IntroductionTM  with the widely used Mazor-XTM Stealth Edition robotic platform  in terms of workflow and lumbar pedicle screw placement accuracy.There has been a surge in robot utilization in spine surgery over the past five years with the rapid development of new spine robotic platforms. This study aimed to compare a new robotic spine platform from Fusion RoboticsMethodsTM system. These four procedures were compared to four propensity-score matched cases with single-level lumbar pedicle screw placement using the Mazor-XTM Stealth Edition. A single surgeon performed all surgeries. The cases were matched in terms of demographics  and comorbidities (Charlson Comorbidity Index score). The primary outcome measure was the operative workflow efficiency (duration as measured with a stopwatch by an independent observer). The secondary outcome measures were pedicle screw accuracy and accuracy to plan.A cadaver lab was conducted, which included four procedures with single-level lumbar pedicle screw placement using the Fusion RoboticsResultsTM platform had a significantly shorter duration in terms of the system set-up time, planning to in-position time, and total procedure time (p<0.05). However, there was no significant difference between the robotic platforms in terms of creating a sterile barrier, scanning and importing images, creating a plan, screw placement, screw accuracy, and screw accuracy to plan.After propensity-score matching, there were four cases in each group with no significant between-group differences in terms of sex, race, BMI, or surgical levels; however, there were significant differences in terms of age (p=0.01) and comorbidities (p<0.001). The workflow efficiency measurement showed that the Fusion RoboticsConclusionTM platform had a significantly shorter procedure workflow duration while maintaining the same accuracy as the most commonly used robotic platform (Mazor-XTM). This is the first study to directly compare different spine surgery robotic systems.Based on our findings, the Fusion Robotics TM Stealth Edition  [TM Stealth Edition [TM Stealth Edition is the most widely used system and the one that is predominantly featured in the literature, as Mazor was the first to develop a robotic spine platform [TM  to the widely used Mazor-XTM robotic platform in terms of workflow and screw placement accuracy.There has been a surge in robot utilization in surgeries in the last 20 years in many surgical disciplines such as general surgery, urology, and gynecology . In 2004Ireland) . Current Edition . The Mazplatform . All of platform . Accordiplatform . Given tWe have adhered to the Strengthening The Reporting of OBservational studies in Epidemiology (STROBE) guidelines in conducting this study and reporting our findings .DemographicsTM GroupFusion RoboticsWe conducted a lab study using two cadavers, and each specimen underwent two separate procedures (L2-3 and L4-5 pedicle screw placement). The cadavers’ demographics  and comorbidities (Charlson Comorbidity Index score) were collected from the donor summary report.TM Stealth Edition GroupMazor-XThe electronic health records of patients who had undergone a single-level robotic-assisted lumbar fusion between May 2017 to April 2018 were reviewed after obtaining approval from our institutional review board (IRB). Patients’ demographics  and comorbidities were collected retrospectively from the charts. A total of 18 patients met our inclusion criteria. After propensity-score matching, four patients were included in our final analysis.OutcomesPrimary OutcomesTM group and during the surgery in the Mazor-XTM group using a stopwatch.The primary outcome measure of this study was the robotic workflow efficiency (duration) for both groups. The duration of the system set-up, creating a sterile barrier (ready for the scan), scanning and importing images, creating a screw plan Figures , 1B, plaSecondary OutcomesTM group  Figure  and the p Figure  or a posl et al. . If the l et al. . The scrStatistical analysisTM was matched to a case that was performed using the Mazor-XTM Stealth Edition with a nearly similar propensity score. The primary outcomes (workflow durations) were compared using Student’s t-test and the secondary outcomes (screw placement accuracy and accuracy to plan) were compared using the chi-squared test. The statistical analysis was performed using the SPSS Statistics software version 26.0 , and statistical significance was set at a p-value of less than 0.05.The baseline characteristics  of both groups were compared using Student's t-test and chi-squared tests. The propensity-score matching analysis was performed to compare the intraoperative workflow parameters as well as screw placement accuracy. The propensity score was calculated using the patient baseline characteristics  as independent variables. Furthermore, one-to-one matching was performed to conform to baseline demographics. A case that was performed using Fusion RoboticsDemographicsTM cases being performed on cadavers of older age and higher comorbidity scores  and comorbidities (p<0.001) between both groups, and this was attributed to the Fusion Robotics and comoPrimary outcomeTM group (p=0.1); however, the original system set-up time (p<0.05) and the planning to in-position time (p<0.05) were significantly longer with regard to the Mazor-XTM. Furthermore, the total procedure time was significantly longer in the Mazor-XTM group (p<0.05). There was a similar workflow duration in both groups during creating a sterile barrier (p=0.5), scanning and import of images (p=0.9), creating a plan (p=0.8), and screw placement (p=0.9)  Table .Secondary outcomesAccording to grading based on Iampreechakul et al., all the screws in both groups were determined to be grade A on the post-placement images with no significant difference between groups Figure . FurtherFusion is one of the most common surgical procedures for the lumbar spine -13. The TM system to those of the Mazor-XTM system for single-level lumbar pedicle screw planning and placement. The primary outcome measure was workflow efficiency. The study results showed that the Fusion RoboticsTM platform had a significantly shorter workflow duration in terms of system set-up time, planning to in-position time, and total procedure time. There was no significant difference between the systems in terms of creating a sterile barrier, scanning and importing images, creating a plan, and screw placement. Regarding the secondary study outcomes, there was no significant difference between the platforms in terms of screw placement accuracy and screw accuracy to plan.This study aimed to compare the efficiency and accuracy of the Fusion RoboticsPrimary outcomesTM group (36.6 ± 4.4 minutes) compared to the Mazor-XTM group (55 ± 1.9 minutes). This significant difference was attributed to longer system set-up time (2.4 ± 0.2 versus 7.8 ± 1.2 minutes) and plan to in-position time for the first screw (3.8 ± 0.5 versus 12.7 ± 5 minutes). The pre-placement of the screw (system set-up) time was non-significantly shorter in the Fusion RoboticsTM group (21 ± 2.1 versus 35.8 ± 14.9 minutes). Also, compared to published data regarding the ExcelsiusGPS® robotic platform, the pre-placement screw time (28.1 ± 5.2 versus 21 ± 2.1 minutes) was significantly shorter for the Fusion RoboticsTM platform while the total operative time (41.4 ± 8.8 versus 36.6 ± 4.6 minutes) was non significantly shorter [TM, Mazor-XTM, and ExcelsiusGPS® robotic platforms [In this study, where all procedures were performed by the same surgeon, there was a significantly shorter total procedure time for the Fusion Robotics shorter . With relatforms . There iSecondary outcomesTM and Mazor-XTM groups in this study was 100%. There are many published clinical studies evaluating the screw placement accuracy of the Mazor Robotics platform, with screw accuracy ranging from 92.6 to 99.5% [Lumbar pedicle screw placement accuracy for both the Fusion Roboticsto 99.5% -24. Furtto 99.5% . No signto 99.5% .Future robotic platformsTM system is that it includes approximately 90% manual surgeon control of the robot. This enhances the range of motion of the robot and makes it more applicable to single-position surgery, whereas other robotic platforms have trouble getting into position. After the manual positioning is completed by the surgeon, the final trajectory is targeted using controls that can be positioned inside or outside the sterile field. This makes the Fusion RoboticsTM platform a hybrid system between a shared-control system and a telesurgical interaction system. Other advantages of the Fusion RoboticsTM platform include a near-field localization camera attached to the operating room table, which minimizes line-of-sight issues. The small sizes of the robot and camera afford the surgeon more room to move and work throughout the procedure.Three robotic control systems are currently used in surgeries: (1) the shared-control system, where the robot and the surgeon perform the surgery simultaneously; (2) telesurgical interaction, where the surgeon remotely controls the surgery done by the robot (such as with the da Vinci system); and (3) the supervisory-controlled system, where the surgeon pre-plans and supervises the operation done autonomously by the robot . All of LimitationsTM cohort to the four procedures in the Fusion RoboticsTM group. Despite the small sample size, there was a significant difference between the groups in terms of the total procedure time, the system set-up time, and the plan to in-position time for the first screw. Furthermore, even though the same surgeon performed all procedures, this was a comparative study between cadaveric surgeries in a lab setting and live cases in an operating room setting.As with all retrospective studies, there is an information bias risk because this study was partially based on chart review. However, this bias was minimized by propensity score matching of the Mazor-XTM Stealth Edition system involving hundreds of procedures at the time of data collection, whereas the same surgical team had undergone only an approximately 30-minute training session with the Fusion RoboticsTM system prior to completing the procedures.Another important difference between the groups was that the surgical team had extensive experience using the Mazor-XTM system but was not available for comparison with the Mazor-XTM system was post-procedure breakdown time. From an overall operating room efficiency and logistics perspective, this metric will be of value to compare robotic platforms in the future. The breakdown time for Fusion RoboticsTM System was 2.4 ± 0.3 minutes.One metric that was collected for the Fusion RoboticsTM platform has a significantly shorter procedure workflow duration (better efficiency) with the same accuracy as the most commonly used spine robotic platform (Mazor-XTM). In addition, the Fusion RoboticsTM system involves manual surgeon control of the robot, thereby enhancing its range of motion compared to other robotic platforms. Thus, it takes a step closer to telesurgical interaction and supervisory-controlled systems. Further clinical studies with a large sample size are required to validate our findings.Based on our findings, the Fusion Robotics


