that explores this molecular phenomenon in lice and other insects.28. These data are whole-genome shotgun, 160 bp paired-end reads generated from an Illumina HiSeq2500 with insert sizes of ~400 bp. The samples were extracted from single or pooled (between 3–50) individuals collected from wild or captive birds. Before assembly, we trimmed adapters and low-quality bases using Trimmomatic v.0.3638  and removed reads <75 bp. We then checked the qualities of the trimmed reads using FastQC v.0.11.7 (Babraham Bioinformatics). To assemble the mitogenomes, we followed previously developed pipelines that use a combination of targeted, de novo, and read mapping assembly20. First, we assembled protein-coding genes (PCGs) for each species using aTRAM v.2.3.139. We converted the trimmed paired-end libraries into BLAST-formatted databases with 50 shards, and assembled the genes using ABySS v2.240 as the de novo assembler, 3 iterations, 10% of the library, and protein sequences of mitochondrial PCGs from the published Campanulotes compar mitogenome as targets13. Because mitochondrial reads are much more prevalent than nuclear reads in shotgun sequence data, using 10% of the library ensures against assembling sequencing errors or nuclear mitochondrial DNA (numts), while also having more than adequate coverage for confident assemblies (often >100X)20. We also attempted to assemble mitochondrial sequences using a completely de novo approach using SPAdes v.3.11.141, using the default parameters and 10% of the reads. We identified mitochondrial assemblies by BLASTing scaffolds against published mitochondrial gene sequences from either C. compar or Ibidoecus bisignatus. However, de novo assemblers can have difficulties assembling through the non-coding control regions of mitogenomes, primarily because of repeats or partially conserved sequences between different genome fragments42. To avoid this issue and assemble full mitogenomes, we used the iterative-mapping approach in MITObim v.1.843. For each trimmed library, we ran MITObim with the –quick option using each assembled gene (from aTRAM) or contig (from SPAdes) as a starting reference. As with the de novo assemblies, we used 10% of the trimmed read libraries for each MITObim run. We then created majority consensus sequences from the MAF files using the “miraconvert” command in MIRA v.4.0.244. We combined these outputs using the “Medium-Low Sensitivity” setting in the Geneious de novo assembler in Geneious Prime v. 2020.1.2 . We considered the resulting contigs as candidate complete mitochondrial chromosomes. We tested for the circularity of these assemblies using AWA45, which splits a contig, flips the two halves, and maps paired-end reads to the adjacent 5’ and 3’ ends of the contigs to test for circularity. We considered strong evidence for complete circularity to have a match score >95%, an alignment score > −2, and continuous connection coverage >20× across the 5′ and 3′ ends of an assembled contig. Finally, we annotated each assembly using the MITOS2 webserver46, with subsequent manual adjustments using open reading frame information and compared against published mitochondrial genes from Campanulotes compar or Ibidoecus bisignatus13.We assembled the mitogenomes from existing genomic data of 24 genera in the louse Parvorder Ischnocera28. We tested three different models of ancestral reconstruction: an Equal-Rates (ER) model, an All-Rates-Different (ARD) model, and a custom model that did not allow for transitions from fragmented mitogenomes to a single mitogenome. We assessed model fit with the corrected Akaike Information Criterion (AICc). We ran ancestral state reconstructions with the ace function in the APE v.5.447 package in R and assessed model fit with the fitDiscrete function in the GEIGER v.2.0.748 R package.To reconstruct the ancestral patterns of mitogenome structure (fragmented vs. single-chromosome) in parasitic lice, we used an existing phylogeny based on nuclear data and mitogenome information from 36 ingroup and 2 outgroup taxa (the 24 mitogenomes from this study combined with existing mitogenomes)Columbicola passerinae 2 (the number reflects likely cryptic species diversity in C. passerinae)31. We assembled the mitogenome fragments from 11 individuals of C. passerinae 2 with aTRAM v.2.3.139, using genes from the mitogenome of C. passerinae 220 as targets, and running aTRAM with 10 iterations, between 10% and 100% of the libraries, and ABySS v.2.240 for de novo assembly. We then annotated the assembled contigs using MITOS2. We included data from two other species as outgroups (C. passerinae 1 and C. columbae). We then compared the differences in gene order among the lice using a presence/absence matrix of gene boundaries. To compare the distance matrix of gene boundaries to genetic distances, we calculated uncorrected and corrected (JC and K80) genetic distances from the mitochondrial gene sequences using the dist.dna function in APE. We then compared the gene boundary and genetic distance matrices with Mantel Tests (999 permutations) using the mantel.randtest