In a human cadaver study, this material, when applied with wire cerclage, was able to significantly reduce sternal instability. However, the material has yet to be tested in pertinent animal models. Here, after a series of physical and mechanical tests to confirm suitability of the experimental material for implantation, three samples of GPC+ were implanted in either the tibia or femur of three different rabbits, alongside sham defects, for two different time modalities. A further seven samples of GPC+ and one poly(methyl methacrylate) control (PMMA) were implanted in either the tibia or femur of two different sheep. The sheep containing the PMMA was sacrificed at 8 weeks and the other at 16 weeks, to evaluate time dependent biological response. Upon sacrifice, microCT images were acquired and histology slides prepared for analysis. All three GPC+ samples implanted in the rabbit model, for the two time modalities, were characterized by minimal bone resorption along with a mild inflammatory response. Five of the seven GPC+ materials implanted in the sheep model  were associated with mild to moderate immune response, comparable to that observed with PMMA, as well as mild bone resorption. The remaining two GPC + materials (implanted in the sheep model for 16 weeks) exhibited no bone resorption or inflammatory response and appeared to stimulate increased bone density at the implant site. These results suggest that GPC + can be a viable bone adhesive for use in hard tissue applications such as sternal fixation and stabilization.Wire cerclage remains the standard method of care for sternal fixation, following median sternotomy, despite being beset with complications. An emerging treatment option has been to augment the wires with an adhesive. A patented ionomeric glass (mole fraction: SiO Median sternotomy, first introduced in 1953 . Upon co™ bone cement , effectively a mixture of castor oil and calcium carbonate, was investigated under an Investigational Device Exemption (IDE) as an adhesive for augmentation of current wire cerclage techniques for sternal fixation [Poly(methyl methacrylate) (PMMA) has been used as a cement in orthopaedic applications since the 1950s, however it is not suitable for sternal applications due to its lack of chemical adherence to bone . In addifixation . Kryptonfixation . Howeverfixation  and a 50fixation , renderifixation .The authors are developing a bone adhesive for sternal fixation augmentation, based on Glass polyalkenoate cement (GPC) chemistry. First developed by Wilson and Kent in 1971 for dental restorative and luting applications , GPCs co2:0.48, ZnO:0.36, CaO:0.12, SrO:0.04), where the Al component is replaced with zinc (Zn) [Commercial GPCs all contain aluminum (Al) in the glass phase, which is a neurotoxin , 27. To inc (Zn) ; incorpoinc (Zn)  as well inc (Zn) , 31. Strinc (Zn)  and stiminc (Zn) . Zn/Sr-Ginc (Zn)  in a hum2:0.24, GeO2:0.24, ZnO:0.36, CaO:0.16) contained germanium (Ge) [The first in-vivo investigation of an Al-free GPC was reported in 2016 by Pierlot et al., where four GPC samples were implanted in two rabbits . The glaium (Ge) . The tisium (Ge) . In Pierium (Ge) . Rabbit ium (Ge)  when comA sheep model is more suitable than a rabbit model for mimicking human bone, although differences still exist –40. PrevIn this study then, both rabbit and sheep models were used to evaluate the in-vivo response of a GPC (GPC+) formulated from the patented zinc and strontium-containing ionomeric glass . Following a series of physical and mechanical tests to confirm clinical suitability of this batch of GPC + , the bioadhesive was implanted into non-critical defects in a bilateral tibiofemoral rabbit model. In the second study, a comparable bilateral tibiofemoral defect in Texel Cross sheep was employed. The desired response in both models was an absence of bone resorption, minimal fibrous encapsulation, and evidence of healthy bone turnover adjacent to the implant.2:0.48, ZnO:0.36, CaO:0.12, SrO:0.04) was prepared at Ryerson University  using specified mole fractions. Correct amounts of analytical grade reagents  were weighed out, thoroughly mixed in a container, and then transferred to a platinum crucible and melted in a furnace  at 1480 °C for 1.5 h. The glass melt was water quenched at room temperature (23 ± 1 °C) and the resultant frit dried in an incubator (37 °C) for 24 h. The frit was then ball-milled and sieved to 45–63 µm. The resultant glass powder was then annealed for 12 h at 640 °C, having reached the annealing temperature in 3 h. Following annealing, the glass was furnace cooled to room temperature. The formulation of the resultant GPC + was: 3 g of glass, 0.9 ml of DI water and 0.9 g of PAA . The handling and mechanical properties of GPC + have been reported elsewhere [The patented ionomeric glass  of the GPC + samples were recorded at room temperature (23 °C ± 1°C). The working time was measured, using a stopwatch, from the start of mixing and was considered complete once the material began to exhibit elastic properties.The working and setting times (The setting time was measured in accordance with ISO 9917-1:2007 . A mold n = 5) of 6 mm height × 4 mm diameter were created by filling plastic tubular molds with mixed cement. The molds were sandwiched between two stainless steel plates protected by a cellulose acetate sheet, and incubated (37 °C) for 1 h. Samples were then removed from the molds, individually placed in 10 ml of DI water and incubated (37 °C) for 1 day.The compressive strengths of the GPC + samples were measured at room temperature (23 ± 1 °C) in accordance with ISO 9917-1:2007 . Cylindrρ was the maximum load (N) at failure and d the sample diameter (mm).Compressive loads were applied to the samples using an Instron Universal Testing Machine  fitted with a ± 10 kN load cell at a crosshead speed of 1 mm/min. The load cell error was calculated at 0.005% at 50 N to 0.04% at 100 N, within which these test samples fracture. The maximum load reached by each sample was recorded. The compressive strength C  of the GPC + samples were evaluated using the method described by Williams et al. . Cement Determination of Compressive Strength at a crosshead speed of 1 mm/min. The GPC + discs were placed on a 3-ball support, and the load was applied as a point in the middle of the disc. The BFS , and F is the recorded load at failureThe BFS testing was performed using the same Instron Universal Testing Machine  described in Section  BFS Eq.  was calcAll animal procedures were approved by an institutional animal care committee  operating under the guidelines of the National Council on Animal Care. All procedures were carried out using an aseptic technique under general anesthesia with intra- and postoperative analgesia.n = 3, 3.0 ± 0.1 kg) were group housed in a 2 × 5 m pen and conditioned for 1 week prior to the commencement of the study. On day 0, each rabbit was sedated with ketamine (10 mg/kg) and dexmedetomidine (0.01 mg/kg) followed by endotracheal intubation with isoflurane general anesthesia. Hair was removed from the tibial and femoral surgical sites and a skin incision was performed on both hind stifle joints. Blunt dissection was used to clear the subcutaneous tissue. Defects (∅ = 3.5 mm) were made in the left and right distal femur and proximal tibia, allowing for a maximum of four implants per animal. Care was taken to avoid migration into the medullary canal. Sterile saline was used to irrigate the defects, removing osseous drilling particles. One GPC+ cement was implanted in each of the three rabbits (n = 3) in accordance to their allocated defect site. In addition, one sham defect was implanted in two of the three rabbits (n = 2). One rabbit was sacrificed at 8 weeks post implantation (n = 1/3) while the other two rabbits were sacrificed at 12 weeks (n = 2/3) for subsequent microCT  and analysis of undecalcified histology sections.New Zealand White rabbits  were group housed in a 3 × 4 m pen and conditioned for 1 week prior to study commencement. On day 0, both sheep were anesthetized with diazepam (0.3 mg/kg) and ketamine (5.0 mg/kg), followed by endotracheal intubation with isoflurane general anesthesia. Wool was removed from the tibial and femoral surgical sites followed by aseptic skin preparation. Upon incising the skin, blunt dissection was used to clear the subcutaneous tissue. Defects (∅ = 6.5 mm) were made in the left and right distal femur and proximal tibia of each animal. Care was taken to avoid migration into the medullary canal and saline irrigation was used to remove osseous drilling particles from the defects. One PMMA cement  and 7 GPC+ cements (n = 7) were implanted in the two sheep used in this study. One sheep was sacrificed at 8-weeks post implantation, and the other at 16-weeks post implantation, for subsequent microCT and histology analysis. The PMMA control was included as one of the four implants in the sheep sacrificed at 8 weeks.Two Texel cross sheep . GPC+ was prepared by placing the PAA component in a glass mixing dish followed by the addition of the DI water component. These were then mixed using a metal spatula until fully homogenous, at which point the ionomeric glass component was added. The cement was mixed for ~30 s until completion, determined by visual confirmation of consistent, homogenous material. The PMMA samples were mixed in a sterile bowl according to the manufacturer’s specifications. Freshly mixed cements were rolled into a cylinder before being lightly packed into the defect by hand. Any excess adhesive around the defect site was removed before closure, where the opening of the defect site was covered with the periosteum, muscle and fascia, subcutaneous tissue and a bandage.Each implant in the sheep model was imaged at day zero using clinical resolution computed tomography (CT)  for confirmation of appropriate defect fill. Clinical CTs were not recorded for the rabbit model given the smaller implant size rendering the image resolution too low for effective analysis. Following animal sacrifice of both the rabbits and sheep, microCT imaging was performed on a General Electric Medical Systems Locus Explore Scanner at 45-micron isotropic pixel resolution using an 18 min protocol . Image slices were reconstructed into 3D images after calibration using a hydroxyapatite (HA) phantom and subsequent image analysis was performed using MicroView .After microCT imaging, all implants were processed for undecalcified ground section histology analysis by light microscopy . Extraneous tissue not required for analysis was stripped from the samples upon their extraction. The samples were placed on ice prior to processing and fixed in 70% ethanol. They were then were embedded in methyl methacrylate, and sections in the transverse plane to the cylindrical defect were cut with a thickness of ~15 µm. The resulting sections were stained with Sanderson’s Rapid Bone Stain and Van Gieson’s picrofuchsin. Images were digitally captured  using standard brightfield techniques at objective magnifications of ×4, ×10, and ×40. The resulting images were analyzed to assess the response of the tissues to the implanted materials. The color of the various specimen components upon Sanderson’s Rapid Bone Stain and Van Gieson’s staining are presented in Table n = 5) working and setting times of the GPC+ samples were 4:48 (stdev: 0:07) minutes and 34:06 (stdev: 0:22) min, respectively. The handling properties of this specific batch of GPC+ were in line with those reported for the same material previously [The average  presented an excellent bone/cement interface, no evidence of bone resorption and some evidence of bone remodelling. Sample microCT images of a GPC+ implant in a rabbit model at 8 weeks are presented in Fig. There was no apparent difference in tissue response at 8 or 12 weeks  had an intact bone-cement interface but no enhancement of the surrounding bone mineral density Fig. . A contiConsidering an example of the GPC+ implants at the 16-week time point  giving artifice.In two of the four GPC+ samples implanted for 16 weeks some lucency was also visible at the interface; in the other two samples, an excellent tissue response was observed, with no bone resorption. There was little evidence of fibrous encapsulation or an inflammatory response around any of the samples implanted for 16 weeks. As can be seen in the CT scans in Fig. 2+ by GPC+, providing local administration of Sr2+ to the surrounding tissues. There are no examples in the literature of Sr-doped GPCs evaluated in animal trials. However, a review of other Sr-enriched biomaterials indicate that they outperform similar materials free of Sr in bone healing, bioactivity and osteo-integration at the bone cement interface [2+ concentrations. Metals coated with SrO have also been shown to increase bone formation, providing evidence for the positive effect of Sr independent of Ca, Si or Zn release [This increase in bone density seen in Fig. nterface . Furthernterface , HA’s [5nterface  and CaP nterface , have be release . Despite release . The tis2+ <400 µm have been reported to cause cell death [Bone resorption, as it relates to GPCs, can be caused by a number of factors. An excess of ions can potentially disrupt normal cell function. For example, in-vitro concentrations of Znll death . Bone rell death . PAA is ll death . They hyll death , 58. Erbll death . Early vll death –62. PE wll death . It therUpon histological analysis of the rabbits in this study (representative images included in Fig. The moderate inflammatory response coupled with the evidence of bone resorption upon GPC+ implantation into the sheep model was only evident after 8 weeks and not after 16 weeks. Sheep bone turns over much more slowly than both rabbit and human bone . FlautreGPCs have potential advantages over PMMA in sternal fixation and stabilization as they exhibit greater radiopacity , have thTo ensure the success of GPC+ in sternal fixation and stabilization, the appropriate ion release concentrations needed to achieve a healthy tissue response and ensure consistent release across samples will be identified. Further, steps should be taken to improve the homogeneity of the material, ideally by the use of a delivery kit that can ensure reproducibility in mixing and application, comparable to that used for the storage, mixing and delivery of commercial PMMA bone cements. In addition, improving the cohesive strength of GPC+ using higher molecular weight PAA should be investigated.A sheep sternotomy trial where the sternum is dissected and then fixated using GPC+ augmented techniques has been designed and will now be conducted to ensure the effectiveness of the cement in this novel application.