Due to the stochastic and time-dependent character of the ice embryo formation and growth , the heterogeneous ice nucleation on atmospheric aerosols or macroscopic solid surfaces is still shrouded in mystery, regardless of the extremely active research and exponential progress within this scientific field. For instance, whether the icing appears from outside-in or inside-out is a subject of intense controversy, with practicability in designing passive icephobic coatings or improving the effectiveness of the cryopreservation technologies. Here, we propose an artful technique for quantitative analysis of the different modes of water freezing using super-nonwettable soot-coated quartz crystal microbalances (QCMs). To achieve this goal, a set of 5 MHz QCMs are loaded one at a time with a 50 μL droplet, whose bulk or contour solidification is detected in real-time. The obtained experimental results show that our sensor devices recognize explicitly if the ice nuclei form predominantly at the liquid–solid interface or spread along the droplet’s entire outer shell by triggering individual reproducible responses in terms of the direction of signal evolution in time. Our results may serve as a foundation for the future incorporation of QCM devices in different freezing assays, where gaining information about the ice adhesion forces and ice layer’s thickness is mandatory. Apart from being pernicious to our hectic daily routine, by blocking the road infrastructure and the operability of the telecommunication towers, high-voltage power lines, wind turbines, aircraft, air conditioners etc. ,3,4,5,6,Of particular scientific interest appears to be the mechanism of ice nucleation around the individual soot particles and associated solid aggregates/agglomerates, and the decades of extensive research in this field reveal that the water uptake ability of the aerosols, their wettability and the porosity of the individual soot particles are dominating the freezing process ,11,12,13Nevertheless, the role of the interfacial contact line in regulating the water freezing remains poorly understood and provokes scientific discussions with many open questions, mainly related to elucidating whether the droplet’s heterogeneous nucleation takes place from outside-in or inside-out ,16,17,18Nowadays, the initiation of freezing events at the droplet’s free surface holds very important practical applications in developing sustainable passive icephobic coatings, whose operation principle relies on the concentric inward motion of the freezing boundary from the contour (outer shell) of the droplet towards the slurry liquid bulk, inducing volumetric expansion and incompressibility of the droplet and its subsequent lifting (“self-dislodging”) from the solid surface . MoreoveEsmeryan et al. that the cryopreservation of human spermatozoa via soot is successful due to outward osmosis, as a result of the contour freezing, and balanced dehydration of the cells [Recently, our research group has accidently discovered a “hybrid version” of the outside-in freezing phenomenon, noticed by assessing the freezing temperature depression of water droplets resting on a variety of soot coatings (see the next sections). Wehave found that the simultaneous cooling of the coating and the liquid, in order to determine the droplet’s solidification temperature, leads to freezing of both the contact interface and the droplet’s entire outer shell. Meanwhile, when the soot surface is preliminary cooled , the aforementioned contour freezing mode is missing, and the droplet freezes in the conventional two-steps’ way: partial solidification at the contact line  followed by isothermal freezing of the remaining bulk liquid (referred to as a bulk mode). These findings validate the hypothesis of he cells . Considehe cells ,18,19,202) [Undoubtedly, the quartz crystal microbalance (QCM) is one of the appropriate piezoresonance sensors that can track any water freezing events in a highly sensitive and reproducible manner, since it reacts instantly to negligible changes in the mass added to its surface (absolute sensitivity of ng/cm2)  and to v2) ,24,25 or2) ,27,28. F2) ,30,31 an2) .The primary objective of this study is to represent a new platform for accurate analysis and facilitated future interpretation of the bulk and contour ice nucleation modes occurring in water droplets through soot coated QCM sensors. We show, experimentally, that the proposed sensor configuration can unambiguously distinguish whether the inception of ice nuclei is solely at the solid–liquid interface or along the droplet’s entire shell, by generating recognizable resonance responses concerning the signal trend. A substantial superiority of our approach, compared to the commonly used high-speed video imaging, is the possibility of calculating the thickness of the ice layer formed throughout the recalescent freezing stage—a task that has not been implemented yet.The soot used as an interfacial coating for a series of six 5 MHz QCMs  is obtained during the controlled combustion of rapeseed oil at atmospheric pressure ,33. The The occurrence of bulk and contour ice nucleation is traced using a QCM 200 analytical instrument  consisting of a digital controller with a built-in frequency counter and ohmmeter, a sensor oscillator SRS 25 and a quartz crystal holder intended to accommodate the QCM device. The real-time sensor signal is continuously recorded on a personal computer at 1 s gate time by means of LabView Stand Alone computer software .Prior to starting the experiments, there was one technical drawback that had to be overcome. The operating thermal range of our measurement system is within 0 to +40 °C , while tThe as-developed holder is composed of an upper and lower part connected to one another by two M4 screws. An appropriate nest, where the 1-inch quartz crystal should be mounted, is carved at the lower (bottom) part, whereas a technological clearance among both metal details is ensured when assembling them, with an aim of eliminating short circuits in the electrical branch of the sensor and its subsequent damage. Major advantages of our custom-designed holder, compared to the SRS’ and other companies’ counterparts, are the wide working thermal range (−200 °C to +1000 °C), the excellent thermal conductivity of the copper and the large contact surface minimizing the temperature gradients between the surrounding environment, the holder’s fuselage and the QCM sensor. The lack of such gradients is verified with a thermal imaging camera FLIR P640  and the maximum observed temperature deviations are ±0.2 °C. Moreover, the length of the coaxial cable see  is identambT~22 ± 1 °C, RH~25 ± 4%), the evaporation rate of the liquid nitrogen is registered to be steady, allowing well-controlled cooling if the needed amount of coolant is added in a timely manner. A transparent lid, whose roof has two windows for dispensing the droplets, is mounted on the base and used to isolate the liquid loaded QCMs from externalities . The undesired temperature gradients are further minimized due to the high thermal conductivity of the copper base and copper crystal holder, and by setting the sensors next to the 2K-type probe of a Signstek 31/2 6802 II dual channel digital thermometer , supporting thermal measurements with an accuracy of ±0.1–0.4 °C.The bulk and contour ice nucleation discerned while studying the freezing time delay and the freezing temperature depression of sessile water droplets (T~0 °C), respectively, are analyzed in-situ in a home-made environmental chamber, shown in pictorial view elsewhere . Concisef~±1 Hz/s), the copper rod was immersed in liquid nitrogen and the chamber was cooled from room temperature to −30 °C for the case of bulk nucleation analysis. Then, a 50 μL water droplet was gently fixed in the middle of the QCM’s surface and its solidification and subsequent thawing at room temperature were contemporaneously tracked by means of the quartz sensor and a photo camera. The contour ice nucleation study was performed in the same way, but the droplet was placed on the soot coated QCM before initiating the cooling, executed with a rate of ~3.5 °C/min . BotAs seen, upon simultaneous cooling of the soot coating and the droplet, the recalescent freezing encompasses solidification of the liquid’s outer shell b, which f~1 MHz; R~5000 Ω), similarly to what is observed when a frost layer densifies on a soot coating . Secondtion see a,b. In ation see b. These The left side of The as-prepared QCMs react differently to the bulk and contour freezing and, as demonstrated in Although useful and exciting, the real-time graphical dependencies cannot be used to extract an additional information concerning the exact elapsed time before the QCMs get out of resonance, nor the precise values of the frequency, resistance and displaced mass shifts upon recalescent freezing, initial and full thawing . Therefore, we analyzed in detail the automatically saved numerical data sheets (see the Excel file in the SI), highlighted each milestone in the file in red color, and then summarized the data in FTf in FTf in R at bulk icing is up to 9 times larger than at contour freezing  impedance of the quartz crystal, n—number of sides in contact with the liquid, ω—angular frequency at series resonance, and L—inductance at unloaded (dry) sensor.When the QCM sensor operates in air, the strong acoustic reflectivity at the substrate–air interface localizes the wave energy in the crystal itself, whose volume (thickness) plays the role of a resonance cavity in which a standing wave is created . As soonf is the resonance frequency (Hz); Δf—frequency shifts (Hz); A—active electrode area (cm2); Δm—mass changes upon binding events (g). However, Equation (3) is valid for film thicknesses up to 1 μm [If part of the water freezes, the solid–liquid sensing interface is replaced by a solid–solid (ice) one, creating strong quartz-ice elastic coupling , extensi to 1 μm  and mass to 1 μm . Beyond  to 1 μm .IFm − ΔTFCm = 25 μg/cm2), and knowing that the density of ice is ~0.92 g/cm3, the ice layer’s thickness at that particular moment is calculated as ~273–556 nm (1st–3rd experiment). On the other hand, the unexpected positive frequency and negative mass shifts during the contour freezing may have two explanations: As stated elsewhere, “the abnormally large increase in the resonance frequency upon the freezing of the water droplet is attributed to changes in the surface stress of the quartz crystal substrate. The increase in the contact radius during the freezing of the water droplet induces an increase in the surface stress of the quartz crystal, which increases the resonance frequencyand decreases theQ-factor of the quartz crystal” [R towards the baseline, over the droplet’s liquefaction, at the contour mode compared to the bulk analogue , the droplet deposition on the cooled soot coated QCM triggers wave penetration in the liquid and frequency downshifts (resistance upshifts) proportionally to the viscosity and density of the water (Equations (1) and (2)). Since the non-wettable surfaces delay the liquid–solid phase transitions by reducing the contact area and the heat transfer rate ,45, the crystal” . While scrystal” . In turnTFCm ~−78 ÷ (−81) μg/cm2 or ΔTFCm ~1.4÷1.5 μg/cm2, the displaced mass upon initial freezing (ΔIFm) differs up to a factor of 4 , but noof 4 see . In otheThe first systematic analysis of the ice nucleation dynamics of sessile water droplets by means of soot coated QCMs revealed that the formation of ice embryos prevalently at the liquid–solid interface instigated consistent resonance frequency decrease and series resistance increase, in conformity with the mass loading effects theoretically predicted by the Sauerbrey’s equation. The compact interfacial icing ceased the continuous oscillations immediately and the non-wettable piezoresonance devices got out of resonance until partial droplet liquefaction occurred. Excitingly, when the heterogeneous ice nucleation was driven jointly at the three-phase contact line and droplet’s contour, the QCM sensors were capable of vibrating at resonance for about 60–150 s. Moreover, unanticipated frequency increase was regularly identified during the contour freezing, complemented by faster recovery of the resonance upon room temperature thawing of the icy droplet. The collected experimental results suggested for different ice adhesion forces maintained by the bulk and contour freezing modes, which is of potential relevance to explicating (in the future) the physical processes governing the nascency of ice nuclei on solid particles or functional coatings. The complexity of this task could be alleviated by using the QCM technology, whose efficiency was undisputedly demonstrated herein.A Bulgarian patent application (No. 113331) was submitted on 26 February 2021 as a result of the reported work.


