These results are in agreement with EARL reference values for fluorine-18 PET. The results in this work showed that accurate quantitative scandium-43/44 PET/CT is achievable in commercial devices. This may promote the future introduction of scandium-43/44-labelled radiopharmaceuticals into clinical use.Scandium-44 has been proposed as a valuable radionuclide for Positron Emission Tomography (PET). Recently, scandium-43 was introduced as a more favorable option, as it does not emit high-energy γ-radiation; however, its currently employed production method results in a mixture of scandium-43 and scandium-44. The interest in new radionuclides for diagnostic nuclear medicine critically depends on the option for image-based quantification. We aimed to evaluate and compare the quantitative capabilities of scandium-43/scandium-44 in a commercial PET/CT device with respect to more conventional clinical radionuclides . With this purpose, we characterized and compared quantitative PET data from a mixture of scandium-43/scandium-44 (~68% scandium-43), scandium-44, fluorine-18 and gallium-68, respectively. A NEMA image-quality phantom was filled with the different radionuclides using clinical-relevant lesion-to-background activity concentration ratios; images were acquired in a Siemens Biograph Vision PET/CT. Quantitative accuracy with scandium-43/scandium-44 in the phantom’s background was within 9%, which is in agreement with fluorine-18-based PET standards. Coefficient of variance (COV) was 6.32% and signal recovery in the lesions provided RC In this regard, the availability, transport, dosimetry, and safety are critical aspects to be addressed FDG.From the images obtained, it can be seen that the noise , noise (COV) and effect of scatter corrections were initially evaluated, as well as in the average residual lung error. For the COV, with similar activity ratio between spheres and background (rSC, gallium-68 with aSC, scandium-43/44 with rSC, scandium-43/ 44 with aSC, scandium-44 with rSC and scandium-44 with aSC, respectively. All levels were <15%, as recommended for fluorine-18 [With regard to the PET/CT capabilities, the device quantification (background calibration—ckground , all radorine-18 ,13,14,17BGcal, based on the acceptable deviation of 10% between measured and known background activity concentration [aSC and scandium-44 with rSC were outside tolerance levels. Interestingly, in contrast to Lima et al., the Biograph Vision produced acceptable results for scandium-44 when using aSC compared to their results with the Biograph mCT. As discussed by Lima et al. and confirmed by the current results (SC overcorrects the scatter contribution, due to the presence of the high-energy γ-radiation (SC. Overall, using the optimal SC, a BGcal below 10% was achieved with 2.7%, 3.1%, 8.0% and 2.9% for fluorine-18 and gallium-68 with rSC, for scandium-43/44 with rSC and for scandium-44 with aSC, respectively.Regarding the ntration , only sc results , relativadiation , which rCOV results, aSC tended to under correct for scatter events. This was also observed in the average residual lung error, where all reconstructions with aSC showed an increased lung error in comparison with the rSC. Notably, even for the worst results, which were obtained for the scandium-43/44 mixture with aSC, the average residual lung error was <15%, which is comparable to acceptable performance for fluorine-18 [Additionally, the impact of these corrections was evaluated in the lung region of the phantom. Knowing that there is no activity in the lung region, measurements of average residual lung error provide an assessment on the impact of the scatter and attenuation corrections in the final image. As shown previously in the meanRC and maxRC were superior when using fluorine-18 compared to the other radionuclides. This can be explained by the pure positron emissions and corrections tailored to this radionuclide. Comparing the scandium-43/44 mixture to pure scandium-44, the RC results were comparable to gallium-68—a more widely used radionuclide.As a final parameter, the recovery coefficients obtained for all radionuclides were investigated. Both COV and RCs.Although having differences in phantom preparation and data analysis between the current measurements and NEMA or EARL, namely, the activity concentration between background and lesions, it is felt that the comparison is still meaningful for 177Lu or 90Y-labeled counterparts. Importantly, delivery of scandium-44 and scandium-43 to PET centers without a cyclotron will be feasible over long distances and, thus, be of particular interest in countries in which the hospitals are far away from the production centers. The final optimization step refers to the use of a mixture of scandium-43/44 versus the use of scandium-44 only, as it can add to the reduction of an unnecessary dose to the producing personnel as well as the patient. Based on the current investigation, there is no obvious disadvantage of using a mixture of scandium radioisotopes. Hence, the concept could be translated to clinics without concern.In this work, the PET/CT quantification capabilities for scandium-43/44 mixture in relation to radioisotopically pure scandium-44, fluorine-18 and gallium-68 were evaluated using a high-end SiPM PET/CT device. It was demonstrated that it could be achieved with either a mixture of scandium-43/44 or pure scandium-44 using a SiPM similar quantitative performance to clinically used radionuclides such as gallium-68 and fluorine-18. This situation will not only enable the clinical translation of scandium-44; it also raises hopes that scandium-43 can be used clinically, in spite of the fact that it cannot easily be prepared in radionuclidically pure form. Scandium radioisotopes benefit from longer half-lives than conventionally used PET nuclides such as gallium-68 and fluorine-18, which may benefit from clinical evaluation where a scan start can occur at later time points when background activity is already cleared. Moreover, scandium may be used for prospective dosimetry prior to therapy with


Purpose: Ketamine is an anesthetic in clinical, but it has also been used as an abusing drug due to its low price and hallucinogenic effects. It is proved that ketamine abusing would cause multiple system damage including the urinary system, which is called ketamine-induced cystitis (KIC). Bladder fibrosis is late stage in KIC and threaten abusers’ life. This study aimed to investigate the molecular mechanism of ketamine-induced bladder fibrosis.Methods: Female Sprague Dawley (SD) rats were randomly divided into 3 groups. 2 groups were treated with tail vein injection of ketamine  for 12 weeks, whereas the control group was treated with normal saline solution. In each group, rat bladders were extracted and samples were examined for pathological and morphological alterations via hematoxylin and eosin (HE) staining, Masson’s trichrome staining and immunohistochemistry (IHC). SV-HUC-1 cells were treated with different concentrations of ketamine solution . Rat bladder and SV-HUC-1 cells were extracted protein and RNA for Western blot and RT-PCR detection. Metadherin (MTDH) siRNAs and overexpression plasmids were used to knock down and overexpress the relative genes. P38 mitogen-activated protein kinase (MAPK) inhibitor was utilized to inhibit the MAPK pathway.Results: Rats in the ketamine group exhibited fibrosis compared to rats of the control group and fibrosis were also markedly upregulated in SV-HUC-1 cells after treated with ketamine, which were ketamine concentration-dependent. After treating with ketamine in SV-HUC-1 cells, there was an increase expression of MTDH, epithelial-mesenchymal transition (EMT) markers, P38 MAPK. MTDH knockdown would suppresses P38 MAPK/EMT pathway to inhibit fibrosis, however, MTDH overexpression could promote the pathway in SV-HUC-1 cells.Conclusion: In rats and SV-HUC-1 cells ketamine-treated models, MTDH can regulate EMT through the P38 MAPK pathway to regulate the process of bladder fibrosis. Ketamine, also known as K powder, is an N–methyl–D–aspartate receptor antagonist . KetaminMetadherin (MTDH), which is also known as astrocyte upregulation factor −1 (AEG-1), was found in primary human fetal astrocytes after being treated with TNF-α or infected with HIV-1 . As a muThe function of MTDH is gradually being recognized in fibrotic diseases. In the study of the unilateral ureteral occlusion (UUO) model, it was found that MTDH can promote renal fibrosis by regulating the EMT . PreviouThe specific mechanism of bladder fibrosis after ketamine abuse is unknown. We hypothesize that MTDH is a key regulatory molecule, and it can regulate the process of bladder fibrosis by regulating EMT. In this study, the expression of MTDH and EMT marker proteins were detected in human bladder cell biochemistry Pillon (SV-HUC-1) cells and bladder tissue of rat models. Then, the