Alpha, Beta, Gamma, Delta, and Lambda increase the capacity of infection and transmission of SARS‐CoV‐2, which might disable the in‐used therapies and vaccines. The COVID‐19 has now put an enormous strain on health care system all over the world. Therefore, the development of animal models that can capture characteristics and immune responses observed in COVID‐19 patients is urgently needed. Appropriate models could accelerate the testing of therapeutic drugs and vaccines against SARS‐CoV‐2. In this review, we aim to summarize the current animal models for SARS‐CoV‐2 infection, including mice, hamsters, nonhuman primates, and ferrets, and discuss the details of transmission, pathology, and immunology induced by SARS‐CoV‐2 in these animal models. We hope this could throw light to the increased usefulness in fundamental studies of COVID‐19 and the preclinical analysis of vaccines and therapeutic agents.Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is the etiology of coronavirus disease 2019 (COVID‐19) pandemic. Current variants including  Animal models that recapitulate characteristics and immune responses observed in COVID‐19 patients are urgently needed. These animal models such as mouse, hamster, nonhuman primate, and ferret, have provided robust platforms for studying the transmission, pathogenesis, and immunology induced by SARS‐CoV‐2, and for evaluating the immunomodulatory and antiviral drugs and vaccines against COVID‐19. Clinical features are always initially characterized by fever, fatigue, dry cough, diarrhea, and chest pain.Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) with zoonotic origin emerges in 2019 and causes mild to severe respiratory illness in humans.Testing medical countermeasures, including therapeutic agents and vaccines that reduce COVID‐19 morbidity and mortality, becomes an emergency scientific event.2Mus musculus) model has been well used for many viral investigations, such as Middle East respiratory syndrome coronavirus (MERS‐CoV)Mouse (2.1Generation of human ACE2 (hACE2) receptor expressing mouse is considered as a forthright strategy for SARS‐CoV‐2 infection in mice. To date, several methods including introducing hACE2 into mice via adenoviral/adenoviral‐associated vectors, expressing hACE2 by mouse ACE2 promoter or heterologous gene promoters and clustered regularly interspaced short palindromic repeats (CRISPR) systems, have been used to develop genetically modified mice. Notably, the varied expression of hACE2 among different organs in mice has led to the mild to severe disease in these mouse models, adding further complexity as factor of investigation of SARS‐CoV‐2‐induced pathogenesis and evaluation of therapeutic agents and vaccines.The first SARS‐CoV‐2 infected transgenic mice are Institute of Cancer Research (ICR) background and express hACE2 under the control of mouse ACE2 promoter.K18‐hACE2 transgenic mice, in which hACE2 expression is driven by the human epithelial cell cytokeratin‐18 (K18) promoter,Another genetically modified mice, that is, HFH4‐hACE2 mice, express hACE2 under the control of a lung ciliated epithelial cell‐specific HFH4/FOXJ1 promoter.4 PFU of SARS‐CoV‐2 induces viral replication in lung and intestine tissues, and causes the interstitial pneumonia characterized by alveolar septa thickening and interstitial infiltrates.The CAG‐hACE2 transgenic mice have ever been used for investigating the SARS‐CoV infection.Finally, instead of permanent genetic modification, adenovirus or adeno‐associated virus that expresses hACE2, that is, Ad5‐hACE2 or adeno‐associated virus (AAV)‐hACE2 which can transduce a large proportion of pulmonary epithelial cells, is introduced to mice to sensitize the respiratory tract for SARS‐CoV‐2 replication. The wide‐type mice intranasally2.2Virus can be adapted to infect wide‐type mice by serial passaging, which may more closely resemble the natural host‐pathogen interactions in mice. This strategy is successful because of a swarm of related viral quasi species in the populations of RNA viruses. The rare viruses in the swarm that contain mutations in the key protein that increase their binding affinity to entering receptors of mice are expected to be selected, owing to their higher levels of replication in lung tissues. This strategy has been successfully applied to investigate the SARS‐CoVP30,Recently, evolution in vivo via serial passage of SARS‐CoV‐2 in the mouse lung tissue generates highly infective virus, that is, MASCp6, with increased virulence in both young and aged mice.P0 (precursor of rSARS2‐N501YP30).In addition, some researchers have directly developed mouse‐adapted virus using reverse genetics, including SARS‐CoV‐2 MA (precursor of SARS‐CoV‐2 MA10)Some trials including potential medical countermeasures against SARS‐CoV‐2, have been in progress or done using the mouse model with mouse‐adapted virus.2.3Several studies have applied the immunodeficient mouse to investigate the pathogenesis in human‐specific viral infections, such as SARS‐CoV.The high mortality of SARS‐CoV‐2 infection closely associates with the disease progression to a severe COVID‐19‐induced ARDS.Chronic diseases, such as obesity, cardiovascular disease, hypertension, and diabetes, are closely correlated to the severe COVID‐19 disease and advance the SARS‐COV‐2‐induced mortality.Although appropriate mouse models for studying SARS‐CoV‐2 transmission have not been reported, the emerged variants might impart cross‐species transmission of SARS‐CoV‐2 to naïve mice by enhancing receptor binding, such as the currently reported N501Y, E484K, and K417N variants.In summary, several mouse models have been used or under development for investigating the pathogenesis of SARS‐CoV‐2 infections. These mouse models develop mild to lethal diseases; however, none of these models can replicate all features observed in COVID‐19 patients, especially the unusual aspects such as the pulmonary vascular disease. Furthermore, more efforts on using mouse models need to be made to demonstrate the roles of risk‐related factors, such as cardiovascular disease and obesity in SARS‐CoV‐2 infection.3Mesocricetus auratus) are small mammals that have been used for decades to study the infections of human respiratory viruses, including influenza virus, SARS‐CoV,4 TCID50 (median tissue culture infective dose) of SARS‐CoV‐2 induces approximately 15% of weight loss and viral replication in upper and lower respiratory tracts and organs of lung, heart, brain, spleen, and liver of hamsters.5 TCID50) induces a severe pulmonary injury in hamsters that shares many features with COVID‐19 patients, including bilateral and multilobular ground glass opacity, severe weight loss, and increased mortality.5 PFU) and develop no sign of disease compared to the equal viral quantity of intranasal inoculation.Syrian golden hamsters  between humans. To date, several studies have launched this animal model for the efforts to prevent the SARS‐CoV‐2 transmission. For example, SARS‐CoV‐2 can be transmitted efficiently from infected hamsters to naive hamsters via aerosols or by direct contact; using surgical mask could well reduce the infected proportion of hamsters (from 66.7% to 16.7%),Expression levels of chemokines and cytokines, which link to COVID‐19 in humans including IL‐6, IL‐10, IFN‐λ, IFN‐γ, CXCL‐10, MX‐2, and TNF‐α, and so forth, are robustly increased in hamsters on 4 dpi, and are gradually resolved on 7 dpi.4Macaca mulatta), African green monkeys (Chlorocebus aethiops), and cynomolgus macaques (Macaca fascicularis), for investigating the pathogenesis involved in SARS‐CoV‐2 infection and also for developing the medical countermeasures  are mammals that have been well used as models to study the infection and transmission of human respiratory viruses, including influenza virus, SARS‐CoV.Ferrets .Several studies have demonstrated the transmission from human to domestic cats (6.2Neovison vison) are mammals that present highly susceptive to the infection of SARS‐CoVThe mink (6.3Rousettus aegyptiacus) with SARS‐CoV‐2 induces viral replication in upper and lower respiratory tract including nasal epithelium, trachea, and in lung and lung‐associated lymphatic tissue with no clinical feature.The bats are mammals that have been shown as natural reservoirs for a variety of RNA viruses, including SARS‐CoV and MERS‐CoV.6.4Tupaia belangeri chinensis) are mammals that are genetically closer to humans than to rodents, and have been used as models for viral infections, including hepatitis viruses ,The Chinese tree shrews (6.5Canis lupus familiaris),Sus scrofa domesticus),Gallus gallus domesticus),Anatinae),in silico dock of affinities to SARS‐CoV‐2 for pigs, dogs,Several studies have shown that animal models including dogs  transmission, such as animal‐to‐animal or animal‐to‐human, and the evolution should be warranted.To data, all reported animal models cannot completely recapitulate the characters, especially the immunology changes in COVID‐19 patients. It may be beneficial for investigators to apply appropriate options of animal models depending on the scientific goals. Although several models such as mouse and hamster, show age and gender‐related skewed distribution when infected with SARS‐CoV‐2, other risk factors of chronic disease‐related models that correlate to the severe COVID‐19 disease and SARS‐CoV‐2‐induced mortality, including hypertension, diabetes, obesity, and cardiovascular disease, should be further tailored to explore the advanced pathogenesis and the prevention against SARS‐COV‐2. Taken together, animal models provide a fundamental platform to investigate the transmission, pathogenesis, and countermeasures against SARS‐CoV‐2.All authors declare no competing interests.Z.B., S.L. and X.P. conceived the manuscript. Z.B. collected references and wrote the manuscript. H.Q. and J.Y. designed the figures. S.L. and X.P. revised the manuscript. All authors commented on the manuscript.Not applicable.