Potent inhibitors of p38 MAPKs have been pursued as potential therapies for several disease indications due to their antiinflammatory properties, although none have been approved to date. Here, we provide a brief overview of p38 MAPKs, including their role in regulating myogenesis and their association with disease progression. Finally, we discuss targeting p38 MAPKs as a therapeutic approach for treating facioscapulohumeral muscular dystrophy and other muscular dystrophies by addressing multiple pathological mechanisms in skeletal muscle. Mitogen-activated protein kinases (MAPKs) are serine/threonine kinases that are members of conserved signaling pathways that alter cellular physiology in response to extracellular signals , p38β (MAPK11), p38γ MAPK12), and p38δ  transcription factors, namely Myf5, MyoD, myogenin, and MRF4, collectively referred to as myogenic regulatory factors   . In mdx  wasting . Moreove wasting . p38α/β mdx mice, but not in exercise-trained WT mice or in nonexercised mdx mice , a model for limb-girdle muscular dystrophy, alleviated pathology in both models, decreasing fibrosis and macrophage infiltration and improving endurance on a treadmill  . This form of heart disease is caused by mutations in e lamins . Mutatio failure . In cont failure . Treatme failure . Three s failure . The aut failure –71. Inde failure . Thus, r failure –74. It wInhibition of p38α/β is also being pursued as a potential treatment for FSHD ; therefore, any potential side effects of pharmacological p38α/β inhibition that affect satellite cell regulation or myogenic differentiation are of concern. In particular, chronic inhibition of p38α/β has the potential to result in a reduction in the ability of satellite cells to proliferate as myoblasts and further differentiate to repair and replace damaged myofibers. The effects of long-term treatment with p38 inhibitors on muscle regeneration have yet to be fully explored. As muscle in patients with FSHD shows evidence of active regeneration based on expression of embryonic isoforms of myosin and the presence of centrally located nuclei, chronic p38α/β inhibition as a long-term treatment may not be feasible . As notemdx and Sgcd–/– mouse models of muscular dystrophy (Similar to other muscular dystrophies where p38α/β inhibition has been tested in preclinical models , FSHD isystrophy . Preclinystrophy . These mystrophy –110, anoystrophy –93.Among several p38α/β inhibitors demonstrated to be efficacious in blocking DUX4 functions in cells from patients with FSHD, the p38α/β inhibitor losmapimod was selected for clinical trials in part due to its positive clinical safety profile in humans. In a trial for COPD, losmapimod was well tolerated in more than 3500 participants , 55 but The lack of relevant animal models to test the action of losmapimod on the multiple p38 targets, apart from the human xenograft model for DUX4, limits development of clinical trial protocols, but such information can be generated as part of future p38α/β inhibitor trials. It remains to be determined what levels of DUX4 inhibition are required for effective clinical outcomes, and whether low levels of DUX4 expression are sufficient or if expression must be entirely eliminated to achieve a reasonable outcome for improvement of patient quality of life. It also remains to be determined whether p38α/β inhibitor treatment will significantly reverse muscle damage or merely prevent further disease progression. The results of the ongoing losmapimod trial will provide an indication of the potential of p38α/β inhibitors as therapeutics for FSHD. Moreover, the results will provide an invaluable molecular and clinical platform to enable future FSHD clinical trials, offering hope for treatment of this devastating disease.


Locusta migratoria to dissect the molecular mechanism by which Kr-h1 functions as activator and repressor at these distinct developmental stages.The zinc-finger transcription factor Krüppel-homolog 1 (Kr-h1) exerts a dual regulatory role during insect development by preventing precocious larval/nymphal metamorphosis and in stimulating aspects of adult reproduction such as vitellogenesis. However, how Kr-h1 functions both as a transcriptional repressor in juvenile metamorphosis and an activator in adult reproduction remains elusive. Here, we use the insect 154, a step essential for its dual functions. During juvenile stage, phosphorylated Kr-h1 recruits a corepressor, C-terminal binding protein (CtBP). The complex of phosphorylated Kr-h1 and CtBP represses the transcription of Ecdysone induced protein 93F (E93) and consequently prevents the juvenile-to-adult transition. In adult insects, phosphorylated Kr-h1 recruits a coactivator, CREB-binding protein (CBP), and promotes vitellogenesis by inducing the expression of Ribosomal protein L36. Furthermore, Kr-h1 phosphorylation with the concomitant inhibition of E93 transcription is evolutionarily conserved across insect orders.We report that the kinase PKCα triggers Kr-h1 phosphorylation at the amino acid residue SerOur results suggest that Kr-h1 phosphorylation is indispensable for the recruitment of transcriptional cofactors, and for its anti-metamorphic and vitellogenic actions in insects. Our data shed new light on the understanding of Kr-h1 regulation and function in JH-regulated insect metamorphosis and reproduction.The online version contains supplementary material available at 10.1186/s12915-021-01157-3. Krüppel-homolog 1 (Kr-h1) is a primary JH early-inducible gene coding for a zinc-finger transcription factor that mediates both anti-metamorphic and vitellogenic actions of JH [Broad-complex (Br-C) in holometabolous insects [Ecdysone induced protein 93F (E93), an adult-specifier gene in both hemimetabolous and holometabolous insects [Spok in prothoracic glands of the fruit fly Drosophila melanogaster and thus prevents precocious larval-pupal transformation [Kr-h1 resulted in blocked vitellogenesis and impaired egg development in the migratory locust Locusta migratoria, the rice borer Chilo suppressalis, the oriental fruit fly Bactrocera dorsalis, the cotton bollworm Helicoverpa armigera, and the brown planthopper Nilaparvata lugens [Aedes aegypti, Kr-h1 regulates the developmental phase in preparation for competence acquisition for blood feeding, as well as subsequent vitellogenesis and egg development [Cimex lectularius, depletion of Kr-h1 in adult females caused severely reduced egg hatchability [Juvenile hormone (JH), an arthropod-specific sesquiterpenoid secreted by the corpora allata, plays a central role in insect metamorphosis and reproduction. In juvenile stages, JH maintains the larval/nymphal status by suppressing the metamorphic action of the steroid hormone 20-hydroxyecdysone (20E) –5. In adns of JH –11. Kr-h insects –14. Kr-h insects  in the c insects , 15. In ormation . Stimulaormation , 19. RNAa lugens . In the elopment –27. In thability .Kr-h1 is transcriptionally activated by the JH-receptor complex comprising Methoprene-tolerant (Met) and Taiman, two members of the bHLH-PAS transcription factor family [Kr-h1 transcription in JH-mediated previtellogenic development of Ae. aegypti [Tribolium castaneum, JH represses the expression of Histone deacetylase 1 (HDAC1), leading to increased levels of histone acetylation and consequently promoting Kr-h1 transcription [Kr-h1 is post-transcriptionally regulated by microRNAs [Blattella germanica, miR-2 eliminates Kr-h1 transcripts at final instar nymphs, which crucially contributes to the onset of metamorphosis [L. migratoria, Kr-h1 is downregulated by let-7 and miR-278, whereas JH suppresses the expression of these two miRNAs. This regulatory loop ensures a proper level of Kr-h1 essential for preventing precocious metamorphosis in nymphs and stimulating JH-dependent vitellogenesis in adults [r family , 29–33.  aegypti . In the cription , 35. BesicroRNAs , 36. In orphosis . In L. mn adults –44.L. migratoria, we investigated Kr-h1 phosphorylation and its involvement in transcriptional repression and activation. The migratory locust L. migratoria is a destructive insect pest worldwide as well as a representative of evolutionarily basal insects with hemimetabolous development and JH-dependent vitellogenesis. We found that PKCα triggers Kr-h1 phosphorylation. Phosphorylated Kr-h1 recruited C-terminal binding protein (CtBP), consequently inhibiting E93 expression and nymphal-adult metamorphosis. Phosphorylated Kr-h1 interacted with CREB-binding protein (CBP), which stimulated the transcription of Ribosomal protein L36 (RL36) and reproduction. We also provide evidence that the essential role of phosphorylated Kr-h1 in recruiting CtBP and repressing E93 expression is evolutionarily conserved in other representative insects including the silkworm Bombyx mori, the beetle T. castaneum and the fruit fly D. melanogaster.In an effort to elucidate how Kr-h1 functions in repressing precocious nymph metamorphosis and stimulating adult reproduction in L. migratoria Kr-h1 (GenBank: KJ425482) computationally by DISPHOS (V1.3) software [154, Ser371, and Ser554 were suggested as potential phosphorylation sites, with Ser154 at the highest score  antibody  antibody was also verified by western blot using the recombinant Flag-tagged proteins of wildtype Kr-h1 and mutated Kr-h1S154A(Ser154 to Ala154) expressed in Drosophila S2 cells treated with methoprene as well as the bacterially expressed GST-tagged peptides of Kr-h1(aa1-290) and Kr-h1S154A(aa1-290) incubated with PKCα  in nymphs and adult females caused significant reduction of p-Kr-h1 but not total Kr-h1 abundance  knockdown had no obvious effect on Kr-h1 phosphorylation  and mutated Kr-h1S154A(aa125-159) peptides, followed by incubating them separately with PKCα for LC-MS/MS analysis. As illustrated in Fig. S154A(aa125-159) peptide was incubated with PKCα, a molecular mass of 4238 Da was detected, same as that observed with Kr-h1S154A(aa125-159) peptide alone , Kr-h1S154A(aa1-290), Kr-h1(aa89-312), Kr-h1S154A(aa89-312), and Kr-h1(aa291-591) incubated with PKCα. As shown in Fig. S154A(aa1-290) or Kr-h1S154A(aa89-312). No phosphorylation band was observed with the truncated Kr-h1(aa291-591) . Compared to that on the day of adult emergence, p-Kr-h1 levels increased at 1–4 days PAE and remained high on days 5–6, resembling that of total Kr-h1 Fig. D. As JH E93 transcription [E93 (GenBank: MT081312) in the final instar nymph of