expression of MTDH was regulated to study its regulatory effect. We provided evidence that the pro-fibrosis effect of MTDH is partly attributable to the promotion of the EMT by activating the P38 MAPK signaling pathway.All the experimental animals were performed on specific pathogen-free (SPF) female Sprague Dawley (SD) rats  weighing between 200 and 250 g. A total of 24 SD rats were randomly distributed into three groups, eight rats per group, and received tail vein injection of normal saline solution (control group), tail vein injection of 25 mg/kg/day ketamine hydrochloride solution  (low dose ketamine group), tail vein injection of 50 mg/kg/day ketamine hydrochloride solution (high dose ketamine group) for 12 weeks. The rats were weighed weekly to adjust the dosage of ketamine hydrochloride solution. Animals were raised at the Experimental Animal Center of Central South University  and the animal experiments were approved by ethical review of experimental animal welfare in Central South University (NO: 2018sydw0225).The bladder tissue which was removed from the rats in the ketamine group and control group was fixed in 4% paraformaldehyde overnight, embedded in paraffin blocks, and sliced into a 4 μm section. Hematoxylin and eosin (H&E) and Masson trichrome staining (MTS) were performed to carry out the histopathologic evaluation such as the morphology and inflammatory cell infiltration of the bladder, the distribution of collagen. Immunohistochemical staining (IHC) was performed to evaluate the fibrosis, MTDH expression, EMT markers of the bladder in ketamine group rats compared with the control group. Briefly, embedded bladder tissue was first deparaffinized and then subjected to a microwave oven for antigen retrieval in citrate buffer. The sections were incubated overnight at 4°C in rabbit MTDH antibody , mouse E-cadherin , mouse vimentin , rabbit fibronectin antibody , and rabbit collagen I antibody  after being blocked with 3% H2O2. And then the sections were incubated with biotinylated secondary antibody  for 30 min at room temperature, and development was achieved with 3,3′-diaminobenzidine.The human bladder cell biochemistry Pillon (SV-HUC-1) was obtained from Guangzhou Cellcook Biotech Co., Ltd.  and cultured in F-12K medium  containing 10% fetal bovine serum  at 37°C constant temperature and 5% CO2. SV-HUV-1 was stimulated with ketamine solution  diluted by medium for 48 h. The SB203580  was treated with SV-HUC-1 in the corresponding group.® INC., Suzhou, China) and C1000 Touch Thermal Cycler, CFX96 Real-Time System (Bio-Rad) according to manufacturer’s instructions. The results were calculated by the 2−ΔΔCt method and data are expressed as a ratio of the control gene GAPDH.Total RNA was extracted from SV-HUV-1 cell lines using RNAiso Plus reagent  and cDNA was synthesized using PrimeScript™ RT reagent kit . The primers designed details were shown in ® software.The SV-HUV-1 cells and bladder tissues were lysed with RIPA  containing protease cocktail  to obtain the total protein. The protein was separated and transferred via cataphoresis using 10% SDS-PAGE gel electrophoresis and PVDF membranes. The primary antibodies against rabbit MTDH antibody , mouse E-cadherin , mouse vimentin , rabbit P38 , rabbit P-P38 , rabbit fibronectin , rabbit collagen Ⅰ , rabbit α-SMA , mouse Flag-Tag  and GAPDH  were incubated with the membranes overnight at 4°C. After incubating, blots were detected by secondary antibody and visualized by the ECL assay (Milliporesigma) and the bands were analyzed with Image JSV-HUC-1 was seeded in a 6-well cell culture cluster. MTDH siRNA and MTDH overexpression plasmid were bought from Sangon Biotech  and the details of the sequence are in SV-HUC-1 cells were inoculated into 96-well plates (1 × 104 cells/well) for 24 h, and then stimulated with ketamine solution (0.1–2 mmol/L) diluted by medium for 48 h. After ketamine treatment, CCK-8 solution  was added to the 96-well (10 ul/well) and incubated with SV-HUC-1 for 2 h, the absorbance at 450 nm was detected via a microplate reader .The urination spot test spot test was performed in a metabolic cage. The test paper used for urine spots was a customized test paper that turns red when exposed to urine, which could accurately indicate the liquid infiltration. Twelve weeks after the injection of ketamine and 0.9% saline, the rats in the experimental group and the control group were moved into a metabolic cage to collect urine. The urine spot test was carried out from 8 am to 12 o’clock in the morning.After anesthesia, the rats were fixed in the supine position, and 75% ethanol was used to disinfect the skin of the abdomen and perineum. After connecting each end of the cystometry tube and the three-way tube and venting the air, they were inserted into the bladder along the urethra of the rat. Gently press the rat’s abdomen to empty the bladder of residual urine. After the computer point signal was reset to zero, the micro-injection pump was turned on, and the normal saline preheated to 37°C was continuously perfused at a rate of 6 ml/h. Observe the urinary interval by observing the fluid outflow interval of the external urethra; by observing the number of times the water pressure is higher than the baseline 10 cm water column within 3 min before the leakage of urine to obtain the frequency of unstable contraction of the bladder detrusor.p < 0.05 or less was considered to be statistically significant.SPSS 20.0  was utilized to perform the statistical analyses. Data were shown as mean ± standard error of the mean (SEM) according to the results of three independent repeated experiments. Two-way analysis of variance (ANOVA) was used to analyze the differences. in vitro. After 48 h of treatment, the viability of SV-HUC-1 cells cultured with 1.5 mmol/L ketamine was significantly decreased (p = 0.01). And the urodynamics test showed that in the ketamine-induced cystitis rat model group, the frequency of urination increased, the interval between urination was shortened, and the unstable contraction increased  on SV-HUC-1 cells using a CCK-8 kit to evaluate the cytotoxicity of ketamine ecreased , indicatecreased . With thecreased . And we ecreased , the resecreased . Compareecreased . And we ecreased . In addincreased . The aboTo elucidate the underlying mechanism of ketamine-induced fibrosis, MTDH and EMT markers expression were investigated. Compared with the control group, the protein and RNA expression of MTDH and vimentin were increased, and the expression of E-cadherin was decreased in SV-HUC-1 cells after ketamine treatment . SimilarWe further investigated the role of MTDH in ketamine-induced bladder fibrosis by MTDH knockdown or overexpression. Successful MTDH knockdown was confirmed by WB analysis. MTDH expression in SV-HUC-1 cells was shown an obvious decrease following the knockdown . Small iTo investigate whether MTDH regulates EMT through the P38 MAPK pathway to promote bladder fibrosis, we measured the expression of P38 and phosphorylated (P)-P38 protein in SV-HUC-1 and rat bladder after ketamine treatment. In comparison with the control group, the expression levels of P-P38 protein were shown to be significantly increased in the ketamine treatment group, while there was no significant changes in the expression of P38 protein . When P3In the present study, underlying mechanisms of ketamine-induced bladder fibrosis were studied using SD rats and SV-HUC-1 cells. Our results suggested that EMT is an important process of bladder fibrosis. Furthermore, we also found that a novel pro-fibrotic molecule MTDH can regulate EMT through the P38 MAPK signaling pathway involved in bladder fibrosis induced by ketamine. MTDH might be a potential biomarkers in detection ketamine induced bladder fibrosis.Since it was first reported in 2007, a large number of the literature revealed that long-term use of ketamine can cause damage to the urinary system . DecreasEMT is involved in a variety of biological processes, such as tissue repair and pathology, including cancer and cataract . It alsoMost of the current research on MTDH focuses on its role in tumors. Previous studies have shown that MTDH can activate several classic cancer-promoting signaling pathways, such as EMT, NF- κ B and MAPK to enhance tumor progression and metastasis . It has Studies have found that P38 MAPK is closely related to myocardial fibrosis, pulmonary fibrosis, and liver fibrosis . Pirfeniin vitro, but in vivo, we did not study the mechanism of MTDH as a key molecule of ketamine induced bladder fibrosis and its possible target for the treatment. However, we revealed that MTDH might be a vital biomarker and treatment target of ketamine induced bladder fibrosis.In this study, we demonstrated that ketamine induced bladder fibrosis involves pathway MTDH/P38 MAPK/EMT. But the study has limitations as follows (I) Clinical investigation and data were not included in the present study. And the expression level of MTDH/P38 MAPK/EMT pathway markers were not detected in clinical bladder samples and this awaits further studies (II) We mainly verified the MTDH/P38 MAPK/EMT pathway In conclusion, we revealed that in rats and SV-HUC-1 cells models, MTDH can regulate EMT through the P38 MAPK pathway to partly regulate the process of bladder fibrosis. MTDH may serve as a novel biomarker and therapeutic target in bladder fibrosis induced by ketamine abuse.