Sus scrofa meridionalis) population inhabiting the island of Sardinia , and test for the existence of Isolation‐by‐Distance (IBD), Isolation‐by‐Barrier (IBB), and Isolation‐by‐Resistance (IBR). A total of 393 Sardinian wild boar samples were analyzed using a set of 16 microsatellite loci. Signals of genetic introgression from introduced non‐native wild boars or from domestic pigs were revealed by a Bayesian cluster analysis including 250 reference individuals belonging to European wild populations and domestic breeds. After removal of introgressed individuals, genetic structure in the population was investigated by different statistical approaches, supporting a partition into five discrete subpopulations, corresponding to five geographic areas on the island: north‐west (NW), central west (CW), south‐west (SW), north‐central east (NCE), and south‐east (SE). To test the IBD, IBB, and IBR hypotheses, we optimized resistance surfaces using genetic algorithms and linear mixed‐effects models with a maximum likelihood population effects parameterization. Landscape genetics analyses revealed that genetic discontinuities between subpopulations can be explained by landscape elements, suggesting that main roads, urban settings, and intensively cultivated areas are hampering gene flow  within the Sardinian wild boar population. Our results reveal how human‐transformed landscapes can affect genetic connectivity even in a large‐sized and highly mobile mammal such as the wild boar, and provide crucial information to manage the spread of pathogens, including the African Swine Fever virus, endemic in Sardinia.Patterns of genetic differentiation within and among animal populations might vary due to the simple effect of distance or landscape features hindering gene flow. An assessment of how landscape connectivity affects gene flow can help guide management, especially in fragmented landscapes. Our objective was to analyze population genetic structure and landscape genetics of the native wild boar  population inhabiting the island of Sardinia . A sharp genetic structure into different subpopulations was observed, after removing the confounding effect of hybridization. Isolation by resistance due to main roads and land cover explained the observed genetic discontinuities.Genetic structure and landscape genetics were investigated in the wild boar ( The impact of anthropogenic barriers and habitat fragmentation on gene flow was investigated in different wild ungulates , based on both morphological and genetic evidence, as it is characterized by a relevant genetic differentiation, due to its long‐lasting isolation, reported by Scandura et al. . Sardinian wild boar, a dwarf form of the European wild boar, is believed to have originated during the Neolithic following a human introduction from the mainland , Isolation‐by‐Barrier (IBB), and Isolation‐by‐Resistance (IBR) were tested using a landscape genetic approach by comparing alternative landscape resistances, as suggested by Balkenhol et al.  and Fran22.12). Human population density is relatively low for Europe (around 68 inhabitants/km2) and people mainly live in major cities and along the coasts, while small villages and large uninhabited areas characterize the interior. Climate is Mediterranean‐temperate at low elevations and along the coast, more continental inland and at higher elevations. Temperature is mild and relatively constant throughout the year . Annual precipitations range from less than 400 mm in the dry south to 1500 mm in the eastern mountains. Such climatic conditions are suitable for wild boar all over the island.The island of Sardinia is the second largest in the Mediterranean Sea , and, based on habitat suitability analyses, higher densities were expected to occur in the central and northern part of the island .A total of 393 wild boar samples were obtained from all over Sardinia by local hunters during the period 2001–2019. Tissue or hair samples were collected from hunted animals and stored, respectively, in absolute ethanol or frozen until analysis. Sampling locations . Each PCR was performed in a 10 μl reaction volume, containing 3 μl of DNA solution, 0.5 U of Taq DNA polymerase (Euroclone), 1× PCR buffer (Euroclone), 2.5 mM MgCl2, 100 μM of each dNTP, and 2 pmol of each primer. Forward primer of each pair was labeled with an ABI fluorescent dye . The amplification profile was set up with an initial step of denaturation at 95°C for 3 min, followed by 35 cycles of 92°C for 45 s, annealing T ranging between 62–52°C for 45 s, and 72°C for 30 s, with a further final extension step of 72°C for 10 min. Amplicons were sized using capillary electrophoresis in an ABI PRISM 3100 or 3730XL Avant automatic sequencer (Applied Biosystems) by the BMR‐Genomics sequencing service . Appropriate calibrations were made to standardize microsatellite scoring results when the automatic sequencer was changed. Peak Scanner software v. 1.0 (Applied Biosystems) was used to analyze electrophoretic data.DNA was extracted using GenElute kit  for tissue samples and Instagene Matrix  for hair samples, and then stored at −20°C. All samples were genotyped with a panel of 16 microsatellites: S090, SW122, SW2532, S355, SW1492, SW461, IGF1, SW951, SW2021, SW2496, S026, S215, SW72, SW857, S155, and SW24 (details at 2.3OH) and expected (EH) heterozygosity, mean number of alleles per locus (A), and FIS were computed in GENETIX v. 4.05  and from domestic pigs in Sardinia was already reported by Scandura et al. . As distn = 568) was analyzed by Bayesian cluster analysis in STRUCTURE v. 2.3.4  ranging from 1 to 10, with settings: admixture model, no population information, correlated allele frequencies, 500,000 burn‐in and 500,000 iterations of data collection. Again, the optimal K‐value was chosen according to the ΔK statistics in Structure Harvester  as belonging to the corresponding subpopulation. Genotypes were plotted in a two‐dimensional space based on their genetic proximity. Pairwise Rousset's ar genetic distance  was also performed using Adegenet package in R v, 4.0.2 Jombart,  to detecRousset, , shown tRousset, , was comRousset, .2.4www.hpc.cineca.it/). This approach uses stochastic search algorithms that solve optimization problems by simulating natural selection processes , Isolation‐By‐Barrier (IBB), and Isolation‐By‐Resistance (IBR). To assess the relevance of each driver, the resistance optimization process described by Peterman et al.  was implScrucca,  to find n = 270) using Circuitscape 5.0 implemented in Julia . To test the IBR hypothesis, we optimized a categorical land‐cover grid surface. Land‐cover data were obtained from a digital map of Sardinia .To test the IBB hypothesis, we optimized a binary grid surface with a 500 × 500 m resolution where cells crossed by main roads had a value equal to 1 while all other cells had a value equal to 0. Main roads were identified as those with an Average Daily Traffic (ADT) higher than one standard deviation of the mean ADT from all the sampling stations in Sardinia , at all loci observed heterozygosity (Ho) was lower than expected heterozygosity (eH), thus revealing an excess of homozygotes that was confirmed by FIS values. Not surprisingly, GENEPOP analysis detected a significant deviation from Hardy‐Weinberg equilibrium due to heterozygote deficiency in the overall population , except for locus S026 (p = .031) when performing HW test for each locus. Several pairs of loci resulted in linkage disequilibrium .The total number of alleles detected in the Sardinian wild boar sample was 154, ranging from 6 to 16 per locus and an average of 9.63 ± 3.18  per locus. Missing alleles represented 2.17% of the dataset. MICRO‐CHECKER did not find any scoring error in the dataset or evidence of allele dropout. Properties of the 16 microsatellite loci used in this study and the variability observed at each locus are shown in Table From the original dataset of 393 Sardinian wild boar samples analyzed with 16 microsatellite loci, 75 individuals showed a high relatedness  to other individuals in the dataset and were likely to represent full‐siblings or parent/offspring. Therefore, they were removed from the dataset to obtain a cleaned pool of 318 unrelated individuals to perform the following analyses.3.2K = 5 (or higher) the Bayesian analysis in STRUCTURE sharply distinguished the main source populations in the overall sample of 568 individuals . However, in order to identify individuals with a clear signature of genetic introgression in Sardinia, we selected K = 4 as it showed a higher support than K = 5 , as the most likely, but local maxima were detected also at K = 5 and K = 8  and wild boars from the rest of the island (north and east), with the main discontinuity between the two clusters apparently represented by the SS131. At K = 5, five subpopulations were clearly identified  detected a partition in two clusters  were assigned to one of the five subpopulations with q > 0.70, and specifically 23 individuals were assigned to NW, 37 to CW, 39 to SW, 57 to NCE, and 54 to SE. No individual was assigned to a population different from that expected on the basis of its sampling site. Such pattern of population differentiation seemed to identify the presence of genetic discontinuity in coincidence with the SS131 and the Campidano plain. The consistency of results obtained by different analytical approaches points to a sharp structuring in the island with a limited ongoing gene flow between subpopulations.On the basis of their The PCA plot Figure  confirme3.4Comparing the best models obtained through the optimization process revealed that the IBR, accounting for land cover and the presence of main roads, was by far the best‐supported hypothesis Table . The IBBThe resistance surface associated with the best‐supported model under the IBR hypothesis revealed minimum resistance to wild boar movement in coniferous and broadleaved forests, and water bodies  and confirmed expectations (Table p ≤ .001) and were significantly lower in pairs including locations classified in the same cluster than in pairs including locations classified in different clusters .As discussed by Reiner et al. , detectiCervus elaphus, Frantz et al., Lynx rufus, Serieys et al., According to our data, the Sardinian wild boar population should not be managed as a single panmictic unit, rather subpopulations should be treated as separate management units. The lack of gene flow across barriers  should be taken into account in the definition of spatial units for disease prevention. Results may also have implications for the management of other wild species in Sardinia. Given that urban areas, main roads and the most intensively cultivated areas apparently play a role as barriers to gene flow in the wild boar population, they could also represent a cause of fragmentation for other mammals (including endemic and endangered species), promoting isolation and genetic drift. However, the effect on other species should be tested by targeted studies, as landscape features might have various impacts on different species ; Data curation ; Methodology ; Software (lead); Writing – original draft (lead). Olivia Dondina: Conceptualization (supporting); Methodology ; Software (lead); Writing – original draft . Valerio Orioli: Conceptualization (supporting); Methodology ; Software ; Writing – original draft . Daniela Biosa: Data curation ; Investigation ; Writing – original draft . Antonio Canu: Data curation ; Investigation ; Methodology ; Writing – review & editing . Giulia Fabbri: Methodology (supporting); Software ; Writing – review & editing . Laura Iacolina: Data curation ; Investigation ; Writing – review & editing . Antonio Cossu: Data curation (supporting); Investigation (supporting); Writing – review & editing . Luciano Bani: Conceptualization (supporting); Methodology (supporting); Supervision ; Writing – review & editing . Marco Apollonio: Conceptualization (supporting); Funding acquisition (lead); Supervision ; Writing – review & editing . Massimo Scandura: Conceptualization (lead); Data curation ; Funding acquisition (lead); Methodology (lead); Project administration (lead); Writing – original draft ; Writing – review & editing .Appendix S1‐S2Click here for additional data file.