locusts resulted in supernumerary nymphs and delayed adult morphogenesis  of L. migratoria E93 gene  antibody. Knowing that p-Kr-h1 had an essential role in suppressing E93 reporter activity, we next performed in vivo ChIP analysis using anti-phospho-Kr-h1 (Ser154) antibody and nuclear extracts from mid-4th and 5th instar nymphs. The antibodies against Kr-h1 and IgG were used as the positive and negative controls, respectively. As shown in Fig. E93 promoter region covering the KBS motif in penultimate 4th instar nymphs in which JH, Kr-h1, and p-Kr-h1 were in high levels. Conversely, a marginal precipitation of p-Kr-h1 was observed at final 5th nymphal instar when JH, Kr-h1, and p-Kr-h1 levels were low  (GenBank: MT081313) was previously found to express in response to the JH-Met-Kr-h1 pathway [RL36 resulted in blocked ovarian growth and arrested oocyte maturation  comprising a KBS motif  expression was high in nymphs, but significantly decreased in adults  mRNA levels significantly increased after adult ecdysis , a transcriptional coactivator with histone acetyltransferase activity, has been demonstrated to play an important role in JH action , 57, 58.B. mori, T. castaneum, and D. melanogaster for further study. Ser154 of L. migratoria Kr-h1 is homologous to Ser76 of B. mori Kr-h1 (BmKr-h1), Ser124 of T. castaneum Kr-h1 (TcKr-h1), and Ser255 of D. melanogaster Kr-h1 (DmKr-h1). Amino acids at the flanking regions of these serine residues occur in a highly conserved context  antibody  located outside of multiple zinc-finger domains was identified in Ae. aegypti Kr-h1 (AaKr-h1). JH treatment caused dephosphorylation of AaKr-h1 at Ser694. Dephosphorylation mimic mutants (AaKr-h1S694V and AaKr-h1S694C) showed significantly higher transcriptional activity than wildtype AaKr-h1 [+/K+-ATPase involved in patency induction and Vg transportation in vitellogenic female locusts [As a primary JH early-response gene, n adults –11. Prev complex , 29–31.  species , 38. FurH action , 35. Thu aegypti , 43. Morogenesis . Additioarmigera . Recentl AaKr-h1 . Our pre locusts . We spec154 is essential for the transcriptional regulation of E93 and RL36, two representatives of Kr-h1 target genes. Such a phosphorylation was required for Kr-h1 to interact with the corepressor CtBP in inhibiting E93 transcription and with the coactivator CBP in inducing RL36 transcription. The p-Kr-h1 wildtype variant Flag-Kr-h1S154D had similar capability to p-Kr-h1 in binding cofactors and exerting transcriptional activity. However, the Kr-h1S154A mutant was unable to recruit the cofactors, consequently abolishing the repression of E93 transcription and the induction of RL36 transcription. These results together address the importance of Kr-h1 phosphorylation in mediating anti-metamorphic and vitellogenic effects of JH.Kr-h1 is capable of activating or repressing transcription of genes in response to JH bound to its receptor Met , 26, 27.2H2 zinc-finger domains. In addition to potentially recognizing a variety of DNA sequences, the zinc-fingers act as a hub for protein-protein interaction [154 residue is localized at the 3rd zinc-finger domain of Kr-h1. Phosphorylation modification is likely to induce a conformational change that is optimal for Kr-h1 to recruit cofactors. In the present study, CtBP and CBP were found to bind with phosphorylated Kr-h1 in repressing E93 transcription and activating RL36 transcription, respectively. Nevertheless, phosphorylated Kr-h1 could also interact with other cofactors in transcriptional activation or repression of target genes. In Ae. aegypti, Kr-h1 acts synergistically with Hairy, thereby mediating the action of Met in gene repression during previtellogenic development of adult females [N. lugens has demonstrated that Hairy directly interacts with the N-terminus zinc-finger domains of Kr-h1 in modulating gene transcription [cyclin B1 via interacting with CBP, but downregulates cyclin B1 transcription by recruiting HDAC3 [The Kr-h1 sequence contains eight Ceraction , 62. The females , 63. A scription . Dual fucription –70. In mng HDAC3 .B. mori, the coleopteran T. castaneum, and the dipteran D. melanogaster. The requirement of phosphorylation for Kr-h1 action on suppressing E93 transcription was found to be also conserved. The findings provide a clear indication that Kr-h1 phosphorylation and its indispensable role in regulating target gene expression are evolutionarily conserved across distant insect orders. These observations further highlight the significance of Kr-h1 phosphorylation in eliciting transcriptional activity. Previously, JH-dependent Ae. aegypti Kr-h1 dephosphorylation at Ser694 has been demonstrated to enhance the transcriptional activity [694 is conserved in some holometabolous insects but not in L. migratoria. The Ser154 of L. migratoria Kr-h1 is homologous to Ser206 of Ae. aegypti Kr-h1. Thus, Kr-h1 orthologues likely bear multiple phosphorylation sites with differential responses to JH. While evolutionarily conserved Kr-h1 phosphorylation sites occur in divergent insect species, the lineage- and species-specific Kr-h1 phosphorylation residues may exist in some insects. It is of interest to address these questions in future research.We have additionally shown Kr-h1 phosphorylation in other insects belonging to divergent orders, including the lepidopteran activity . The phord zinc-finger domain. While Kr-h1 phosphorylation levels increased along with JH-induced total Kr-h1 expression, more rapid increase of Kr-h1 phosphorylation than total Kr-h1 was observed in locusts treated with methoprene. JH-induced Kr-h1 phosphorylation was also seen in methoprene-exposed S2 cells. Phosphorylated Kr-h1 recruited CtBP in nymphs, which inhibited E93 expression and metamorphosis. Phosphorylated Kr-h1 recruited CBP in adults, consequently stimulating RL36 transcription and vitellogenesis. Kr-h1 phosphorylation and its essential role in recruiting CtBP and repressing E93 expression are evolutionarily conserved in L. migratoria, B. mori, T. castaneum, and D. melanogaster. Thus, our present study fills a knowledge gap of phosphorylation modification of Kr-h1, an intermediate regulator in the JH/Met-response gene expression hierarchy.Kr-h1 functions both as a transcriptional repressor in preventing precocious larval/nymphal metamorphosis and a transcriptional activator in stimulating adult reproduction in insects. PKCα phosphorylated Kr-h1 at a serine residue localized in the 3L. migratoria was maintained as previously reported [The gregarious phase of reported . s-(+)-mS154A(aa125-159) peptides (BiotechPark) were separately incubated with PKCα  in reaction buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 20 mM MgCl2, 1 mM DTT, and 1 mM ATP at 30°C for 30 min. After termination with 1/10 volume 1% formaldehyde and centrifugation at 8000×g for 10 min, the supernatant was desalted by C18Zip-Tip (Millipore), reduced by 10 mM DTT at 56°C for 1 h, and alkylated by 20 mM iodoacetamide (IAA) at room temperature in dark for 1 h. Extracted peptides were then lyophilized and resuspended in 0.1% formic acid, followed by LC-MS/MS analysis.Synthesized Kr-h1(aa125-159) and Kr-h1S154A(aa1-290), Kr-h1(aa89-312), Kr-h1S154A(aa89-312), and Kr-h1(aa291-591) were separately cloned into pGEX-4t-1 vector  for overexpression of recombinant GST-tagged proteins in Escherichia coli Rosetta competent cells (Transgen). Cells were lysed by sonication in lysis buffer with 50 mM Tris-HCl pH 7.5 plus 0.1% Triton X-100 and cleared by centrifugation at 8000×g for 30 min at 4°C. GST-fusion proteins were purified by GST resin (Thermo Fisher Scientific) and incubated with PKCα , followed by SDS-PAGE and Pro-Q Diamond Phosphoprotein Gel Stain (Invitrogen).cDNA fragments for Kr-h1(aa1-290), Kr-h1S154A, Kr-h1S154D, BmKr-h1S76A, BmKr-h1S76D, TcKr-h1S124A, TcKr-h1S124D, DmKr-h1S255A, and DmKr-h1S255D was performed using Q5 Site-Directed Mutagenesis Kit (NEB). S2 cells were transfected with the recombinant vectors using Lipofectamine 3000 (Thermo). Primers used for recombinant vector construction and site-directed mutagenesis are provided in Table S1 , CtBP (nt 1-1332), CBP (nt 1-1728), BmKr-h1 (nt 1-1086), TcKr-h1 (nt 1-1407), and DmKr-h1 (nt 1-2376) were separately cloned into pAc5.1/Flag vectors (Invitrogen). Site-directed mutagenesis for Kr-h1154) (Jingjie PTM-Biolab), VgA [Protein extracts from insects and S2 cells were isolated in ice-cold lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1% Nonidet P-40, 1% Triton-X 100, 0.5% sodium deoxycholate, 1 mM PMSF plus protease, and phosphatase inhibitors (Roche). Lysates were cleared by centrifugation, subjected to 8% SDS-PAGE, and transferred to PVDF membrane (Millipore). Western blots were conducted using antibodies against Kr-h1 , phosphoab), VgA  and Flagab), VgA  was used−ΔΔCt method, normalized by ribosomal protein 49 (Rp49). Primers for qRT-PCR are listed in Table S3 , and first-strand cDNAs were reverse transcribed using FastQuant RT kit with gDNase (Tiangen). qRT-PCR was performed using a RealMasterMix SYBR Green kit (Tiangen) with a LightCycler 96 system (Roche), initiated at 95°C for 15 min, and followed by 40 cycles of 95°C for 10 s, 58°C for 20 s, and 72°C for 30 s. Relative expression levels were calculated using 2cDNA templates were amplified by PCR, cloned into pGEM-T vector (Tiangen), and confirmed by sequencing. dsRNAs were synthesized by in vitro transcription with T7 RiboMAX Express RNAi System (Promega). Locusts were intra-abdominally injected with 15 μg dsRNA, and boosted once on day 5. Phenotypes were photographed by Canon EOS550D camera and Leica M205C stereomicroscope. Primers used for RNAi are given in Table S3  and confirmed by sequencing. S2 cells were co-transfected with these constructs along with recombinant vectors expressing wildtype or mutated Kr-h1 of L. migratoria, B. mori, T. castaneum, and D. melanogaster. Methoprene was applied at 10 μM 48 h post transfection and for 6 h. The luciferase activity was measured using a Dual-Luciferase Reporter Assay System and a GloMAX 96 Microplate Luminometer (Promega).154) or IgG, followed by qPCR. Primers used for ChIP are listed in Table S3 . Briefly, fat bodies collected from nymph and adult females were fixed with 1% formaldehyde to crosslink chromatin for 10 min at 37°C. After addition of 125 mM glycine, chromatin was sonicated to shear into 200-1000 bp DNA fragments. The complexes were then immunoprecipitated with antibody against Kr-h1, phospho-Kr-h1  with Tukey’s post hoc test using the SPSS22.0 software. Significant difference was considered at P < 0.05. Values were reported as mean ± S.E.Statistical analyses were performed by Student’s Additional file 1: Figure S1. Identification of Kr-h1 phosphorylation site. Figure S2. Identification of kinase triggering Kr-h1 phosphorylation. Figure S3. Effect of 15-min exposure of methoprene on Kr-h1 phosphorylation. Figure S4. Effect of E93 knockdown on locust metamorphosis. Figure S5. Responsiveness of Kr-h1 phosphorylation to JH. Figure S6. Effect of RL36 knockdown on locust reproduction. Figure S7. Effect of CtBP or CBP knockdown on E93 or RL36 expression. Figure S8. Alignment of the 3rd zinc-finger domain of Kr-h1 and the partial promoter sequences of E93 with KBS motifs. Table S1. Primers used for cloning and gene expression. Table S2. Primers used for site-directed mutagenesis. Table S3. Primers used for qRT-PCR, RNAi and ChIP.Additional file 2. The individual data values for Fig. Additional file 3. Original Western blot data