To determine the international reliability and validity of the modified Radiographic Union Scale for Tibial fracture (mRUST) scoring method for open tibial shaft fractures based on ratings of radiographs by separate groups of North American and Tanzanian surgeons.Seven North American and 9 Tanzanian surgeons viewed 100 pairs of AP and lateral radiographs of open tibial shaft fractures obtained in Dar Es Salaam, Tanzania. The radiographs showed 25 patients’ fractures at 4 time points postfracture after treatment with either external fixation or intramedullary nailing. Surgeons evaluated each fracture using the mRUST scoring method and indicated their confidence that the fracture was healed on a scale from 1 to 10. Reliability of mRUST was determined using inter-rater agreement among North American and Tanzanian surgeons. Validity was determined via analysis of correlation between mRUST scores and EQ-5D-3L index scores at each time point postfracture.mRUST scores demonstrated strong reliability overall (ICC = 0.64) as well as within each group of North American (ICC = 0.72) and Tanzanian (ICC = 0.69) surgeons. Reliability was stronger for external fixation than for intramedullary nailing cases. mRUST scores were significantly correlated with overall healing confidence at all time points and with quality of life at 6 months and 1 year postfracture. mRUST scores also correlated significantly with patients’ quality of life scores (EQ-5D index) at 6 months and 1 year postfracture.North American and Tanzanian surgeons exhibited strong agreement in rating open tibial shaft fractures. Using mRUST scores is a valid means of assessing radiographic healing of tibial fractures in austere environments like Tanzania. A crucial consideration in treatment of tibial fractures is evaluation of fracture healing. However, defining and measuring healing status remains controversial. Currently, there are no universally accepted “gold standard” measures of union or nonunion. This contributes to significant variability among surgeons in terms of the methods they use to determine nonunion and how quickly they elect to perform corrective surgery for nonhealed fractures. The lack of consensus about healing assessment makes it difficult for physicians to make accurate and unbiased assessments of fracture healing status.Approximately half a million patients suffer from tibial fractures in the US every year. and modified RUST (mRUST) scoring tools to assess healing status of tibial fractures using radiographic analysis. These tools use the presence of bridging callus and obliteration of fracture lines on anteroposterior (AP) and lateral radiographs in order to assign a numerical value for a healing tibial shaft fracture. In the standard RUST instrument, raters are asked to score each of 2 cortices visible on both AP and lateral radiographs of a tibial fracture. Each of the 4  cortices is scored based on the following guidelines: 1 = no callus, 2 = bridging callus, 3 = remodeled. For mRUST, a fourth option  was added to the rating scale to differentiate between nonbridging and bridging calluses. This corresponds to a rating system of 1 = no callus, 2 = callus present, 3 = bridging callus, 4 = remodeled. Scores range from 4 to 12 (for standard RUST) and 4 to 16 (for mRUST), with lower scores indicating a less healed fracture and higher scores suggesting a more advanced stage of healing. The reliability (agreement in scoring among surgeons) and validity (correlation between scores and patient-relevant outcomes) of RUST and mRUST have been demonstrated in several studies.,6,7More recently, researchers developed the standard Radiographic Union Scale for Tibial fractures (RUST)However, important gaps in the literature remain. First, the mRUST scoring system has never been validated internationally, as studies to date have focused on North American surgeons’ assessments of fractures. Secondly, evidence linking mRUST scores to clinically relevant patient outcomes remains limited. Finally, a minimum mRUST score threshold below which fractures can be considered “not healed” with confidence has not yet been established. This lower threshold could inform surgeons’ decisions about whether or not to perform surgery. Moreover, because clinical trials typically follow nonunions as endpoints in fracture repair studies, a nonunion threshold is also useful for research purposes. which randomized patients with Gustilo-Anderson type I-IIIA open tibia fractures to treatment with either definitive external fixation or intramedullary nailing. The purpose of the present study was to evaluate the reliability of mRUST scoring of open tibial shaft fractures between North American and Tanzanian surgeons, and to correlate mRUST scores with both patients’ health-related quality of life and surgeons’ overall assessment of fracture healing, at 4 time points after fracture stabilization. Additionally, we sought to identify upper and lower mRUST score thresholds that correspond with healed fractures and not healed fractures, respectively.This study addresses these gaps in the literature using data from a recently completed randomized control trial in Tanzania,2In this study, AP and lateral radiographs of open tibial shaft fractures were evaluated by 16 experienced orthopaedic trauma surgeons practicing at major urban medical centers in either North America (n = 7) or Tanzania (n = 9). For each pair of radiographs, surgeons determined the mRUST score and provided a rating of confidence that the fractures were healed. Surgeons were recruited by email to participate in the study, which involved completing a 20-minute online survey. Data from surgeons’ assessments of radiographs and from quality-of-life surveys were then used to assess the reliability and validity of mRUST. Informed consent was obtained from all surgeons prior to their participation in the study, and the study was approved by the IRB at UCSF. The UCSF IRB # is 14-14792, PI is Dr. Saam Morshed. This repository of images from patients with fractures was selected because of the standardization and intervals of acquisition for the parent trial, and the great variance of open [versus closed] fracture healing expected at any given time of follow-up. Patients had been treated with either external fixation (n = 14) or intramedullary nailing (n = 11). For each patient, AP and lateral radiographs and quality-of-life surveys were acquired at 4 time points postfracture . Radiographs were included in the study based on the availability of complete data and high-quality radiographs for each patient at all 4 time points postfracture. Seventy-three patients in the study had received both AP and lateral radiographs at each time point. A picture of each radiograph was taken and uploaded electronically for evaluation. All radiographs associated with these patients were then evaluated by a 4th-year orthopaedic surgery resident (HJR) for image quality. The quality of each image was assessed as “Good,” “Poor,” or “Obstructed,” indicating that the view of the fracture was obstructed . Data was excluded from 48 patients whose radiographic images were assessed as “Poor” or “Obstructed” at 1 or more time points. Twenty-five patients were included who had high-quality radiographs available at all 4 time points, yielding a total of 100 pairs of AP and lateral radiographs.Surgeons evaluated 100 pairs of AP and lateral radiographs of 25 patients with Gustilo-Anderson type I-IIIA open tibial shaft fractures who had participated in a recent randomized control trial in Dar es Salaam, Tanzania.The online survey was designed and presented using Qualtrics survey software . After a live tutorial on mRUST scoring, each surgeon viewed a randomly selected subset of 25 pairs of AP and lateral radiographs, presented in random order. Each pair of images was displayed on a separate page of the survey. For each pair of radiographs, surgeons were asked to evaluate the fracture with the mRUST score and estimates of confidence that fractures were healed. For mRUST scoring, surgeons rated each cortex of the fracture as “no callus,” “callus present,” “bridging callus,” or “remodeled,” with each respective choice receiving an associated score of 1–4. Aggregation of scores for the 4 cortices yielded a total mRUST score for each fracture ranging from 4 to 16. Healing confidence was evaluated by asking surgeons to rate their confidence, on an incremental scale from 1 to 10, that the fracture was “not healed” (lower anchor) or “healed” (upper anchor).