In this article, we introduced a case of rare congenital anomalies that was asymptomatic until adulthood and was complicated by infective endocarditis and dissection of aortic valve leaflet. In this article, we introduced a case of rare congenital anomalies that was asymptomatic until adulthood and was complicated by infective endocarditis and dissection of aortic valve leaflet. The incidence of endocarditis in adults with CHD has been reported to be 11 per 100 000 person‐years compared with 1.5‐6 per 100 000 patient‐years in general population. However, CHD‐associated mortality has decreased to 10% because of improvement in the diagnosis of infective endocarditis, antibiotic therapy, cardiac surgery, and interventional procedure.2A 23‐year‐old male patient was admitted to our hospital due to headache and decreased communication with others since 2 weeks ago. The patient also had history of generalized weakness, diarrhea, fever, and decreased appetite from 4 months ago. He did not complain of dyspnea, chest pain, dizziness, paresis, and paresthesia.During physical examination at admission, the patient's vital signs were as follows: blood pressure :110/80 mm Hg, pulse rate: 120 beats per minute, respiratory rate: 18 per minute, temperature: 39.5°c, and O2 saturation: 90% in room air. The patient seemed ill and pail and had Broca aphasia. His skin was normal in inspection; no discoloration or lesion was found. In examination of thorax, we heard a diastolic murmur in precordium at aortic foci, holosystolic murmur in left parasternal border with decreased S1 intensity and S3 gallop. Lung sounds were clear except at upper part of right lung, and no rales were heard. Abdomen was soft with mild tenderness at left upper quadrant. His extremities were warm with no edema.2.1The patient was an active person up to 4 months ago and had no specific medical disorder and no history of trauma.2.2According to patient's history and examination, differential diagnosis was central nervous system (CNS) infection or mass, infective endocarditis with emboli to CNS, and complicated gastrointestinal (GI) infection.2.3Initial laboratory findings showed anemia with hemoglobin: 7.6 g/dL, white blood cells: 10 000 per microliter, erythrocyte sedimentation rate: 48 mm/hr, and lactate dehydrogenase: 552 U/L. After that Brain Magnetic Resonance imaging (MRI) was done and showed hyperintense lesions in left parietal and occipital regions with cortical, subcortical and white matter involvement in T2 sequence in favor of infarction (Figure Chest CT scan revealed prominent cardiomegaly with increased venous congestion and a cavitary consolidation at posterior segment of right upper lobe of lung in favor of septic emboli Figure . The patThe patient was diagnosed infective endocarditis with multiple septic emboli and destructed aortic valve in context of congenital heart disease, and blood culture also was positive for Streptococcus Viridans.2.4Patient received a complete course of antibiotic therapy with vancomycin and gentamycin according to antibiogram and also heart failure medication and then underwent open‐heart surgery of aortic valve replacement with resection of subaortic web and VSD closure successfully.3This was a case of an adult with multiple congenital heart disease including VSD, DCRV, subaortic web, and severe hypoplasia of PMVL that presented with complications of aortic valve endocarditis including multiple emboli to brain, lung, and spleen.The association between infective endocarditis and acute aortic dissection is extremely rare;Absence of PMVL is usually fatal in utero, while hypoplasia of it typically present in childhood with symptomatic mitral regurgitation 3.1After successful heart surgery and patient recovery, he was discharged home, and in 6‐month follow‐up, he was uneventful.None declared.FJ: wrote the initial draft, SAM: collected the patient's data, SJ: reviewed literatures, AS: edited the initial draft, KM: involved in concept, data collection, final edition of manuscript, and revision.Written informed consent for publication of figures was obtained from patient.Video S1Click here for additional data file.Video S2Click here for additional data file.Video S3Click here for additional data file.Video S4Click here for additional data file.


We encountered a patient with severe coronavirus disease 2019 (COVID‐19)‐related pneumonia, who died of progressive respiratory acidosis after 2 months of treatment with mechanical ventilation. The autopsy revealed diffuse alveolar damage (DAD) without any active signs of fungal or bacterial infections, suggesting prolonged and over‐activated immune responses against COVID‐19 infection. When COVID‐19 patients develop acute respiratory distress syndrome, it is essential to remember that the infection can progress to DAD a few months after the disease onset. We experienced a fatal case of coronavirus disease‐2019 (COVID‐19) pneumonia, who died of progressive respiratory acidosis after 2 months of treatment with mechanical ventilation. The autopsy revealed diffuse alveolar damage (DAD) without any active signs of fungal or bacterial infections, suggesting prolonged and over‐activated immune responses against COVID‐19 infection. When COVID‐19 patients develop acute respiratory distress syndrome, it is essential to remember that the infection can progress to DAD a few months after the disease onset. The coronavirus disease 2019 (COVID‐19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has become a global pandemic, resulting in innumerable deaths. In severe cases of COVID‐19‐related pneumonia, acute respiratory distress syndrome (ARDS), histologically appearing as diffuse alveolar damage (DAD), is known to develop because of cytokine storms.We report a fatal case of COVID‐19 with an autopsy finding in which the patient slowly developed progressive pulmonary fibrosis and respiratory acidosis after methylprednisolone therapy. The patient had died 58 days after the onset of COVID‐19 infection.A Japanese man in his 70s with a history of type II diabetes mellitus, hypertension and hyperlipidaemia, and a 15‐pack‐year smoking history, presented with a 1‐week history of cough and dyspnoea. He was diagnosed with COVID‐19 via a SARS‐CoV‐2 antigen test, which was verified by a polymerase chain reaction (PCR) test. He was intubated and admitted to our emergency room department for rapidly progressing respiratory failure.2]: 1.0). Coarse crackles were noted on physical examination. Laboratory findings revealed elevated a white blood cell count of 30,400/μl, haemoglobin level of 13.1 g/dl, platelet count of 110,000/μl, creatinine level of 1.36 mg/dl, haemoglobin A1c level of 9.0%, KL‐6 of 591 U/ml, C‐reactive protein level of 15.2 mg/dl, ferritin level of 2124 ng/ml and d‐dimer level of 22.0 μg/ml. Arterial blood gas testing showed a pH of 7.32, partial pressure of carbon dioxide (PaCO2) of 40.9 mmHg, partial pressure of oxygen (PaO2) of 62.0 mmHg, HCO3− of 20.4 mEq/L and elevated lactate level of 2.1 mmol/L. Bilateral consolidation with lower lung predominance was observed on chest computed tomography (CT), consistent with the symptoms of COVID‐19 infection.On arrival, the patient was lethargic with a Glasgow Coma Scale score of E4VTM5, temperature of 37.2°C, blood pressure of 94/62 mmHg, pulse rate of 89 bpm, respiratory rate of 12/min and oxygen saturation of 88% on mechanical ventilation . His chest CT revealed mediastinal emphysema, which improved spontaneously by lowering positive end‐expiratory pressure (PEEP) and tapering the methylprednisolone to 10 mg/day. From day 39, he developed fever, and his FiO2 and PEEP requirement increased. Additionally, his tidal volume decreased to below 300 ml/breath, resulting in respiratory acidosis. Chest CT taken on day 44 demonstrated diffuse ground‐glass opacities along with bronchial dilatation and volume loss of bilateral lungs. On day 49, his blood pressure dropped, requiring vasopressor support, and his renal function deteriorated as respiratory acidosis progressed. The patient eventually died on day 58  and placed in a prone position. Continuous infusion of neuromuscular blocking agent and deep sedation were introduced. He was treated with pulsed methylprednisolone (1000 mg once daily) for 3 days and favipiravir (800 mg twice daily), in addition to the antibiotic therapy. Despite the decrease in his FiOAfter his death, his family agreed to allow post‐mortem examination. The gross lung findings included increased lung weight  with solid appearance. Microscopic lung findings showed collapsed alveolar spaces filled with collagen fibres, consistent with the organizing phase of DAD. There was no evidence of active infection or embolism Figure .In this report, the patient received pulsed methylprednisolone against ARDS due to COVID‐19. Although his respiratory condition temporarily improved, he eventually died of respiratory acidosis 58 days after the onset of COVID‐19, due to progression of pulmonary fibrosis.It has been reported that in the ICU setting, approximately 60%–80% of COVID‐19 patients develop ARDS, with an average onset time of 8–9 days after the initial onset COVID‐19 symptoms. In previous autopsy reports, DAD has been the predominant histological finding, followed by organizing pneumonia, pulmonary embolism and alveolar haemorrhage.In our patient's case, respiratory acidosis developed 40 days after the onset of symptoms, indicating that this restrictive change progressed over 1.5 months. In addition, SARS‐CoV‐2 PCR testing was continuously performed on tracheal aspirate during hospitalization, and the PCR continued to be positive for 30 days after disease onset, indicating that the SARS‐CoV‐2 viral load was persistently high, and this could have led to a sustained inflammatory response. We used corticosteroids and favipiravir as the initial treatment, followed by early reduction of corticosteroids. To improve the prognosis, we may have needed to use long‐term potent anti‐inflammatory treatment such as baricitinib (a JAK inhibitor) or tocilizumab (an IL‐6 inhibitor) as