function in the ade4 v. 1.749 R package.To compare mitogenome structure and arrangements in a single species of louse, we used data from the dove louse taxon C. passerinae 2, we used Bowtie2 v.2.3.5.150 to map 10% of the paired-end reads against the mitogenome fragments to test for A) validation of assemblies and B) evidence for heteroplasmy . We viewed resulting read alignment BAM files in Geneious Prime v.2020.1.2  to assess coverage and screen for sequence variants associated with variable gene arrangements. Finally, we compared tRNA secondary structure with the Vienna package RNAfold (http://www.tbi.univie.ac.at/~ivo/RNA/RNAfold.html) in Geneious for fold prediction, using the Turner energy model51 rescaled to 37 °C. We confirmed these inferred structures by eye in comparison to those inferred for Bothriometopus52.For any variable gene arrangements within t-tests in the GGPUBR v. 0.40R package53.We tested for differences in nucleotide composition in all available full mitogenomes of parasitic lice  phylogeny using IQTree v.2.1.156  models to the louse mitogenome dataset. For lice with fragmented mitogenomes, we used the average length among the different fragments. We fit models using both total AT% and AT% of fourfold degenerate sites. For PGLS, we used the ML tree estimated previously in the pgls function from the CAPER v.1.0.1R package, with both Brownian and Pagel’s λ correlations.Before comparing AT% between different groups of lice, we first estimated phylogenetic relationships among the lice to account for phylogeny in our comparisons. We extracted mitochondrial PCGs from the full mitogenomes, translated each gene to amino acids, and aligned them using the –auto option in MAFFT v.760. These data are subsets of mitochondrial PCGs from lice in the genus Columbicola  and Physconelloides . Columbicola have highly fragmented mitogenomes20, whereas Physconelloides have a single mitogenome. We aligned the genes according to amino acids and extracted fourfold degenerate sites. We then estimated substitution rate matrices for these sites in MegaX v.10.1.861 using GTR models and Γ distributions with four categories.We also wanted to test the hypothesis that there will be more C to T substitutions in fragmented mitogenomes compared to single mitogenomes. To do this, we used the mitochondrial data from refs. 62. RELAX tests whether dN/dS ratios significantly shift toward 1  in specific phylogenetic branches relative to the other background branches. We first identified branches involved in these transitions based on our ancestral state reconstructions (described above), using the transition branches as test branches and the remaining branches as references . We ran all RELAX tests on the Datamonkey webserver63.We tested for evidence of relaxed selection associated with transitions from single to fragmented mitogenomes using RELAX in the HyPhy packageFurther information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Reporting Summary


Acinetobacter baumannii is an emerging pathogen that is increasingly resistant to antibiotics and is mainly responsible for pneumopathy in fragile patients. This germ is frequently responsible for epidemics in hospitals. We aimed to describe the steps of the investigation of an outbreak of Acinetobacter baumannii affected our hospital, the measures implemented and the follow-up of the actions.Following alerts issued by the microbiology department concerning 5 swabs detecting Acinetobacter of the same strain and the same antibiotic resistance profile in 3 different departments of intensive-care units (ICU), a team of the prevention and healthcare security department went onsite for an investigation in the hospital.We identified five cases with identical strains of multi-resistant Acinétobacter. The field visit allowed to identify some deficiencies in professional practices. All the patients were hospitalized in ICU . The synoptic table showed that there was an overlap of hospitalization periods.A crisis cell was set up to validate, coordinate and implement control measures in accordance with CTINILS recommendations. Indeed, we proceeded to a technical isolation of the cases in their hospitalization sector, reinforced the basic hygiene and bio-cleaning measures and sensitized the medical and paramedical. Given that the three ICU departments shared the same medical staff during night shifts, the assumption that the germ was carried by the caregivers was the most likely hypothesis. We proceeded with a swab of the elements of the environment in the services concerned. Results showed that Acinetobacter was found on the nursing cart (visibly clean). A training about bio-cleaning and hygiene standard precautions is programmed.Continuous surveillance, continuous hygiene trainings, combined with a rapid reaction capacity in case of identification of a new case, is essential to control the spread of nosocomial germs.• Multidrug-resistant Acinetobacter baumannii (MRAB) is an emerging cause of intensive care unit (ICU) outbreaks.• Enhanced infection control measures limited the outbreak.