With the susceptibility of sea ice ecosystems to climate change, there is a pressing need to fill knowledge gaps surrounding sea ice habitats and their microbial communities. Of fundamental importance to this goal is the development of new methodologies that permit effective study of them. Based on outcomes from the DiatomARCTIC project, this paper integrates existing knowledge with case studies to provide insight on how to best document sea ice microbial communities, which contributes to the sustainable use and protection of Arctic marine and coastal ecosystems in a time of environmental change.Sea ice continues to decline across many regions of the Arctic, with remaining ice becoming increasingly younger and more dynamic. These changes alter the habitats of microbial life that live within the sea ice, which support healthy functioning of the marine ecosystem and provision of resources for human-consumption, in addition to influencing biogeochemical cycles (e.g. air–sea COThe online version contains supplementary material available at 10.1007/s13280-021-01658-z. The Arctic marine system represents a diverse collection of water bodies that cover the continental shelves and deep-water basins of the northernmost latitudes on our planet Fig. . A definUnderstanding the critical role of sea ice microbial growth in Arctic marine ecology and biogeochemistry requires accurate knowledge on which microbial communities are present, how they function, as well as information on how abundant and active they are. However, analysing biological and biogeochemical properties in sea ice is fundamentally complicated by its inherent heterogeneity and multiphase nature  is widely used to approximate ice algal abundance or even represent the primary productivity of a given location. Caution should be used interpreting the chl a proxy in isolation, as the concentration of this pigment relative to carbon can vary substantially with environmental conditions, algal acclimation state, and species  inherent to sea ice algae  to spatial variations in light availability.Assessing the response of sea ice microbial communities to environmental change that is driven by amplified global warming in the Arctic  the adaptation of the Biogeochemical Flux Model (BFM-SI) of Tedesco et al. (Effective ecosystem-based management of Arctic waters must bring together current knowledge across trophic levels (i.e. from microorganisms to megafauna), while planning for data collection in the future that addresses critical knowledge gaps and the challenges of a rapidly changing system. Here we provide a summary of sympagic microbial communities of the Arctic, particularly on the abundance, function and production of prominently studied algal and bacterial groups of microbial life Fig. . We higho et al.  for repro et al. , with: ma biomass on the order of 5 m . The pan-Arctic variability in chl a and production, which is especially evident when comparing the magnitude of chl a and production between basins of the central Arctic (low chl a and production) and continental shelf regions like Baffin Bay (high chl a and production), is reflective of differences in ice dynamics and limitations on algal growth. For example, while Resolute Bay in the Canadian Arctic is distanced from nutrient-rich Pacific and Atlantic waters entering the comparatively nutrient-depleted surface waters of the Arctic Ocean, chl a and maximum algal production in the first-year ice present is still high due to local topography that mixes the water column and replenishes nutrients for algal growth , conditions of water column stability  and heterotrophic (O2 consumption and CO2 production) conditions have been documented during the spring bloom . Through this assessment we found that dinoflagellates, which typically account for 4–16% of algal communities in sea ice . We found that one of the most abundant reconstructed bacterial genomes belonged to the Saccharospirillaceae family, and it encoded genes for both facultatively anaerobic denitrification as well as dissimilatory nitrate reduction to ammonia pathways  and the subsequent reconstruction of metagenomic assembled genomes (MAGs) was also performed on the samples of northwestern Hudson Bay  represents 27% of all measurements in Fig. a have the potential to improve efficiency of sampling and ultimately the spatio-temporal coverage of sea ice biogeochemical studies.Studies on sea ice microbial communities in the Arctic are limited by the logistics of accessing its more remote regions. For example, the concentrated number of primary production values within the vicinity of the Resolute Bay, Canada, research facility . Chlorophyll a concentration can be related to a change in the shape of the transmitted PAR spectrum, as a result of the pigment’s preferential absorption of certain wavelengths  of particular wavelengths correlated to traditionally core-based chl a values. Here, NDIs are calculated according to the equation:Tz(λx) represents transmitted irradiance or transmittance at a given wavelength of PAR ) was determined through Pearson Correlation analysis. A linear regression between ice core-based chl a and transmittance , and as a result, our understanding of spatial variability in the ice algal bloom was improved.The value of this method is highlighted by recent work of the DiatomARCTIC project in the Lincoln Sea of the High Arctic Fig. , 4c. Herloe Fig. . Througha for the entire ice column, which provides an integrated value for surface, interior and bottom-ice communities. However, the distribution of sea ice algae is also known to vary horizontally across fine (millimetres) spatial scales with the presence or absence of brine channel features , variability in the vertical distribution of cells through the ice that is observed as a change in location of the coloured band of pigment Fig. , may be Bay Fig.  we relatNumerical models of biogeochemical processes have been developed to better study pan-arctic variability and potential future changes in sea ice primary production, nutrient and gas dynamics , but results in similar peak magnitudes. The model setup used here includes a single group of algae with fixed photophysiological parameterisations. Including additional algal groups that were potentially more efficient in utilising nutrients under varying light levels could result in the modelled chl a better following observed concentrations. This is an important next step in development of the BFM-SI.Total modeled chl oom Fig. d decreasoom Fig.  in the s. a Fig. d shows t2 levels in the atmosphere . Through photosynthetic activity, ice algae also impact the global cycling of carbon and help remove rising anthropogenic COa using optical or photometric approaches we have demonstrated the potential to assess ice algal chl a biomass over scales of significantly greater spatial coverage or detail that are not ordinarily possible with collection of ice cores alone. Finally, our ice algal bloom simulations using the BFM-SI model highlight the critical importance of validating environmental conditions parameterized by models to ensure accurate representation of bloom development for process studies of individual field sites. The complexity and severity of conditions in the Arctic marine system presents an enormous challenge in scientific investigations. In addition, strong regional and seasonal differences will require more advances in up-scaling observations for accurate representation of biogeochemical processes in model settings and basin-wide estimates. Through development of innovations to tackle these challenges we can create an accurate benchmark of understanding, from which the consequences of future change to the system may be evaluated by researchers and governing bodies alike.Global warming has created a pressing need to effectively characterise the habitats and microbial communities of Arctic marine ecosystems. To best inform on the management of the Arctic marine ecosystem it is critically important that this work includes the unique environment of sea ice, which hosts an active and microbially diverse community that provides a number of services for northern latitudes and the global community. Our ability to prepare for change and manage resources accordingly is hindered by the sparse coverage of sea ice biogeochemical studies across the vast Arctic, and to some extent, the caveats of traditional methodologies that may be inaccurate, time consuming, or limited by gaps in our knowledge of such complex systems. The compiled work of the DiatomARCTIC project, which drew upon data over a broad geographical range in the Arctic, has demonstrated through case studies how innovative approaches of combining in-situ, laboratory and numerical studies may advance our understanding of microbial function and growth within sea ice. The use of molecular tools like metagenomics and metatranscriptomics has the capacity to reveal complex and novel metabolic strategies within sea ice environments. Here we have shown that this includes the potential for facultative anaerobic activity by bacteria, which has important implications for nutrient cycling in the ice and potentially on nutrient availability to algal communities. Through representation of chl Supplementary file1 (PDF 318 KB)Below is the link to the electronic supplementary material.