well as antiviral treatment such as remdesivir. Furthermore, in cases where fibrosis becomes a problem due to prolonged disease course, antifibrotic therapies are worth considering in addition to antiviral and anti‐inflammatory treatments. Clinical trials of antifibrotic therapies such as nintedanib and pirfenidone for progressing pulmonary fibrosis are currently underway, and these treatments may be an option in the future.Although candidemia occurred during the acute phase of ARDS, we consider that it only slightly contributed to the post‐acute pulmonary fibrosis, since antifungal therapy was initiated immediately with good response, and the progression of pulmonary fibrosis and respiratory acidosis was observed more than 1 month after its diagnosis. The recommended duration of therapy for candidemia without complications is 2 weeks. In this case, however, we had to continue administration of the antifungal drug. The reason for this was that the patient was still immunocompromised due to the continued use of corticosteroids for the treatment of pulmonary fibrosis, and his respiratory condition worsened around day 33; hence, we had to consider the possibility of a fungal breakthrough infection. As for bacterial infections, we empirically administered broad‐spectrum antibiotics when his respiration worsened, considering the risk of secondary bacterial infections. As it turned out, autopsy findings of the lungs did not show the presence of fungal organisms by Grocott's methenamine silver stain, and no neutrophilic infiltration or purulent exudates suggestive of fungal or bacterial infection were observed; therefore, the infection was considered to be well controlled. Furthermore, the involvement of ventilator‐induced lung injury should be considered in this case. Although preventative strategies were used such as limiting tidal volume inspiratory pressure, prone positioning and limit spontaneous respirator effort, lung injury may have progressed due to long‐term ventilator management. Certainly, we have not completely ruled out other infections or the effects of positive pressure ventilation. Based on the PCR results and lung autopsy findings, we think that SARS‐CoV‐2 itself was more involved than other infections in the progression of DAD.In conclusion, we reported a lethal case of COVID‐19 with the prolonged progression of lung injury and respiratory acidosis over 2 months from symptom onset. It is important to keep in mind that DAD may develop over a long period of time after COVID‐19 infection.None declared.Akiko Tamura, Ryosuke Imai, Yutaka Tomishima and Naoki Nishimura managed the patient. Akiko Tamura and Ryosuke Imai drafted the manuscript, and Yutaka Tomishima and Naoki Nishimura edited and revised it critically.The authors declare that appropriate written informed consent was obtained for the publication of this manuscript and accompanying images.


BCS1L‐related disease, provide better understanding of the genotype–phenotype correlations and identify reliable prognostic disease markers.To delineate the full phenotypic spectrum of BCS1L variants were considered eligible. Clinical, laboratory, neuroimaging and genetic data were analysed. Patients were stratified into different groups based on the age of disease onset, whether homozygous or compound heterozygous for the c.232A>G (p.Ser78Gly) variant, and those with other pathogenic BCS1L variants.We performed a retrospective multinational cohort study of previously unpublished patients followed in 15 centres from 10 countries. Patients with confirmed biallelic pathogenic BCS1L variants were more frequent in those with later symptom onset.Thirty‐three patients were included. We found that growth failure, lactic acidosis, tubulopathy, hepatopathy and early death were more frequent in those with disease onset within the first month of life. In those with onset after 1 month, neurological features including movement disorders and seizures were more frequent. Novel phenotypes, particularly involving movement disorder, were identified in this group. The presence of the c.232A>G (p.Ser78Gly) variant was associated with significantly worse survival and exclusively found in those with disease onset within the first month of life, whilst other pathogenic BCS1L‐related disease comprises a continuum of clinical features rather than a set of separate syndromic clinical identities. Age of onset defines BCS1L‐related disease clinically and early presentation is associated with poor prognosis. Genotype correlates with phenotype in the presence of the c.232A>G (p.Ser78Gly) variant.The phenotypic spectrum of  Disorders of mitochondrial oxidative phosphorylation (OXPHOS) represent one of the most common groups of inherited metabolic diseases, with a combined minimum birth prevalence of 1 in 4300 live births.c reductase) catalyses the transfer of electrons from reduced coenzyme Q10 to cytochrome c whilst simultaneously pumping protons from the mitochondrial matrix across the inner mitochondrial membrane to the intermembrane space.UQCRB, UQCRQ, UQCRC2, UQCRFS1 and CYC1) or assembly factors  have been reported to be associated with mitochondrial CIII deficiency and human disease.An important subgroup amongst mitochondrial disorders comprises the mitochondrial complex III (CIII) deficiencies.BCS1L gene encoding BCS1, a mitochondrial inner membrane protein that acts as a translocase for insertion of the Rieske FeS subunit into the precomplex of CIII to facilitate assembly of the holoenzyme complex.BCS1L are GRACILE and Björnstad syndromes. Classical GRACILE syndrome (MIM 603358) is an autosomal recessive disease characterised by Growth Restriction, Aminoaciduria as a sign of tubulopathy, Cholestasis with Iron overload in the liver, Lactic acidosis and Early death.BCS1L gene.pili torti) and sensorineural hearing loss.The most frequent cause of mitochondrial CIII deficiencies is due to defects in the BCS1L disease, provide better understanding of the genotype–phenotype correlations and identify reliable prognostic disease markers using data from the largest known multinational cohort of patients with confirmed biallelic pathogenic BCS1L variants.We aimed in this study to improve the clinical recognition of patients with BCS1L variants who had been diagnosed and followed up at the participating centres were considered eligible.We conducted a multinational retrospective study of patients from 15 centres in 10 countries: Finland , United Kingdom , United States , France , Spain , Sweden , Norway , Austria , Italy  and Oman . Previously unpublished patients with confirmed biallelic pathogenic Detailed clinical, biochemical, muscle biopsy, neurophysiological, neuroimaging and genetic data were obtained using a standardised case report form completed by the responsible investigator(s) at each centre and reviewed by the study principal investigator (O.H). Data entry was completed in December 2020.The date of disease onset was defined as the date of the first symptom(s) requiring medical evaluation. End of follow‐up was defined as the date of the patient’s last follow‐up visit or death. Available clinical and laboratory longitudinal data, both at disease onset and later during the disease course, were collected. Small for gestational age was defined as newborns with birth weight below the 10th percentile for the gestational age.Proximal renal tubulopathy included Fanconi syndrome and was defined as having two or more of the following: generalised aminoaciduria, glycosuria, low molecular weight proteinuria, bicarbonate loss resulting in renal tubular acidosis and renal salt wasting.Liver involvement was defined by the presence of two or more of the following parameters in at least two different time points: elevation of aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), gamma‐glutamyltransferase (GGT), bilirubin or ammonia; low serum albumin; or pathological histological findings on liver biopsy.Movement disorders included dystonia, athetosis and tremor.BCS1L variants.Initially, detailed clinical, laboratory, neuroimaging and genetic data for the whole cohort were analysed to study the phenotypic spectrum of the disease regardless of the age of disease onset or genotype. To study the disease spectrum beyond the neonatal period, the patients were classified into those with disease onset within the first month of life and those with later onset. The study cohort was further stratified into those who were homozygous or compound heterozygous for the c.232A>G (p.Ser78Gly) variant and those with other pathogenic BCS1L) PDB:6UKP and PDB:6UKS.https://github.com/matteoferla/BCS1_analysis. Interactive page was created using Michelaɴɢʟo.Models were prepared with PyRosettap value less than 0.05 was considered to be statistically significant. For survival analysis, the endpoint was time to death which was defined as the time from the date of disease onset to the date of death. Univariate survival analysis was performed using the log‐rank test (Kaplan–Meier) to compare differences in survival time between categories.Detailed descriptive data analysis was performed using SPSS , Version 26.0. A two‐sided Ethical approval for the study was obtained from the Regional Committee for Medical and Health Research Ethics, Western Norway (REK 2017/625). Each participating centre had obtained approval by the local ethical committee. The study was registered as an audit at Great Ormond Street Hospital, London, UK (Registration Number: 2224).BCS1L variants were identified. Ten were diagnosed in Finland, five in the United Kingdom, four in the United States, three each in France and Sweden, two each in Austria, Norway and Spain and one each in Italy and Oman. The majority of patients were of European ancestry (n = 23), whilst three were from the Middle East, three Turkish, two Pakistani and two Black American.Thirty‐three patients,  with biallelic pathogenic n = 24/33) of patients included in this study cohort had disease onset either at birth or within the first month of life. Fourteen patients were born prematurely with