–11 at each of the 4 time points postfracture. Patients rated 5 dimensions of their health on 3-level scales , as well as their overall health status using a 100-point visual analogue scale. An overall index score was then calculated for each patient and time point using the “eq5d” package in R. Scores were adjusted for country using the package's built-in parameters for Zimbabwe, since no parameters are currently available for Tanzania in the package.Patients in the randomized control trial completed EQ-5D-3L quality-of-life surveys which estimates ICCs and confidence intervals using the variance components from a one-way ANOVA while accounting for intra-rater and intra-patient measurement groupings. Results of ICC calculations were interpreted based on the work of Landis and Koch and following the example of Litrenta et al. ICC values below 0.2 were defined as “slight agreement,” 0.21–0.40 as “fair agreement,” 0.41–0.60 as “moderate agreement,” 0.61–0.8 as “substantial agreement,” and values above 0.81 as “nearly perfect agreement.”,15 Validity of mRUST was evaluated using linear regression models to determine the correlation between mRUST scores and patients’ quality of life (EQ-5D index) scores and surgeons’ evaluations of healing status at each time point postfracture.Inter-rater reliability was assessed using intraclass correlation coefficients (ICCs) of mRUST scores. ICCs were calculated using the “ICC” package in R,3Results of mRUST reliability analyses, stratified by country and treatment type, are displayed in Tables P = .014, r2adj = 0.280) and 1 year  postfracture. However, no significant correlations were found between mRUST scores and EQ-5D index scores at 6 weeks and 3 months postfracture.Table Correlations between mRUST scores and surgeons’ reported confidence of fracture healing are displayed in Table 4 both overall and within each subgroup of North American and Tanzanian orthopaedic trauma surgeons. Reliability was higher for fractures treated with external fixation compared to those treated with intramedullary nailing. In terms of validity, mRUST scores at later time points (6 months and 1 year) postfracture were significantly associated with patients’ self-reported general health (EQ-5D index scores). Finally, mRUST scores correlated significantly with surgeons’ estimated confidence that a fracture had healed at all 4 time points postfracture.While the mRUST scoring system has previously been validated in the North American context, this study is the first to evaluate the reliability and validity of mRUST in surgeons and patients from under-resourced countries. In the present analysis, inter-rater reliability values fell within the range of “substantial” agreement, according to the criteria outlined by Landis and Koch,,15,16 The present results build on this prior research. Mitchell et al and Litrenta et al identified overall ICC values of 0.71 and 0.68, respectively, for mRUST scoring of lower extremity fractures by North American trauma surgeons. By comparison, the overall ICC value of 0.64 identified here suggests that the reliability of mRUST scoring is relatively stable for surgeons operating in vastly different cultural contexts. However, inter-rater reliability was slightly higher within each subgroup of North American and Tanzanian surgeons than for the overall group of raters, which hints at possible differences across medical institutions and cultures in how surgeons applied the mRUST scoring technique for this study.The reliability of mRUST has previously been validated among North American surgeons and patients. found greater reliability in mRUST score distributions for distal femur fractures treated with intramedullary nailing (ICC = 0.74) compared with those treated with plate fixation (ICC = 0.59). Mitchell et al also found that tibial fractures treated with intramedullary nailing were associated with more reliable mRUST score distributions (ICC = 0.75) than those treated with external fixation (ICC = 0.62). By comparison, the results of our study indicate that mRUST scores were more reliable for fractures treated with external fixation (ICC = 0.72) compared with intramedullary nailing (ICC = 0.57). This discrepancy in findings, while unexpected, may be due in part to differential effects of image exposure levels on the visibility of radiographs depicting fractures treated by intramedullary nailing versus external fixation.Several previous studies have also investigated the reliability of mRUST across different treatment modalities. Litrenta et alCompared to reliability measures, validity measures of mRUST have received relatively less attention in the literature. Here, construct validity of mRUST was assessed by comparing average mRUST scores for a given patient and time point with health-related quality of life as assessed by the EQ-5D score at the same time point. The EQ-5D index score is a relevant clinical outcome measure designed to assess a patient's self-reported health and wellness at various points in the recovery process. The direct link that we discovered between a reasonably “objective” clinical tool (mRUST) and self-reported patient health at 6 months and 1 year postfracture is compelling evidence of construct validity because there is currently no gold standard method of assessing the healing status of open tibial fractures. However, we also found that mRUST scores were not significantly associated with EQ-5D index scores at earlier time points (6 weeks and 3 months). This negative finding may stem from the fact that many tibial fractures, and particularly those that are open, may take more than 3 months to show radiographic signs of healing. Meanwhile, quality-of-life measures at earlier time points may depend less on fracture stability than other factors such as wound healing or regaining range of motion.Another important finding of this study was that mRUST scores correlated significantly with surgeons’ estimated confidence that a fracture had healed at all 4 time points postfracture. These data support the content or face validity of the instrument. Furthermore, mRUST scores explained an incrementally greater proportion of variance in “healed” confidence ratings at each successive time point postfracture, suggesting a stronger association between these 2 methods of assessment of union at later time points. The confidence intervals displayed in Table This study had several important limitations. First, the radiographs available in our database were of varying quality (compared with North American standards), which could have affected the consistency of mRUST scores. We addressed this shortcoming by having an orthopaedic surgery resident (HJR) exclude poor-quality images and those in which the view of the fracture was obstructed. While this method resulted in greater consistency in image quality across the stimuli, it could have introduced some degree of selection bias into our findings. Another important limitation was that many of the surgeons who evaluated the radiographs had limited prior familiarity with the mRUST scoring method beyond the brief training that was administered at the start of each survey. This limitation could have yielded lower reliability scores than might be expected if the study were administered to surgeons with more prior training in the mRUST scoring method. Finally, our sample size was limited by the number of patients (25) who received complete radiographs at all 4 time points postfracture, due to loss to follow-up.In conclusion, this was the first study to validate the mRUST scoring method outside of the North American context. While no “gold standard” exists for evaluating the radiographic healing status of tibial fractures, our findings suggest that mRUST is reliable in diverse clinical contexts and aligns closely with patient-relevant clinical outcomes, particularly at later stages in the healing process. This work paves the way for further research into mRUST and other tools that can improve surgeons’ assessments of fracture healing in diverse and international clinical contexts.