Introduction: Current high-accuracy image-guided systems for otologic surgery use fiducial screws for patient-to-image registration. Thus far, these systems have only been used in adults, and the safety and efficacy of the fiducial screw placement has not yet been investigated in the pediatric population.Materials and Methods: In a retrospective study, CT image data of the temporal region from 11 subjects meeting inclusion criteria (8–48 months at the time of surgery) were selected, resulting in n = 20 sides. These datasets were investigated with respect to screw stability efficacy in terms of the cortical layer thickness, and safety in terms of the distance of potential fiducial screws to the dura mater or venous sinuses. All of these results are presented as distributions, thickness color maps, and with descriptive statistics. Seven regions within the temporal bone were analyzed individually. In addition, four fiducial screws per case with 4 mm thread-length were placed in an additively manufactured model according to the guidelines for robotic cochlear implantation surgery. For all these screws, the minimal distance to the dura mater or venous sinuses was measured, or if applicable how much they penetrated these structures.Results: The cortical layer has been found to be mostly between 0.7–3.3 mm thick (from the 5th to the 95th percentile), while even thinner areas exist. The distance from the surface of the temporal bone to the dura mater or the venous sinuses varied considerably between the subjects and ranged mostly from 1.1–9.3 mm (from the 5th to the 95th percentile). From all 80 placed fiducial screws of 4 mm thread length in the pediatric subject younger than two years old, 22 touched or penetrated either the dura or the sigmoid sinus. The best regions for fiducial placement would be the mastoid area and along the petrous pyramid in terms of safety. In terms of efficacy, the parietal followed by the petrous pyramid, and retrosigmoid regions are most suited.Conclusion: The current fiducial screws and the screw placement guidelines for adults are insufficiently safe or effective for pediatric patients. Image guidance provides a technological solution for accurate anatomy and instrument localization through anatomy-to-image registration . RegistrOtorhinolaryngological applications to date are mainly focused on rhinology. In endoscopic sinus surgery (ESS), the use of image guidance has demonstrated a potential to improve surgical outcomes . AchieviRecent guidelines for cochlear implant surgery recommend implantation in pre-lingual phase as early as 6 months old in order to obtain optimal hearing outcomes . The bonAnother obstacle posed by the most recently described IGS techniques that can be applied in pediatrics for CI surgery is the need of a dynamic reference screw placement to bear the tracker as described in previous studies , 8, 10. Hence, the primary aim of this study is to investigate the efficacy and safety of screw placement in pediatric cases. The efficacy is measured in terms of the thickness of the cortical layer of the temporal bone  derived from computed tomography data of pediatric cases. The safety is measured in terms of distance to dura mater or venous sinuses (DDVS – distance to dura or venous sinuses), i.e. sigmoid and transverse sinus. The secondary aim is to further evaluate the safety of screw placement by implanting screws in patient-specific 3D-printed models, and subsequently investigating their distance to the dura and the venous sinus (4 mm thread length screws).n = 20 sides included in the study protocol. The two remaining sides were discarded due to image quality issues. An algorithm was used to estimate the thickness of the cortical bone and the distance of the fiducial screws to anatomical structures (dura and venous sinuses).With permission of the local institutional review board (KEK 2017-01722), CT image data from 11 subjects meeting inclusion criteria  were selected, resulting in Using medical image analysis software  the temporal bone, dura and sigmoid sinus were segmented . Surface3, XCAT XL, Xoran, MI, USA) were acquired. By means of registration through a mutual information approach (Amira), the original CT was co-registered to the phantom cone-beam CT scan (CBCT) and the relative positions of the screws were added to the segmented temporal bone anatomy.Each of the phantoms were implanted with four titanium reference cone screws  on the mastoid tip, posterior to MacEwen's triangle  and parallel to the temporal line and superior and posterior to the spine of Henle . The fifA study was conducted to investigate the suitability of cortical bone thickness in the mastoid region at the potential sites for screw implantation. Available datasets were divided into four groups of different ages . Between all the aforementioned IGS techniques for CI surgery, we chose to use the most recent guidelines for screw placement provided by a commercially available otological surgical system . These techniques require the placement of five 4 mm thread length fiducial screws around the mastoid area.To investigate bone thickness, measurements were conducted in digital reconstructed models of the temporal bone, and the results analyzed statistically, as well as presented as thickness maps over the region of interest. Similar measurement methodologies in digital reconstructed models have been used in the literature , 16, 17.The dura mater and sigmoid sinus segmentation used the already segmented temporal bone, in a way that only the medial (interior) surface was segmented. The interior surface of the bone also contains channels from nerves and vessels, which were removed manually from the three-dimensional mesh. The fiducial screws were also segmented, and the axes were manually determined using the tip and screw head. All the data was exported as STL files providing a 3D representation.The available cortical bone thickness in the temporal bone region, the CLT, was measured in the surface mesh data from the segmented temporal bones with a custom-developed script in MATLAB . Thickness was calculated as the minimal distances from grid points projected onto the surface of the temporal bone to the nearest opposite surface. These opposite surfaces can be either an air cell or the interior surface of the temporal bone.The distance from the surface of the temporal bone to either dura mater or the venous sinuses, the DDVS, was calculated as the minimal distances from projected grid points onto the surface of the temporal bone to a manually segmented dura and sigmoid sinus surface mesh.The measurements of the distance of the fiducial tip to the dura or sigmoid sinus were carried out by first determining the screws head and tip points. Then, either the minimal distance was measured from the screw tips to the dura mesh, or how much the tip lies beyond the dura mesh along the axis of the screw.Without prior knowledge the screws can easily be placed in areas where the distance to the dura or the venous sinuses is short . In factth to the 95th percentile), depending on the location.The results show a great anatomical variance between the 11 subjects, even unrelated to age. A result overview is displayed in The mastoid region and petrous pyramid can be clearly seen due to the long distance to the dura and venous sinuses (th to the 95th percentile) in the region of interest for screw placement in all four age groups. In all age groups there are areas that are thinner than 0.7 mm, and areas that are thicker than 3.3 mm  create an additional obstacle. Causing the more superior screw to be often localized in the middle fossa region. Furthermore, to keep accuracy, a certain distance between the screws must be respected. Creating a smaller area for middle ear access trajectory placement in IGS surgery, while obeying the limits posed by the facial recess and cochlear angles on this age group. Although the mastoid bone itself present adequate thickness for the screws , the surth to the 95th percentile) in the region of interest for screw placement across all subjects. Even if only the thickest regions  are considered, the thickness values are still in a similar range. Hence, it is likely that a thin cortical layer is encountered during screw placement, and the screws could become loose during surgery, leading to navigational errors and potentially dangerous situations. The results of the distance to dura mater or the venous sinuses are mostly 1.1–9.3 mm with a median of 3.3 mm. Thus, the screw placement with the thread lengths of the current IGS systems (i.e. around 4 mm) run into the risk of penetrating the dura mater or venous sinuses.The results show thin cortical layers in this pediatric population, which are mostly 0.6–3.2 mm thick . Especially in the data for the cortical layer, there are zero thickness values. These values stem mainly from the cranial suture lines, where the cortical layer thickness can be understood to be zero on the map. Although our sample size is small, some clinical considerations can be drawn regarding expected complications. Meningitis, fistula, thrombophlebitis, subdural empyema, otogenic suppurative thrombophlebitis, brain abscess and CSF  leakage can be expected from damage to the dura. Venous air embolism, thrombosis, infarction, thrombophlebitis and death can happen if the sigmoid or the transverse sinuses (at risk with the reference screw positioning) are damaged. Although these complications are rare in daily CI surgical procedures, but with the introduction of new technology and its new requirements, care must be taken to avoid them.This study demonstrated potential damage to the dura and venous sinuses in all screw positions for the pediatric population with the currently used fiducial screws and screw placement guidelines. The measurements taken show thin cortical layers, and short distances from the temporal bone surface to the delicate anatomy underneath.Due to the thin cortical layer and distance to vital anatomical structures , the current fiducial screws and the screw placement guidelines might pose a challenge for safety and efficacy in image-guided surgery for patients younger than 48 months old. Therefore, an adaptation of current fiducial screws and/or their placement is necessary. Additionally, the use of image-guided technology (e.g. with navigated surface matching), or technologies such as ultrasound for screw placement could potentially assist in increasing safety and efficacy of the procedure.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.GO'T and JH created the study design. SW and MC reviewed the study design. MC provided the data used in the study. JH and FM developed the software for the analysis of the virtual surface meshes of the anatomy. GO'T, FM, and JH conducted the phantom study on the additively manufactured models. GO'T and JH analyzed the collected data and wrote the manuscript. All authors reviewed the manuscript and approved the submitted version.This work was supported by the Swiss National Science Foundation SNF (Project 176007).SW is cofounder, shareholder, and chief executive officer of CASCINATION AG, Bern, Switzerland, that commercializes the robotic cochlear implantation technology. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Takifugu flavidus. The T. flavidus hydrolysates (TFHs) with the maximum degree of hydrolysis (DH) and angiotensin-I-converting enzyme (ACE)-inhibitory activity were selected and then ultra-filtered to obtain fractions with components of different molecular weights (MWs) . The components with MWs < 1 kDa showed the strongest ACE-inhibitory activity with a half-maximal inhibitory concentration (IC50) of 0.58 mg/mL. Purification and identification using semi-preparative liquid chromatography, Sephadex G-15 gel chromatography, RP-HPLC, and LC–MS/MS yielded one new potential ACE-inhibitory peptide, PPLLFAAL . Molecular docking and molecular dynamics simulations indicated that the peptides should bind well to ACE and interact with amino acid residues and the zinc ion at the ACE active site. Furthermore, a short-term assay of antihypertensive activity in spontaneously hypertensive rats (SHRs) revealed that PPLLFAAL could significantly decrease the systolic blood pressure (SBP) and diastolic blood pressure (DBP) of SHRs after intravenous administration. These results suggested that PPLLFAAL may have potential applications in functional foods or pharmaceuticals as an antihypertensive agent.Alcalase, neutral protease, and pepsin were used to hydrolyze the skin of  Hypertension, which is a common and serious chronic medical condition, is a major risk factor for developing cardiovascular diseases . Hyperte50 values of 1.3 and 2.8 μmol·L−1, respectively  (pH 8.3) with 0.3 M sodium chloride. Then, 50 µL of the sample solution and 150 µL of the substrate HHL were added to a centrifuge tube, mixed, and incubated at 37 °C for 5 min. The ACE solution (50 mU/mL) was added to start the reaction, and the sample was then incubated at 37 °C for 45 min. The reaction was terminated by adding 250 μL of 1 M HCl. The absorbance of the mixture was measured at 220 nm using an RP-HPLC system  on a SunFire C18 column (4.6 × 250 mm). The mobile phase comprised distilled water/acetonitrile  with a flow rate of 1 mL/min. External standard hippuric acid (HA) was used to calculate the concentration of HA. Distilled water was used as a control and the ACE-inhibitory activity (%) was calculated according to Equation (1): S is the peak area of hippuric acid with distilled water and T0 is the peak area with the sample.The ACE inhibition was measured using a modified spectrophotometric technique according to the method of Jianpeng Li et al. . ACE and50 value was defined as the peptide concentration of a sample that inhibited 50% of the ACE activity under the assay conditions.The IC18, 3 μm, 100 Å). Then, 2% ACN  and 80%ACN  were used as mobile phases A and B, respectively. Elutions were performed with a gradient of 6–95% B at a flow rate of 300 nL⋅min−1. The raw mass spectrometry test file was searched using PEAKS Studio software for the corresponding database.The separated peptides, obtained via the RP-HPLC, with the highest ACE-inhibitory activity were analyzed with a Q Exactive mass spectrometer . The parameters that were adopted for the instrument and methods were described by Tu et al. The sample was desalted and loaded onto a chromatographic column  using a solid phase method. The purity of the peptide was 98%, which was verified by HPLC, and the sequence of the synthesized peptide was determined using HPLC–MS/MS. Synthetic PPLLFAAL was used for the validations of the ACE-inhibitory activity in vitro and in vivo and the determination of the ICT. flavidus peptides were tested using the method of Lin et al. [y-axis) versus the reciprocal of the substrate concentration (x-axis). The Vmax and Km were calculated, respectively, as shown in the y- and x-axis intercepts of the primary plot.The kinetic inhibition models of the n et al. . Brieflyhttps://www.rcsb.org/structure/1O8A, 20 October 2021). Water molecules were removed and the zinc ion was retained in the ACE model [Molecular docking was performed to predict the conformation between ACE and PPLLFAAL using Discovery Studio 2019 software . The crystal structure of ACE (PDB ID: 1O8A) was downloaded from the Protein Data Bank  with the parameters set according to Jiang et al. [The initial coordinates of the ACE–PPLLFAAL complexes for molecular dynamics (MD) simulations were chosen using the criterion of the lowest energy of the complex combined with visual inspection . The MD g et al. . A subseAnimals were obtained from Vital River Laboratory Animal Technology , who also performed the blood pressure measurements in spontaneously hypertensive rats (SHRs) with a tail systolic blood pressure (SBP) of over 180 mmHg. Animals were fed a normal diet, and tap water was freely available. SHRs  were raised at room temperature (25 °C) under a 12 h light/dark cycle. The antihypertensive effect of PPLLFAAL was evaluated by measuring changes in the SBP and DBP after intravenous injection with a single dose of 5 mg/kg body weight. Captopril (5 mg/kg) was used as a positive control, and the negative control group was injected with an equal volume of saline. The SBP and DBP of the rats were measured using the tail-cuff method before administration and at 2, 4, 6, 12, and 24 h post-administration, using a BP-98A blood pressure monitor  after heating the SHRs at 37 °C for 30 min in a warming chamber. All the rats received humane care conforming to the National Institutes of Health Guide for Care and Use of Laboratory Animals. This study was approved by the Ethics Committee of Guangdong medical laboratory animal center (No. 20211001).Statistical analysis was performed using SPSS 19.0 software . Experimental data are presented as means ± standard deviations (SD) and were statistically evaluated using one-way analysis of variance (ANOVA) with Duncan’s test. Origin 9.0  software was used to process the data.T. flavidus were used to prepare potential ACE-inhibitory peptides. The TFHs that were prepared using different proteases displayed diverse ACE-inhibitory activities in vitro. The highest ACE-inhibitory activities were observed in the TFH that were prepared with alcalase. The components that were obtained using its ultrafiltration showed that the low-MW peptides (<1 kDa) conferred the strongest ACE-inhibitory activity. One novel potential ACE-inhibitory peptide (PPLLFAAL) was isolated from the peptide fraction with MW < 1 kDa using semi-preparative HPLC, a Sephadex G-15, and RP-HPLC and identified using LC–MS/MS. PPLLFAAL showed high ACE-inhibitory activity with an IC50 value of 28 μmol·L−1 and appeared to be a non-competitive inhibitor. The molecular docking and molecular dynamics simulation results indicated that the peptides could firmly bind to ACE at the active site, as well as to zinc ions. Our results indicated that PPLLFAAL exhibited effective ACE-inhibitory activity in vitro, which makes it a potential candidate for the development of functional foods or anti-hypertensive drugs in the future.In this study, the skins of


Bacillus megaterium 1259 (BM1259) on growth performance, nutrient digestibility, rumen fermentation, and blood biochemical parameters in Holstein bull calves. The results demonstrated that the addition of BM1259 to the diets can significantly improve the growth performance and elevate the apparent digestibility of crude protein and neutral detergent fiber. Moreover, supplementation with BM1259 ameliorated rumen fermentation and reduced the emission of both ammoniacal nitrogen and sulfuretted hydrogen in feces and urine. In addition, adding 12 g/head/day of BM1259 had no adverse effect on blood biochemical parameters and the health status of Holstein bull calves. This study demonstrates that BM1259 can be applied as a potential microecologics to improve production performance and nitrogen utilization in Holstein bull calves.This study was conducted to investigate the effects of dietary supplementation with Bacillus megaterium is an ideal microecologics in the feed industry. BM1259 was already isolated from chicken manure and the whole-genome sequencing was also analyzed in our previous study. However, few studies concentrated on dietary supplementation with BM1259 in young ruminants and especially its effect on Holstein bull calves have not been reported. Hence, this experiment was conducted with the aim to evaluate the effects of BM1259 on growth performance, nutrient digestibility, rumen fermentation, and blood biochemical parameters in Holstein bull calves. Twenty-four healthy Holstein bull calves with the initial age of 90 days old and a similar body weight (115 ± 6.5 kg) were selected and randomly allocated into two groups with one Holstein bull calf in each pen (2.5 m × 2.2 m). Holstein bull calves in the control group (COG) were fed a basal total mixed ration (TMR), while experimental treatments (BMG) were fed with the TMR diet supplemented with 12 g/head/day of BM1259 powder (1 × 1010 cfu/g) separately. Results showed that (1) the average daily gain and dry matter intake of the BMG were significantly higher than those of the COG (p < 0.01), increased by 12.5% and 8.79%, respectively, during the 4–8 weeks after the addition of 12 g/head/day of BM1259; from 0 to 8 weeks, ADG (p < 0.05) and DMI (p < 0.05) in the BMG were significantly higher than those in the COG, increased by 14.9% and 6.04%, respectively. (2) At the end of the fourth week, the apparent digestibility of crude protein and neutral detergent fiber in the BMG was significantly higher than that in the COG (p < 0.05), increased by 5.97% and 6.70%, respectively; at the end of the eighth week, the apparent digestibility of crude protein and neutral detergent fiber was significantly higher than that of the COG (p < 0.01), increased by 5.88% and 10.26%, respectively. (3) At the end of the eighth week, the rumen fluid pH (p < 0.05), MCP (p < 0.05), and acetate (p < 0.05) in the BMG were significantly higher than those in the COG, increased by 9.03%, 19.68%, and 12.74%, respectively; at the end of the fourth and eighth week, NH3-N concentration in the BMG was significantly lower than that in the COG, with a decrease of 21.81% and 16.40%, respectively. (4) At the end of the fourth (p < 0.05) and eighth week (p < 0.05), the glutamate content of the rumen fluid of the Holstein bull calves in the BMG was significantly higher than that in the COG, increased by 13.21% and 14.32%, respectively; at the end of the fourth week, the contents of glutamate in the serum (p < 0.05), urine (p < 0.05), and feces (p < 0.05) of the Holstein bull calves in the BMG were significantly lower than those in the COG, decreased by 25.76%, 33.87%, and 9.23%, respectively; at the end of the eighth week, the contents of glutamate in the serum, urine, and feces of the Holstein bull calves in the BMG were significantly lower than those in the COG (p < 0.01), decreased by 26.69%, 27.94%, and 11.11%, respectively. (5) After adding 12 g/head/day of BM1259, the urine ammonia–nitrogen content of the BMG was extremely significantly lower than that of the COG at the end of the fourth and eighth week (p < 0.01), decreased by 54.60% and 40.31%, respectively. (6) After adding 12 g/head/day of BM1259, there was no significant effect on the level of blood biochemical parameters of the Holstein bull calves. This study demonstrates that BM1259 can be applied as a potential microecologics to improve growth performance, nutrient digestibility, rumen fermentation, and nitrogen utilization in Holstein bull calves. Bacillus megatherium as probiotics has become a research hotspot due to its unique characteristics, such as its strong resistance to stress, high temperature resistance, and easy storage [3, and improve the air environment of pig pens to a certain