gestational age less than 37 completed weeks and 19  patients were small for gestational age. A symptom‐free period ranging from 3 months to 7 years was observed in nine patients.The majority , failure to thrive (n = 27), proximal renal tubulopathy (n = 24), hepatopathy (n = 24), early death (n = 14), sensorineural hearing loss (n = 12), iron overload (n = 10), movement disorders (n = 9), cholestasis (n = 7) and seizures (n = 6). Other features observed in our cohort include global developmental delay (n = 9), attention deficit hyperactivity disorder (n = 3), cataract (n = 3), learning difficulties (n = 2), optic atrophy (n = 1) and hypertrophic cardiomyopathy (n = 1). A summary of the clinical phenotypes observed in our cohort is provided in Table Patients included in this cohort presented with a spectrum of clinical phenotypes including lactic acidosis  and those presenting later , we found that features consistent with GRACILE syndrome such as growth failure, lactic acidosis, tubulopathy, hepatopathy, iron overload and early death were more frequently observed in those with onset within the first month of life as compared to those with later onset. In contrast, features such as movement disorders  and seizures occurred across all ages but were more frequently identified in our study cohort in those with onset after the first month of life . Elevations of alanine aminotransferase  and aspartate aminotransferase  levels were also frequently observed, together with hypoglycaemia  and hypoalbuminaemia . Major laboratory findings, both at disease onset and later, are summarised in Table Laboratory investigations revealed elevated serum lactate in the majority of patients , in liver biopsy  and in cultured skin fibroblasts .Respiratory chain enzyme activities Table  showed Cn = 8/10, 80%) showed abnormalities at the time the first imaging was performed. The most common cerebral MRI finding was T2/FLAIR hyperintensities scattered in the deep white matter, thalamus and dentate nucleus. Detailed description of the brain MRI findings is provided in Table MRI of the brain was available for 10 cases. The majority  who were homozygous for the c.232A>G (p.Ser78Gly) variant presented at birth whilst those who were compound heterozygous for c.232A>G (p.Ser78Gly) with another variant (n = 4) presented within the first month of life. Those with other BCS1L pathogenic variants (n = 20), whether homozygous or compound heterozygous, presented from birth up to 7 years of age. Features consistent with GRACILE syndrome were more frequently reported in patients with homozygosity or compound heterozygosity for the c.232A>G (p.Ser78Gly) variant, whilst features such as movement disorders and seizures were predominantly observed in patients with pathogenic BCS1L variants other than c.232A>G (p.Ser78Gly)  followed by sepsis . The median survival time of patients with disease onset within the first month of life was 27 days (range 1 day–4.5 years), compared to 8 years (range 2–14 years) for those with disease onset after 1 month . The main cause of death was multi‐organ failure (BCS1L variants (Table p < 0.001) worse survival compared with those having other pathogenic BCS1L gene variants  variant was 4 days (range 1 day–3 months), compared to 2 years (range 1 month–14 years) for those with other pathogenic https://michelanglo.sgc.ox.ac.uk/r/bcs1).The consequences of the variants at the protein level were assessed in both ATP‐bound and unbound conformations  are predicted to be extremely destabilising in both conformations  due to severe clashes and most likely do not result in any folded protein, similar to the truncations. In support of this, these two mutations and the truncations are found solely as compound heterozygous variants in combination with structurally less severe mutations Table . It is ap.Ser78Gly affects the interface  between different chains, but is not predicted to be destabilising, therefore may result in weaker oligomerisation.Several of the affected residues are key to the conformational switch of the protein. Two mutations, p.Glu163Lys and p.Arg183His, are predicted to be neutral overall in both conformations, but substantially alter the chemical characteristics and are found in key positions for the conformational switch; specifically, the angle of the loop of Glu163 allows it to form a salt bridge with Arg184 in the apo form, but not in the ATP‐bound form.BCS1L variants, and demonstrate the breadth of clinical manifestations and the natural history of the disease. As far as we can ascertain, this is the largest cohort of patients with BCS1L disease so far described. Our studies also extend the phenotypic spectrum of the disease by identifying novel phenotypes other than the classic GRACILE and Björnstad syndromes.We present the detailed description of 33 patients with confirmed pathogenic biallelic BCS1L.n = 18/33) fell into other categories as summarised in Table BCS1L‐related disease and shows that it clearly includes features associated with mitochondrial dysfunction involving the central nervous system such as movement disorders, seizures and Leigh‐like phenotype. Amongst those with onset after the first month of life, we observed patients with a disease‐free period.We systematically reviewed our detailed clinical, laboratory, neuroimaging and genetic data, both at disease onset and later during the disease course. GRACILE and Björnstad syndromes are the most frequently reported phenotypes associated with pathogenic variants in n = 9) of the patients presented with movement disorders, in contrast to higher rates reported in other series of patients with mitochondrial disorders.BCSL1‐related disease have a high mortality in the neonatal period, before movement disorders can develop.Interestingly, movement disorders including dystonia, athetosis and tremor, were frequently observed in our study cohort, regardless of the age of disease onset. Patients with mitochondrial disorders often show movement disorders associated with involvement of the basal ganglia.Since we were able to collect a large number of patients for such a rare disease, we could assess the prevalence of non‐classical clinical features and follow disease development. By stratifying patients into those with onset within the first month of life and those with later onset, we could identify clear phenotypic, genetic and prognostic differences. Features consistent with GRACILE syndrome such as lactic acidosis, hepatopathy, tubulopathy, growth failure and early death were more frequently reported in patients with disease onset within the first month of life as compared to those with later disease onset. In contrast, neurological features including movement disorders and seizures were more frequently reported in those with onset after the first month of life Table .BCS1L variants located throughout the gene were identified  presented at birth with features consistent with GRACILE including early death. Those who were compound heterozygous for c.232A>G (p.Ser78Gly) and another variant presented within the first month of life with features consistent with GRACILE syndrome but with variable additional features including movement disorders and seizures. Patients with other pathogenic BCS1L variants could have a symptom‐free period, from birth to their first presentation, for as long as 7 years. Neurological features such as movement disorders and seizures were more frequently reported in patients with pathogenic variants in BCS1L other than c.232A>G (p.Ser78Gly), however features such as lactic acidosis, tubulopathy, hepatopathy and growth failure were also reported in this group. Further analysis revealed that the presence of c.232A>G (p.Ser78Gly), in homozygous or compound heterozygous state, was significantly (p < 0.001) associated with worse survival as compared to other pathogenic BCS1L variants. This clear genotype–phenotype correlation has not only an essential prognostic value, but also an important implication in genetic counselling, especially for families seeking prenatal diagnosis.Twenty‐three pathogenic d Figure . Genotypn = 3/4) and fibroblasts (n = 1/3). Previous published studies reported CIII deficiency predominantly in the liver,BCS1L disease, as five of the patients in our cohort in whom respiratory chain analysis was performed  had normal results.Respiratory chain analysis showed CIII deficiency (CII+CIII or CIII) in skeletal muscle (10/12), liver  that may not trigger the clinical suspicion of BCS1L‐related disease. Thus, the possibility of BCS1L‐related disease needs to be considered in phenotypes beyond the classical GRACILE and Björnstad syndromes, and this may improve early clinical recognition of the disease.Our data also lead us to believe that The authors declare no financial or other conflict of interest related to this work.O.H and S.R designed the study, were responsible for data collection, analysed the data and drafted the initial manuscript and approved the final manuscript as submitted. P.I, N.K, M.F, E.F, M.A, M.B, N.D, D.D, D.G, H.H, F.I, N.J, M.K, E.M, K.N, J.D.O.E, S.P, A.P, G.B., K.T, D.B, S.W, J.T, L.B and V.F were responsible for data acquisition and analysis, revising the manuscript critically and approving the final manuscript as submitted. All authors are responsible for accuracy and integrity of the work.Table S1. Major laboratory findings of patients included in this study cohort.Click here for additional data file.Table S2. Respiratory chain enzyme activities.Click here for additional data file.Table S3. Summary of neuroimaging findings.Click here for additional data file.Table S4. Genetic findings: BCS1L variants observed in the cohort.Click here for additional data file.Table S5. Allele frequencies of pathogenic variants of BCS1L.Click here for additional data file.Table S6. Predicted consequences of variants at the protein level assessed in both ATP‐bound and unbound conformations.Click here for additional data file.Table S7. Published cases with BCS1L mutations and clinical phenotypes (n = 87).Click here for additional data file.