After the lockdown of Wuhan on January 23, 2020, the government used community-based pandemic prevention and control as the core strategy to fight the pandemic, and explored a set of standardized community pandemic prevention measures that were uniformly implemented throughout the city. One month later, the city announced its first lists of “high-risk” communities and COVID-19-free communities. Under the standardized measures of pandemic prevention and mitigation, why some communities showed a high degree of resilience and effectively avoided escalation, while the situation spun out of control in other communities? This study investigated: 1) key factors that affect the effective response of urban communities to the pandemic, and 2) types of COVID-19 susceptible communities.This study employs the crisp-set qualitative comparative analysis method to explore the influencing variables and possible causal condition combination paths that affect community resilience during the pandemic outbreak. Relying on extreme-case approach, 26 high-risk communities and 14 COVID-19 free communities were selected as empirical research subjects from the lists announced by Wuhan government. The community resilience assessment framework that evaluates the communities’ capacity on pandemic prevention and mitigation covers four dimensions, namely spatial resilience, capital resilience, social resilience, and governance resilience, each dimension is measured by one to three variables.The results of measuring the necessity of 7 single-condition variables found that the consistency index of “whether the physical structure of the community is favorable to virus transmission” reached 0.9, which constitutes a necessary condition for COVID-19 susceptible communities. By analyzing the seven condition configurations with high row coverage and unique coverage in the obtained complex solutions and intermediate solutions, we found that outbreaks are most likely to occur in communities populated by disadvantaged populations. However, if lacking spatial-, capital-, and governance resilience, middle-class and even wealthy communities could also become areas where COVID-19 spreads easily.Three types of communities namely vulnerable communities, alienated communities, and inefficient communities have lower risk resilience. Spatial resilience, rather than social resilience, constitutes the key influencing factor of COVID-19-susceptible communities, and the dual deficiencies of social resilience and governance resilience are the common features of these communities. The global outbreak of COVID-19 has made prevention and mitigation of the pandemic an issue of urgent concern internationally  and its Wuhan is the capital of Hubei Province. The city is composed of 15 administrative districts and 1107 communities. Until 2020, Wuhan has a permanent resident population of 12,32 million, ranking the sixth-largest city in China . BesidesIn the Chinese context, communities are the first state-backed organizations  and hencOn February 24, the Wuhan New Crown Pneumonia Pandemic Prevention and Control Headquarters announced the first list of 278 “high-risk” communities and residential blocks in Wuhan  . On MarcResilience is originally a concept from physics that refers to the ability of a substance or system to return to its original position after being displaced , 14. In Like community resilience definition, there is no consensus as to how it should be measured. Depending on different conceptualization approaches and specific resilience domains to be assessed, measurement tools vary from quantitative methods relying on preexisting datasets, to qualitative and participatory framework using focus groups, interviews, and mixed approaches which combine quantitative and qualitative data . Among tDeveloping a proper and adequate community resilience measurement framework remains a complex challenge. Clark-Ginsberg et al. point out that “community resilience measurement is less about developing measures that can satisfy all design criteria and more about minimizing the negative elements of measurement choices and maximizing the positive” , and thaDespite their important theoretical and empirical contributions, the current literature presents some limitations. First, studies on community resilience mostly focus on the community’s ability to adapt to natural disaster risks in the context of climate change , researcTo complete the theoretical and methodological deficiencies of existing research, this study adopted a qualitative comparative method to investigate the influencing factors and their dynamic interactions that affect community resilience during the pandemic outbreak. Through the analysis of 40 urban communities in Wuhan, we identified key factors and common features that determine communities’ capacity for pandemic prevention and mitigation, as well as types of communities susceptible to the COVID-19 pandemic. This study aims to provide theoretical inspiration for urban communities to respond to the same type of public health risk.Qualitative comparative analysis (QCA) is a research method oriented toward small and medium sample case studies (10–40). Through a comparative analysis of causal conditions in multiple cases, it seeks to reveal multiple conjunctural causation relationships between conditional variables and outcome variables . Multipli configurations in total, i = number of condition variables). A specific configuration is the combination of some condition variables and an outcome variable, which might correspond to several observed cases. In this sense, QCA is useful for data exploration and case interpretation in a synthetic way, as a relevant tool for typology-building [At present, QCA has developed three research methods: crisp-set qualitative comparative analysis (csQCA), fuzzy-set qualitative comparative analysis (fsQCA), and multi-value qualitative comparative analysis (mvQCA). Among them, crisp-set QCA is the most widely used method by researchers –39. Its building . It can building .To examine whether there is a necessary and sufficient relationship between a given condition variable and an outcome variable, one should measure the consistency and coverage of a single condition variable in the overall sample cases.The univariate necessity and adequacy analysis are assessed by the consistency index. The formula for calculating the consistency index is as follows:If variable X  is a sufficient condition for Y (outcome variable), the consistency index should be greater than 0.8 . At the This indicator describes how well the condition (or combination) X interprets the outcome Y. The larger the value of the coverage index, the stronger the explanatory power of X to Y.This study chooses csQCA as the analysis method. The reason is as follows: First of all, the boundary of the condition variables and outcome variables involved in the study is relatively clear, and can be divided into two categories by “true” or “false”. Second, with the combination comparison and calculation of multiple concurrent conditions in multiple cases, a csQCA study can help identify the necessary conditions and sufficient conditions combination paths that lead to the emergence of COVID-19-susceptible communities. This can partly compensate for the limitations of current quantitative or qualitative researches on this issue. In addition, through the analysis of multiple concurrent configurations between condition variables and outcome variables, qualitative comparative research can also help reveal the complex dynamic interactions between factors that influence community resilience.This study integrated and improved the assessment frameworks put forward by Bruneau et al. and Peacock et al., and developed a community resilience assessment framework covering the four dimensions of spatial resilience, capital resilience, social resilience, and governance resilience. Each dimension is measured by 1–3 variables. As the total number of variables in a QCA is usually limited from four to seven , the stuThis term refers to the resilience of community physical space, building environments, and public infrastructure, such as community space planning, public facilities, characteristics of buildings and residential houses, and disaster prevention facilities , 23, 43 Communities incorporate a stock of human- and economic capital that defines their capital resilience. This resilience is defined by the community population characteristics, such as education and skills, the employment rate, and the average income level of families , 23. ComIt involves the quality and stock of a community’s social capital, which is reflected through the number of community social organizations, the relationship between residents, and their participation in community affairs , 23. ResIt refers to the level of a community’s governance capabilities and the abundance of governance resources. It is reflected through the completeness of the community governance structure, the community decision-making and implementation capabilities, and the adequacy of human and institutional resources on community governance , 33, 54.Lastly, as for the outcome variable “Whether it is a COVID-19 susceptible community”, we assign a value of 1 to high-risk communities and 0 to COVID-19 free communities . The data for the condition variable “whether community administrators have received complaints due to poor implementation of anti-pandemic measures” are taken from the “Leadership Message Board” of People’s Daily Online (liuyan.people.com.cn) and Weibo (weibo.com). Some information that is difficult to verify