extent [In recent years, research and application of microecologics has attracted extensive attention from scholars in domestic fields and abroad. As a new kind of green and environmental protection feed additive, microecologics have been widely used in animal husbandry due to its green, safe, and residue-free function, which can be used to replace antibiotics, to promote the healthy growth of animals, and improve the environment ,2. At pr storage ,5. BM125 storage ,7,8,9,10 storage . Huo et n extent . Yu et an extent .However, few studies concentrated on dietary supplementation with BM1259 in young ruminants and especially the study and application of BM1259 on Holstein bull calves is still unknown. Hence, this work was conducted with the aim to evaluate the effects of BM1259 on growth performance, nutrient digestibility, rumen fermentation, and blood biochemical parameters in Holstein bull calves. Together, these data could represent a useful basis for future applications of BM1259 as ruminant microecologics.All Holstein bull calves used in this study were strictly cared in accordance with the principles of Yangzhou University, the Institutional Animal Care, and the Use Committee (SYXK (Su) IACUC 2016-0019).10 cfu/g) separately. TMR were adjusted as isonitrogenous and isoenergetic diets , from April to July 2020. Twenty-four healthy Holstein bull calves of 90 days old with similar body weights (115 ± 6.5 kg) were selected and randomly allocated into two groups with 12 replicates in each group and 1 Holstein bull calf in each pen (2.5 m × 2.2 m). Holstein bull calves in the control group were fed a basal total mixed ration (TMR) without BM1259 and the BMG were fed with the TMR supplemented with 12 g/head/day (recommended dose for the preliminary experiment) BM1259 powder  were mixed and stored in a refrigerator at −20 °C for the determination of the apparent digestibility of nutrients. Feed and feces were dried and ground using a pulverizer for the subsequent determination of dry matter, crude protein, neutral detergent fiber, acid detergent fiber, ether extract, and crude ash content. For both TMR and fecal samples, DM was detected using AOAC method 930.15; EE was measured using AOAC method 920.85; CP was tested using the method described by Kjeldahl with an azotometer ; NDF and ADF were measured using the methods described by Van Soest and a fiber analyzer ; and ash content was measured according to AOAC method 938.08 [At the 7th day, the digestion and metabolism test was conducted at the end of the formal period, in which the first two days were the adaptation period and the last five days were sampled. Fecal samples were collected 3 times a day with an interval of 8 h for each time. After the collection, 10% Hg, after which supernatants were removed to centrifuge tubes containing 25% metaphosphoric acid and then stored at −20 °C for the analysis of volatile fatty acid concentration using a GC Ultra gas chromatograph  as described by Guo et al. [On the last day of the experiment, the rumen fluid (250 mL) was collected through an oral collector and filtered with a four-layer sterilized gauze two hours after the morning feeding. The pH  value was determined immediately. Then, 10 mL aliquots were added with 0.1 mL of 6 mol/L HCl and stored at −20 °C for the determination of the ammonia–nitrogen concentration according to the method of Weatherburn . Anothero et al. . The yieo et al. .g at 4 °C for 10 min to collect serum, which was then frozen at −20 °C for further analysis. Serum samples were analyzed for the quantification of the total protein (TP), albumin (ALB), globulin (GLOB), aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and uric acid (UA) according to methods described in previous studies [Jugular blood samples of approximately 10 mL were collected from each Holstein bull calf in vacuum plasma tubes at 3 h after the morning feeding on the third day of weeks 4 and 8 . The col3-N in the urine was determined by method of phenol–sodium hypochlorite colorimetry [The ITC pre-column derivatization method was used to determine glutamate. The specific method was in reference to the research report of J.L. Zhang et al. . The conorimetry .The numerical data were analyzed by one-way analysis of variance (ANOVA) with orthogonal data and the significance of differences between the groups was determined by Tukey’s post-hoc test, assuming significance levels of 0.05 and 0.01. The tables present the means and standard error of the mean (SEM). The calculations were made using SAS 9.4 software .p < 0.01), increased by 12.5% and 8.79%, respectively, during the 4–8 weeks after the addition of 12 g/head/day of BM1259; from 0 to 8 weeks, ADG (p < 0.05) and DMI (p < 0.05) in the BMG were significantly higher than those in the control group, increased by 14.9% and 6.04%, respectively. The feed conversion rate, withers height, body length, and chest girth were higher in the BMG but the differences were not significant (p > 0.05).As shown in p < 0.05), increased by 5.97% and 6.70%, respectively, after adding 12 g/head/day of BM1259. At the end of the eight week, the apparent digestibility of CP and NDF was significantly higher than that of the control group (p < 0.01), increased by 5.88% and 10.26%, respectively. Compared with the COG, the apparent digestibility of ADF and EE in the BMG was higher but there was no significant difference (p > 0.05).As is presented in p < 0.05), microbial protein (p < 0.05), and acetate (p < 0.05) in the BMG were significantly higher than those in the COG, increased by 9.03%, 19.68%, and 12.74%, respectively, after adding 12 g/head/day of BM1259. At the end of the fourth and eighth week, NH3-N concentration in the BMG was significantly lower than that in the COG, with a decrease of 21.81% and 16.40%, respectively. The content of sulfuretted hydrogen, total volatile fatty acids, propionate, butyrate, and acetate:propionate showed no significant differences between the COG and BMG at each stage of experiment (p > 0.05).As is shown in p < 0.05) and eighth week (p < 0.05), the glutamate content of the rumen fluid in the BMG was significantly higher than that in the COG, increased by 13.21% and 14.32%, respectively; at the end of the fourth week, the contents of glutamate in the serum (p < 0.05), urine (p < 0.05), and feces (p < 0.05) of the Holstein bull calves in the BMG were significantly lower than those in the COG, decreased by 25.76%, 33.87%, and 9.23%, respectively; at the end of the eighth week, the contents of glutamate in the serum, urine, and feces in the BMG were significantly lower than those in the COG (p < 0.01), decreased by 26.69%, 27.94%, and 11.11%, respectively.In this study, in order to explore the effects of adding 12 g/head/day of BM1259 on the glutamate content, the glutamate content was measured in the rumen fluid, serum, feces, and urine of the Holstein bull calves in the BMG and COG . Resultsp < 0.01), decreased by 54.60% and 40.31%, respectively. H2S content in the urine of the BMG decreased overall but there was no significant difference between the two groups (p > 0.05).As is shown in As is presented in 10 cfu/g bacillus subtilis improved the milk yield of Holstein cows by 11.61% [It was found that adding bacillus to the diet of Holstein bull calves could improve the performance of Holstein bull calves. Youssef et al. showed that feeding bacillus subtilis BSN fermentation significantly improved the feed conversion rate and milk yield but had no significant effects on the milk fat percentage and milk protein percentage . Hu et ay 11.61% . Deng ety 11.61% . It was y 11.61% . In this3-N is the main nitrogen source (about 20–100%) in the process of rumen microbial flora growth and reproduction of ruminants. The concentration of NH3-N should be moderate in order to facilitate microbial growth and promote microbial MCP generation. Too high or too low concentration is not suitable for microbial growth [3-N concentration was significantly decreased after the addition of 12 g/head/day of BM1259, which was in the range of 8.46–12.03 mg/dL, although lower than that of the 10 g/head/day of BM1259 (15.95–25.32 mg/dL) in Yu [3-N concentration. VFA is one of the energy substances used to maintain the normal life activities of ruminants, which can provide more than 70% of the digestive energy and participate in different metabolisms in the body [3-N contents were significantly increased [Saccharofermentans in our previous study [3-N and H2S in the urine not only affected the health of the livestock and humans, but also reflected the nitrogen utilization capacity of animals [3-N in the urine of Holstein bull calves significantly decreased and the content of H2S in the excreta was significantly reduced.The rumen pH value is one of the evaluation parameters of rumen microbial growth, metabolism balance, and fermentation degree, which is mainly affected by the amount of saliva buffer secretion of ruminants . The ruml growth ,32,33. IL) in Yu . Howeverthe body . Diana encreased . Ding etncreased . However animals . In thisThe level of the blood biochemical parameters can reflect the status of nutrient metabolism and the acid-base balance of the animal body to a certain extent. Meanwhile, the level of serum albumin and total protein is an indicator for measuring the health status of the animal ,40,41. I3-N and H2S in the feces and urine, indicating a possible amelioration in the rumen fermentation and nitrogen utilization. In addition, adding 12 g/head/day of BM1259 had no adverse effect on blood biochemical parameters and health status. However, further research is necessary to uncover the mechanism by which BM1259 improves nitrogen utilization in Holstein bull calves.In summary, the addition of BM1259 to the diets of Holstein bull calves can significantly improve the growth performance and elevate the apparent digestibility of CP and NDF. Moreover, supplementation with BM1259 increased C2 and C2:C3 in the rumen, and reduced emission of NH


Clinicians should be aware that biologic agents, which include polysorbates, can cause delayed local skin reactions at a previous injection site. This case highlights that biologic agents, which include polysorbates, can cause delayed local skin reactions at a previous injection site. A 30‐year‐old woman presented with severe asthma, poorly controlled despite treatment with fluticasone furoate 200 μg, umeclidinium bromide 62.5 μg, vilanterol 25 μg and frequent prednisolone burst administration; we initiated treatment with dupilumab. The respiratory symptoms improved dramatically with no adverse events. Three days after the second dose of dupilumab, a skin rash appeared at the site of the first dose. The rash gradually worsened; however, it resolved spontaneously without treatment. Twelve days after the second dose, a similar rash was observed at the site of the second dose, which also resolved spontaneously Figure . DupilumNone declared.Toshiyuki Sumi: conception and design. Toshiyuki Sumi, Yuta Nagahisa, Keigo Matsuura, Motoki Sekikawa and Yuichi Yamada: acquisition of clinical, radiological and pathological data, and drafting the article. Hisashi Nakata and Hirofumi Chiba: revision of intellectual content and final approval of the version to be published. All authors read and approved the final manuscript.The authors declare that appropriate written informed consent was obtained for publication of this case report and accompanying images.