Background: Hemophagocytic lymphohistiocytosis (HLH) is a rare clinical syndrome with a high mortality rate. There is no biomarker to predict the early therapeutic response.Objective: Our study explores the significance of serum ferritin in predicting the response of induction therapy.Methods: We retrospectively analyzed the clinical data of 102 adult patients with HLH admitted to our hospital. These patients received HLH-94 regimen for induction therapy. The patients were divided into remission group and non-remission group according to the response of induction therapy. Results: Ferritin values between 1–4 weeks post induction were predictive of remission (p<.05), which were higher in the non-remission group than in the remission group. Ferritin obtained 2 weeks post-induction had the highest ROC for predicting remission, with a cut-off value of 1188.5 µg/L. And patients with ferritin lower than 1188.5 µg/L had better response of induction therapy.Conclusion: Our study suggests that serum ferritin is a good indicator to predict the efficacy of induction therapy for adult HLH. Serum ferritin is a good indicator for predicting the efficacy of adult HLH induction therapy.Serum ferritin two weeks after treatment may be a better indicator to judge the early curative effect.Serum ferritin after treatment also had a predictive significance for the survival of HLH. Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory syndrome caused by abnormally activated macrophages and cytotoxic T cells , which cSerum ferritin is an intracellular storage protein, which is mainly found in macrophages . The incThis is a retrospective study with 150 patients who were admitted to the hospital with a preliminary diagnosis of HLH between January and December 2017. Inclusion criteria: (1) Met the HLH-2004 diagnostic criteria  [1]; (2)We analysed retrospectively the patients' general conditions , aetiology, baseline biochemical parameters, all parameters included in the HLH-2004 Diagnostic Criteria  cytotoxic activity, sCD25, ferritin) and induction therapy regimens. The NK cell activity assay applied in this paper is a patented technology of our research centre. The type of ferritin assay employed at the Hospital is Beckman Coulter. The normal reference value of SF is 10–120 µg/L for females and 20–300 µg/L for males. We mainly analysed the predictive effect of ferritin on the response of the induction treatment.2 in patients aged 18–39 years; 50–75 mg/m2 in patients aged 40 years and older). Dexamethasone, 10 mg/m2/d for one to two weeks, 5 mg/m2/d for three to four weeks, 2.5 mg/m2/d for five to six weeks, 1.25 mg/m2/d for seven weeks, reduced to discontinuation for eight weeks. We adjusted the dose and duration of dosing based on individual patient characteristics. And we performed an assessment for initial induction therapy response after two to four weeks of induction therapy with the HLH94 regimen. We continued the current HLH94 regimen of induction therapy for eight weeks for patients who achieved remission. For unrelieved patients, we switched to a DEP regimen for induction therapy, which includes liposomal doxorubicin treatment combined with etoposide and methylprednisolone.All patients enrolled in this study received HLH-94 strategies for induction therapy, which included dexamethasone, etoposide, cyclosporine, and intrathecal methotrexate and dexamethasone. We made age-related adjustments for the use of etoposide in adults due to the lower need for and tolerance of etoposide , malignancies (N = 28), autoimmune disorders (N = 9), primary HLH (N = 6) and no identified underlying disorder (N = 24) (n = 53) and non-remission group (n = 49) based on the response of induction treatment. All patient races were Chinese, and fever was observed in all patients. A comparison was made between the two groups by non-parametric test. And results showed that compared with remission group, the levels of sCD25 (p = .035), direct bilirubin (p = .034) and urea (p = .030) upon admission were significantly higher and the values of platelets (p = .003) were significantly lower in non-remission group. It also showed there was significant difference in etiology. Regarding to race, age, gender, clinical signs, laboratory variables , there was no significant difference between the two groups  (p > .05)  . Patientp > .05) . In ordep > .05) .p = .013, p = .004, p = .005, p = .002). There was no statistical difference between the two groups in ferritin levels before treatment (p = .526)  .p = .004), and the best cut-off value was 1188.5 µg/L. Ferritin at three weeks was the second indicator to predict the induction response with 85.7% sensitivity and 59.1% specificity , and the best cut-off value was 1149.0 µg/L. Ferritin at four weeks was the third indicator to predict the induction response with 84.6% sensitivity and 64.5% specificity . Ferritin at one week after treatment had the lowest AUC  with 57.8% sensitivity and 68.9% specificity, and the best cut-off value was 2602 µg/L . Ferritin at three weeks less than 1149.0 µg/L after induction treatment  or ferritin at four weeks less than 846.5 µg/L  were also significantly associated with response, while results showed that ferritin at one week after treatment was not statistically significant to predict the response of induction    and 5.p = .009) and four weeks (p = .017) were significantly associated with long-term outcome. While the results indicated that the ferritin at one week (p = .508) and two weeks (p = .348) after treatment might did not predict survival. ROC curves were employed to analysed the ability of ferritin at three and four weeks after induction therapy to predict two-year survival. The results showed that ferritin at three weeks after treatment was the first indicator to predict the overall survival with 68.4% sensitivity and 76.9% specificity , and the best cut-off value was 1442.5 µg/L. Ferritin at four weeks after treatment was the second indicator to predict the overall survival with 74.4% sensitivity and 70.6% specificity , and the best cut-off value was 803 µg/L. Kaplan–Meier curve showed that ferritin at three  and four weeks after treatment  might have a predictive effect on long-term outcome (We followed the patients for two years and analysed the correlation among ferritin values after induction therapy with the overall survival. Results showed that ferritin levels at three weeks ( outcome .HLH is a high-inflammatory factor storm syndrome characterized by an extreme increase in pro-inflammatory cytokines. The early symptoms are atypical, and the diagnosis is difficult. However, the disease progresses rapidly, and many patients die rapidly due to seeking medical treatment too late or unclear diagnosis. Thus, early diagnosis and treatment of HLH are very important. The reactants in the acute phase of HLH are often elevated, and the most relevant one is serum ferritin . FerritiThe current diagnostic criteria for HLH 2004 in adults set “Ferritin ≥500 µg/L” as one of the diagnostic criteria. This result is mainly based on the threshold value for the diagnosis of HLH in children. The threshold value of ferritin is different in patients of different ages, and ferritin diagnostic thresholds have not been verified in adult HLH ; therefoThis article compared the correlation among general conditions and various laboratory indicators at admission with the response of induction treatment by chi-square test and independent samples non-parametric test. We found that statistically significant increases in sCD25, direct bilirubin and urea levels and statistically significant decreases in platelet at admission in the non-remitting group compared to the remitting group . We alsoWe found that ferritin two weeks after treatment had some predictive value for response to induction therapy. Studies have shown that ferritin may be a useful indicator of disease activity and prognosis in adult HLH patients . In thisIn contrast to previous studies, this paper focuses on the predictive value of ferritin for induction response in patients with HLH. Previous investigators have discussed the relationship between ferritin and the prognosis of patients at six months, one year and two years after treatment and conclude that serum ferritin is a good predictor of survival ,22. Our Due to the retrospective nature of the study, we had to face a certain degree of missing data from clinical and laboratory investigations or patients who were not followed up. Although the number of patients was sufficient for the final analysis, multicentre prospective randomized clinical trials with larger sample size are needed to further verify our results. In addition, individual differences in HLH patients and disease management among physicians vary widely.Ferritin post-induction may be a predictor of induction response and long-term outcome. Ferritin values two to four weeks after induction therapy appears to be a useful marker for predicting induction response. And attaining a ferritin below 1188 µg/L at two weeks post-induction is an indicator of a better induction response. Ferritin helps to predict the response to induction therapy early and helps clinicians to initiate salvage therapy in a timely manner.


Focused ultrasound (FUS) mediated blood–brain barrier disruption (BBBD) targets the delivery of systemically‐administered therapeutics to the central nervous system. Preclinical investigations of BBBD have been performed on different anesthetic backgrounds; however, the influence of the choice of anesthetic on the molecular response to BBBD is unknown, despite its potential to critically affect interpretation of experimental therapeutic outcomes. Here, using bulk RNA sequencing, we comprehensively examined the transcriptomic response of both normal brain tissue and brain tissue exposed to FUS‐induced BBBD in mice anesthetized with either isoflurane with medical air (Iso) or ketamine/dexmedetomidine (KD). In normal murine brain tissue, Iso alone elicited minimal differential gene expression (DGE) and repressed pathways associated with neuronal signaling. KD alone, however, led to massive DGE and enrichment of pathways associated with protein synthesis. In brain tissue exposed to BBBD , we systematically evaluated the relative effects of anesthesia, microbubbles, and FUS on the transcriptome. Of particular interest, we observed that gene sets associated with sterile inflammatory responses and cell–cell junctional activity were induced by BBBD, regardless of the choice of anesthesia. Meanwhile, gene sets associated with metabolism, platelet activity, tissue repair, and signaling pathways, were differentially affected by BBBD, with a strong dependence on the anesthetic. We conclude that the underlying transcriptomic response to FUS‐mediated BBBD may be powerfully influenced by anesthesia. These findings raise considerations for the translation of FUS‐BBBD delivery approaches that impact, in particular, metabolism, tissue repair, and intracellular signaling. AnanesthesiaBBBblood brain barrierBBBDblood–brain barrier disruptionCNScentral nervous systemDAMPdamage associated molecular patternDGEdifferential gene expressionFUSfocused ultrasoundGOgene ontologyGSEAgene set enrichment analysisH&Ehematoxylin and eosinIsoisofluraneIso‐FUSisoflurane anesthesia with focused ultrasound treatmentKDketamine/dexmedetomidineKD‐FUSketamine/dexmedetomidine anesthesia with focused ultrasound treatmentLEAleading edge analysisMBmicrobubblesMRgFUSmagnetic resonance‐guided focused ultrasoundNESnormalized enrichment scorePCAprincipal components analysisPNPpeak‐negative pressureQCquality controlRBCred blood cellRINRNA integrity numberSIsterile inflammation1The blood–brain barrier (BBB) is essential to maintaining homeostasis in the central nervous system (CNS). The BBB describes a specialized vasculature, consisting of nonfenestrated endothelium, pericytes, astrocytic