through the Internet, such as whether the physical spatial characteristics of individual communities are high-rise buildings or old-broken-small housing, was verified by residents in Wuhan through telephone interviews.Research data mainly are originally collected from government information disclosure via the Internet and social networks. The data related to the cases are taken from the China Community website; category 2(configurations 2 and 6) is sufficient in capital resilience but vulnerable in spatial, social, and governance resilience; category 3(configuration 7) is sufficient in spatial and capital resilience, but vulnerable in social and governance resilience.Among the seven configurations, six of the configurations are related to insufficient spatial resilience, four of the configurations are related to insufficient capital resilience. Spatial resilience thereby is the main factor that influences the degree of community resilience, the results confirm the findings of the above analysis of the necessary conditions for COVID-19 susceptible communities. Lastly, insufficient social resilience and governance resilience appear in all of the seven configurations, it’s the common feature of all COVID-19-susceptible communities.The above seven condition combination paths involve three types of COVID-19-susceptible communities. This article names them vulnerable communities , alienated communities (configurations 2 and 6), and inefficient communities (configuration 7). See Table Vulnerable communities covered 57.7% of susceptible community cases. Because of its overall weakening characteristics in the four dimensions of spatial resilience, capital resilience, social resilience, and governance resilience, vulnerability is its core feature, so this type of community is named a vulnerable community. Its condition combination is configuration 1, configuration 3, configuration 4, and configuration 5.First, in terms of spatial features, vulnerable communities fall into two categories. One is characterized by high-rise buildings and usually consists of social housing (such as Case 1), resettlement housing (such as Case 5), or urban villages . The other involves large non-gated old communities with poor common facilities and living conditions, namely old-broken-small communities. Second, in terms of human- and economic capital, vulnerable communities are urban areas populated by disadvantaged groups. The elderly, disabled, low-income households, and floating population account for a large proportion of the total community population. Third, the social capital of vulnerable communities is relatively weak. They are usually communities established after the year 2000 and belong to young communities. Residents come from different parts of the country, and their relationship networks and social bonds are weak. The apparent exceptions, such as cases 4 and 12, where the communities were established early on, however, share the common trait that they have only got a few social organizations. Therefore, they lack the channels and platforms for enhancing communication and cooperation among residents. Finally, vulnerable communities also belong to communities with low governance efficiency. These communities often lack governance resources. Except for the establishment of a general Party branch at the community level, there is no other Party branch, and community Party members have not been effectively organized and mobilized. This means these communities lack effective leadership organizations and active participants. In these communities, administrative members often receive complaints from residents in terms of competence and responsibility. Therefore, vulnerable communities lack robust resilience to effectively meet the needs of community residents for pandemic prevention and control.After being designated as high-risk communities, vulnerable communities mainly rely on the support and help of external resources to overcome the pandemic. During the period of COVID-19, the general practice of Wuhan City was to support high-risk communities one-on-one by higher-level official departments, establishing temporary Party branches that played the role of leadership, sending extra Party members and government employees to augment the community’s pandemic response personnel, relying on the authority of higher-level departments and Party organizations to mobilize community residents and organizations to work together to fight the pandemic.The main difference between alienated communities and vulnerable communities is that the former ones are not areas populated by vulnerable groups. The residents’ socio-economic status and human capital are in the middle or even the upper level of society. The reason why they are named alienated communities is that these communities are mostly young communities with a short history of establishment in which residents are strangers to each other, that is, alienated. Its condition combinations are configurations 2 and 6. The alienated communities cover 34.6% of COVID-19-susceptible community cases.The vulnerability of alienated communities is mainly reflected in three dimensions: spatial resilience, social resilience, and governance resilience. They are usually new communities built around the real estate economy after the rise of the commercial housing market. In terms of housing type, they usually involve high-rise modern buildings. The residents are similar in social and economic status, that is, the middle- or upper class. However, these communities are often loosely organized communities, with few social organizations and low participation in community affairs. Party branch building is imperfect. As it is difficult for the homeowners to establish an autonomy committee, the property management agency thereby lacks supervision, and its conflicts with the homeowners are numerous. Community residents’ committees often find it difficult to gain the trust and cooperation of residents because of their bureaucratism in daily work. Due to their high socioeconomic status, community residents have a relatively strong awareness of rights, and they often put higher service requirements on community administrators. Additionally, the existing property management agencies and residents’ committees cannot effectively meet the expectations of residents. As a result, the administrators of this type of community are often vulnerable to complaints and resistance from residents during the pandemic. In short, despite the strong capital resilience of alienated communities, the lack of resilience to the pandemic in the design and planning of physical residential space, as well as insufficient social resilience and governance resilience, leaves this type of community vulnerable to COVID-19.Inefficient communities do not involve high-rise buildings or old-broken-small housing, nor are they inhabited by disadvantaged groups, but are communities lacking both social resilience and governance resilience. Because of their low collective action efficiency and governance efficiency, they are called “inefficient communities.” Their condition combination is configuration 7. Case 6 and Case 14 in this study belong to this type of community, covering 7.7% of risk-susceptible community cases.Case 6 is a good illustration of inefficient communities. It involves a new community located in Qingshan District, Wuhan, established in 2002. According to the community’s information registered on the “China Community Network”: “The community covers an area of 141,700 square meters, with a total construction area of 222,000 square meters and a green area of 2.27 hectares. The community is surrounded by trees and has a beautiful environment. It is composed of five independent courtyards. There are 37 residential buildings, 160 unit doors, 2,289 households with 7,819 people, including 4 low-income households, 46 disabled people, and 243 elderly living alone.” In terms of physical space characteristics, the community has a low floor area ratio and a high greening rate. It is characterized by neither high-rise buildings nor old-broken-small housing, so it has strong spatial resilience. In terms of its capital resilience, the population of disadvantaged groups accounted for approximately 3.7% of the total population of the community. It is not a community populated by disadvantaged groups. It has strong resilience in human capital and economic capital. However, the community was established in 2000, it is a young community. The number of registered social organizations is only 3, which is lower than the average of 40 community cases. The community has only one Party branch composed of three Party members. The number of Party members is limited. The community has not received any reward from high-level governments. Moreover, during the pandemic, the community administrators received complaints from residents for not being “serious and responsible” . The main reason for inefficient communities to become pandemic-susceptible communities is their weak social resilience and governance resilience; therefore it is difficult for them to resist the spread of the virus during the outbreak.The results of analysis of necessary conditions of the seven variables revealed that spatial resilience is the key influencing factor of COVID-19 susceptible communities, and the seven condition configurations showed that the dual deficiencies of social resilience and governance resilience are the common features of these communities. These issues are further discussed below.From the aforementioned univariate necessity analysis, it can be seen that insufficient spatial resilience constitutes the key factor of pandemic-susceptible communities. Among the 26 high-risk community cases, 22 cases lack spatial resilience, with a case coverage rate of 84.6%. Two types of communities are characterized by insufficient spatial resilience: First, residential communities are dominated by large-scale high-rise buildings, with high population density and frequent intercrossing between residents. The vertically enclosed shared facilities of high-rise buildings, such as elevators, ventilation facilities, and sewage facilities, can easily cause the virus to spread in the same building space. The second is the “old-broken-small” communities. Residential buildings usually have poor ventilation and lighting conditions, lack of basic sanitation facilities (it is common for many people to share toilets and kitchens), and poor environmental sanitation  making iAmong the 26 high-risk community cases, a total of 15 communities involved disadvantaged groups inhabited areas, accounting for 57.7%, and 13 of the latter lacked spatial resilience. However, among the 14 COVID-19-free cases, there were also 7 communities inhabited by disadvantaged groups. Among them, two communities lacked spatial resilience, but had strong social resilience and governance resilience; the other 5 communities had strong spatial resilience, social resilience, and governance resilience. The above data show that the key reason why communities inhabited by disadvantaged groups have become pandemic-susceptible communities may be that these groups often live in communities that lack spatial resilience, not their weak human and economic capital resilience. As long as communities lacking capital resilience have sufficient social resilience and governance resilience, they can also be protected from the impact of the pandemic.If we closely examine the 14 cases of COVID-19-free communities, it can be found that communities inhabited by disadvantaged groups with sufficient social resilience and governance resilience are all old neighborhood communities with numerous social organizations. In this type of community, residents are relatively homogeneous in member structure and status. Most of the residents have known each other for decades, they have relatively stable and continuous social networks, and there exist mutual trust and cooperation relationships with each other. Traditional authority and administrative organizations, such as Party branches and residents’ committees, are relatively efficient, and they enjoy a high degree of authority and recognition among residents, so they have strong mobilization capabilities to enforce specific measures. The existing endowments and resources of the community can be converted into effective collective action efficiency and governance efficiency at the critical moment of disaster, providing resilience for efficient disaster response.Although each of the three types of COVID-19 susceptible communities has its unique characteristics, they share common features with a lack of social resilience and governance resilience.Firstly, communities susceptible to COVID-19 are often communities low on organization and resident participation, in other words, lacking social resilience. In these communities, residents have not formed cooperative relationships based on mutual trust. Instead, the residents are unlikely to provide mutual assistance in the face of disasters, and mainly rely on the residents’ committee, property management agencies, and higher-level governments to fight the pandemic,It is worth discussing that the number of social organizations is usually regarded as an important indicator to measure the stock of social capital in a community , 23. CanSecondly, COVID-19-susceptible communities have got insufficient governance resilience. The imperfect governance structure and the replacement of communities’ autonomy with administrative management are common features of most urban communities in China. It is common for old community residents’ committees to take charge of property management, while new commercial housing communities often lack the self-governing organization, namely a homeowners’ committee. An outstanding problem brought about by the governance structure is insufficient human resources for community governance. Once the community was hit by a critical disaster, the community’s emergency management personnel was overwhelmed. Moreover, the extension of the hierarchical administrative management style in communities not only leads to the weak self-government ability of the community residents, but also makes it difficult for the coordination of multiple governance bodies such as the grass-roots formal organizations (residents’ committees), informal organizations , market organizations (property management agencies), and residents. The hierarchical administrative relationship between residents’ committees and higher-level governments has also led to the lack of initiative and autonomy of the former. Thereby, in the process of disaster response, residents’ committees usually show behavioral inertia and passively execute orders and tasks of higher-level governments, cannot act decisively, autonomously, and respond flexibly in the face of a crisis. Lack of decision-making capacity and adaptability of policy implementation to local conditions constitute two important reasons why some communities become high-risk communities.Based on the assessment framework of community resilience, this study adopts a crisp-set qualitative comparative analysis method to examine the pandemic prevention and control capacity of 40 communities in Wuhan during the COVID-19 pandemic. Subsequently, the paper discusses the influencing variables and their configuration paths affecting the risk resilience of urban communities. The study found that vulnerable communities, alienated communities, and inefficient communities constitute three types of COVID-19-susceptible communities. Vulnerable communities inhabited by disadvantaged groups are not uniquely vulnerable to the pandemic as middle-class and wealthy communities can also be high-risk areas. The spatial dimension, rather than the capital dimension, constitutes the key influencing variable that affects the risk resilience of communities. Insufficient spatial resilience constitutes a necessary condition for COVID-19-susceptible communities, and its influence exceeds the human and economic capital dimension.The contributions of this study are double: First, based on the QCA approach, we have identified the key influencing factors of pandemic-related community resilience, which is a missing element in the current literature; Second, through analyzing the consistency of seven condition variables, we discovered that the spatial resilience rather than capital or social resilience constituted the key factor of community resilience in the situation of a pandemic, finding constitutes a novelty compared to existing ideas which tend to emphasize the imbalanced importance of social network or socio-economic capital of the community. This study pointed out the main reason that communities populated by disadvantaged populations are vulnerable to COVID-19 might be the physical structures and environment of these communities usually lack spatial resilience, rather than their disadvantaged socio-economic capital.Our findings suggest city policy-makers in new similar crises pay more attention to communities and places that lack spatial resilience, including identifying these places beforehand, taking appropriate risk precautions such as strengthening pandemic surveillance and prevention, taking locking-down measures in very early stage, transferring populations to places with strong spatial resilience if necessary. Meanwhile, this study confirmed the existing hypothesis that COVID-19-susceptible communities are the result of a comprehensive combination of weaknesses in multiple resilience dimensions. Empowering communities’ social capital and economic capital, as well as improving their governance capacity, constitute indispensable measures to improve a given pandemic susceptible community’s ability to respond to the disaster.