processes, microglia, and basement membrane working in concert to precisely permit nutrient transport while protecting against toxins and pathogens. However, the BBB also presents a significant neuropharmacological obstacle, preventing 98% of small‐molecule therapeutics and nearly 100% of large‐molecule therapeutics from accessing the CNS.Focused ultrasound (FUS) following IV infusion of microbubbles (MB) is a promising approach for BBBD.BBBD with FUS is reversible and may be applied in a manner that yields little to no histological damage after repeated treatment.One such parameter is general anesthesia. Anesthetic protocols, ubiquitous in preclinical FUS BBBD studies, distinctly impact the circulation time of MB and the extent of FUS‐induced vascular damage.22.1n = 4 per group). Contrast‐enhanced MRIs  or ketamine/dexmedetomidine (KD) and treated with Magnetic Resonance‐guided Focused Ultrasound (MRgFUS) targeted to the right or left striatum . After read alignment and QC, principal components analysis (PCA) was performed on transformed transcript counts from each sample to assess global differences between treatment conditions  compared to naïve brain. Regardless of the anesthetic background, FUS and MB produced moderate  and negligible  effects on gene expression, respectively. Iso alone had a marginal effect on the transcriptome, only significantly changing the expression of 26 genes. Next, we performed gene set enrichment analysis (GSEA) to identify biological processes consistent with genes differentially expressed within each contrast  versus KD (KD‐FUS). First, we evaluated the extent and overlap of differentially expressed genes Figure  and diffp‐adj = 0.06).To further compare the effect of anesthesia on FUS BBBD, we performed LEA on selected gene sets enriched by both Iso‐FUS and KD‐FUS. Comparing transcripts in the LEA of the (Iso + MB + FUS)/(Iso + MB) contrast against those in LEA of the (KD + MB + FUS)/(KD + MB) contrast for the same pathway allows us to address whether FUS is achieving the same “end” (pathway enrichment) by similar “means” (transcript regulation) on different anesthetic backgrounds. We performed comparative LEA on gene sets associated with cell–cell junctions and inflammation, as these were the most consistently induced by both Iso‐FUS and KD‐FUS. Out of the 173 genes in the Cell Junction Organization gene set (GO:0034330), Iso‐FUS and KD‐FUS enriched 48 and 50, respectively Figure . 19 tranp‐value <0.1 for Iso + MB + FUS vs Iso, Iso + MB + FUS vs Iso + MB, KD + MB + FUS vs. KD, and KD + MB + FUS vs. KD + MB contrasts). To narrow the scope of immune system‐related LEA overlaps, we repeated this analysis on the Chemokine Activity gene set (GO:0008009) which only contains 34 genes , a broad collection of 1709 genes associated with the immune system Figure . Iso‐FUSs Figure . Iso‐FUS2.6We next evaluated the effects of anesthesia, MB, and FUS on transcripts known to be associated with the BBB.2.7Given the anesthesia‐dependence of BBBD and FUS‐induced gene expression, we next tested whether anesthesia affected the extent of damage in the brain parenchyma after treatment with the same FUS pressure. We performed histological analysis of murine brains treated with combinations of Iso, KD, and FUS Figures . With th3BBBD mediated by FUS‐activated MB has emerged as a promising technique for the image‐guided and noninvasive delivery of therapeutics to the CNS. Though this procedure is safe, our understanding of cellular responses to FUS BBBD at the transcriptional level is still limited. This knowledge gap becomes especially significant when considering that preclinical BBBD studies have been performed on a multitude of different anesthetic backgrounds Table , a factop‐adjusted <0.05) of 3291 genes when compared to naïve controls , leading to increased protein translation.PCA and hierarchical clustering performed on variance‐stabilizing transformed RNA‐seq counts data revealed the relative contributions of Iso, KD, MB, and FUS to intersample variability with respect to CNS gene expression Figure . The moss Figure . WhetherWe observed increases in inflammatory signatures elicited by both anesthetics Figure . Of the SI caused by FUS‐activated MB has raised concerns over its feasibility for repeated clinical application. SI can last for at least 24 h after a single sonication, and is dependent on MB dose and FUS pressure.Despite differential responses at the transcriptional level and in MRI signal enhancement Figure , FUS appTransient SI can provide beneficial effects in certain disease contexts with respect to clearance and regeneration.Our investigation has some limitations. First, RNA‐sequencing only provides transcript‐level information. mRNA may not always correlate proportionally to protein expression.4We investigated how Iso and KD, the two most commonly used anesthetics in preclinical FUS BBBD studies, differentially affect CNS responses to FUS‐activated MB. At the same acoustic pressure, FUS induced similar profiles of MB cavitation and measures of damage regardless of the anesthetic. RNA sequencing performed acutely after treatment with combinations of Iso, KD, MB, and FUS revealed distinct contributions from each. Specifically, while Iso alone produced transcriptomic profiles nearly identical to those of naïve mice, it also elicited stronger signatures of stress in the neurovascular unit when combined with FUS. KD, however, induced sweeping transcriptome changes alone, but blunted markers of SI while promoting gene sets associated with tissue repair upon FUS application compared to Iso‐FUS. These results provide important context for previous preclinical FUS studies, and underscore anesthesia as an important experimental variable to consider for future work.55.1ad libitum. All animal experiments were approved by the Animal Care and Use Committee at the University of Virginia and conformed to the National Institutes of Health regulations for the use of animals in research.11 week old female C57BL/6 mice were purchased from Jackson and maintained on a 12/12 h light/dark cycle. Mice weighed between 22 and 28 g and were given food and water 5.2Mice in groups designated “KD” received 50–70 mg/kg Ketamine and 0.25–0.5 mg/kg Dexmedetomidine via intraperitoneal injection with no additional maintenance or reversal drug given. Mice in groups designated as “Iso” or “Iso‐MA” were placed in an induction chamber and received isoflurane delivered to effect in concentrations of 2.5% in medical air using a vaporizer. For isoflurane groups, anesthesia was maintained via nosecone for a total of 90 min.5.3An MRgFUS system was used to treat brains for RNA‐seq studies. Once mice were anesthetized, a tail vein catheter was inserted to permit intravenous injections of MBs and the MRI contrast agent. The heads of the mice were shaved and depilated, and the animals were then placed in a supine position over a degassed water bath coupled to an MR‐compatible small animal FUS system . The entire system was then placed in a 3T MR scanner . A 3.5 cm diameter receive RF coil, designed and built in‐house, was placed around the head to maximize imaging SNR. Baseline three‐dimensional T1‐weighted MR images were acquired at 0.3 mm resolution using a short‐TR spoiled gradient‐echo pulse sequence and used to select 4 FUS target locations in and around the right or left striatum.5 MBs/g b.w.), formulated as previously described.Mice received an injection of albumin‐shelled MBs  and processed using an in‐house MATLAB (MathWorks) algorithm. Acoustic emissions at the fundamental frequency, harmonics , sub harmonic (0.5f), and ultra‐harmonics  were assessed by first taking the root mean square of the peak spectral amplitude (Vrms) in each frequency band after applying a 200 Hz bandwidth filter, and then summing the product of Vrms and individual sonication duration over the entire treatment period. Broadband emissions were assessed by summing the product of Vrms and individual sonication duration for all remaining emissions over the entire treatment period.5.5Six hours after treatment, mice were euthanized via an overdose of pentobarbital sodium and phenytoin sodium. Immediately following euthanasia, the mouse brains were harvested and the anterior right quadrants (~100 mg) were excised . For FUS‐treated mice, contrast MR images were referenced to confirm extraction of the full volume of sonicated brain. A representative 3D image of the harvested tissue is provided in Figure 5.6A stereotactic tabletop FUS system was used to treat brains for histological studies. Sonications using the stereotactic frame were performed using a 1‐MHz spherical‐face single‐element FUS transducer with a diameter of 4.5 cm (Olympus). FUS  was targeted to the right striatum. The 6‐dB acoustic beamwidths along the axial and transverse directions are 15 and 4 mm, respectively. The waveform pulsing was driven by a waveform generator  and amplified using a 55‐dB RF power amplifier . A voltage‐PNP calibration is provided in Figure 5 MBs/g b.w.) and Evans Blue, followed by 0.1 ml of 2% heparinized saline to clear the catheter. Sonication began immediately after clearance of the catheter. In contrast to the MR‐guided experiments, which targeted four spots, only one location was targeted in these studies due to the increased focal region of the transducer .Once anesthetized, a tail‐vein catheter was inserted to permit i.v. injections of MBs and Evans Blue. The heads of the mice were shaved and depilated, and the animals were then positioned prone in a stereotactic frame (Stoelting). The mouse heads were ultrasonically coupled to the FUS transducer with ultrasound gel and degassed water and positioned such that the ultrasound focus was localized to the right striatum. Mice received an i.v. injection of the MBs  equipped with a PROGRES GRYPAX microscope camera . Ten 20× images from the region of the right striatum with maximal Evans Blue extravasation were taken per section and 2–6 sections were imaged per brain. A researcher blinded to treatment condition assigned a semi‐quantative score of 0 (none—complete absence of RBC extravasation/vacuolation), 1 , 2 , or 3 (severe—multiple large sites of RBC extravasation/vacuolation) to each 20× image for RBC extravasation and vacuolation using a custom MATLAB (MathWorks) script.5.8p‐values <0.05 are presented as significant unless stated otherwise. For GSEA, the fgseap‐values <0.05 are presented as significant unless stated otherwise. For histological analyses, data are presented as mean ± SEM. Statistical significance was detected by one‐way ANOVA followed by comparison against naïve with Dunnett's multiple comparison test.For contrast enhancement and acoustic emissions analyses, data are presented as mean ± SEM. Statistical significance was assessed by Mann–Whitney test. For differential gene expression significance analysis, DESeq2The authors declare no conflict of interest.Conceptualization: Alexander S. Mathew, Catherine M. Gorick, Natasha D. Sheybani, E. A. Thim, and Richard J. Price; Methodology: Alexander S. Mathew, Catherine M. Gorick, Natasha D. Sheybani, E. A. Thim, William J. Garrison, Alexander L. Klibanov, G. W. Miller, and Richard J. Price; Investigation: Alexander S. Mathew, Catherine M. Gorick, Natasha D. Sheybani, E. A. Thim, William J. Garrison, Alexander L. Klibanov, G. W. Miller, and Richard J. Price; Formal Analysis: Alexander S. Mathew, Catherine M. Gorick, Natasha D. Sheybani, E. A. Thim, and Richard J. Price; Writing—Original Draft Preparation: Alexander S. Mathew and Richard J. Price; Writing—Review & Editing: Alexander S. Mathew, Catherine M. Gorick, Natasha D. Sheybani, E. A. Thim, William J. Garrison, Alexander L. Klibanov, G. W. Miller, and Richard J. Price; Supervision; Funding Acquisition: Alexander S. Mathew and Richard J. PriceAppendix S1: Supporting InformationClick here for additional data file.