Wnt1-Cre lineage specific role of Gpr161 during mouse embryonic development. We observed three major gross morphological phenotypes in Gpr161 cKO  fetuses; protrusive tectum defect, encephalocele, and craniofacial skeletal defect. The overall midbrain tissues were expanded and cell proliferation in ventricular zones of midbrain was increased in Gpr161 cKO fetuses, suggesting that protrusive tectal defects in Gpr161 cKO are secondary to the increased proliferation of midbrain neural progenitor cells. Shh signaling activity as well as upstream Wnt signaling activity were increased in midbrain tissues of Gpr161 cKO fetuses. RNA sequencing further suggested that genes in the Shh, Wnt, Fgf and Notch signaling pathways were differentially regulated in the midbrain of Gpr161 cKO fetuses. Finally, we determined that cranial neural crest derived craniofacial bone formation was significantly inhibited in Gpr161 cKO fetuses, which partly explains the development of encephalocele. Our results suggest that Gpr161 plays a distinct role in midbrain development and in the formation of the craniofacial skeleton during mouse embryogenesis.Sonic hedgehog (Shh) signaling regulates multiple morphogenetic processes during embryonic neurogenesis and craniofacial skeletal development. Gpr161 is a known negative regulator of Shh signaling. Nullizygous Gpr161 mice are embryonic lethal, presenting with structural defects involving the neural tube and the craniofacies. However, the lineage specific role of Gpr161 in later embryonic development has not been thoroughly investigated. We studied the  Sonic hedgehog (Shh) signaling is one of the critical mammalian morphogen signaling pathways that regulates dorsoventral neural tube patterning , neural Mammalian neural crest cells (NCCs) arise from the dorsal neural tube which delaminate from their site of origin and subsequently migrate and differentiate into the designated cell types in peripheral organs . SeveralGpr161 hypomorphic mutant mice had both congenital cataracts and spina bifida , and facial nerve ganglia (VII) at E9.5. By E11.5, the Cre expression was widely expanded into the neural tube, including the mid/hindbrain and most of the orofacial and pharyngeal arch regions   or Gpr161f/f (flox control) did not show any similar abnormal phenotypes  . The Gprenotypes . Spinal the face . We alsond E18.5 .Gpr161 cKO fetuses  . The pHH3 positive cells tend to be located in the mitotic zone of VZ  , whereasne of VZ  in WT wh fetuses . In addi fetuses .Gpr161 cKO fetuses, we measured Shh and Wnt signaling activities within dissected midbrain tissues from floxed/Cre controls and Gpr161 cKO fetuses  and β-catenin , were increased, and one of Wnt target genes, CyclinD1, was increased , Gpr161f/+;Cre/+ (Cre control), and Gpr161 cKO  fetuses at E13.5 , Fgf8, Spry1 , Hes3 , Wnt1  and Draxin  (Gpr161 cKO fetuses. The Gene Ontology (GO) analysis further demonstrated that DEGs were highly enriched in the processes involved with neurogenesis, neuronal differentiation, neuronal morphogenesis, and mitotic cell cycle (Gpr161 cKO.To further unbiasedly identify the molecular basis of protrusive tectum phenotypes in at E13.5 . The hea control . The topgulated) . These rll cycle . Taken tGpr161 cKO fetuses. We observed up to 69% of the Gpr161 cKO fetuses had encephalocele  and Alizarin Red S . We observed a significant loss of mineralized skull and facial bones in Gpr161 cKO fetuses at E17.5  during embryonic midbrain morphogenesis.As previously reported  and shownd bones . The brastin-Cre . We also fetuses , indicatncreased , 4B, andncreased  was inhid Nkx6.2 , which wf Gpr161 . Corticaferation . As PtchCyclinD1, was increased  were generated and graciously provided by Dr. Saikat Mukhopadhyay  and the detailed information was previously reported (Wnt1-Cre (#009107), Nestin-Cre (#003771), and Rosa26-lsl-LacZ (#003474), were purchased from Jackson laboratory. The genotypes of the mice and embryos/fetuses were determined by PCR-based genotyping.All mice were housed and handled according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of The University of Texas at Austin. reported . The traWnt1-Cre and Rosa26-LacZ. The harvested embryos were fixed, and β-gal staining was performed according to the manufacturer’s instruction (Millipore Sigma). The images were captured by Leica stereomicroscope with a Nikon digital camera.Embryos were collected at E9.5 and E11.5 from timed mated breeding pairs between Gpr161f/f and Gpr161f/+;Wnt1-Cre/+. Collected fetuses were fixed, paraffin embedded, and sectioned with 4 um-thickness. The paraffin sections were deparaffinized, dehydrated, antigen-retrieved, blocked (blocking solution: Thermo Fisher scientific), and incubated with primary antibodies  diluted with Lab Vision™ Antibody Diluent Quanto (Thermo Fischer scientific) overnight at 4°C. After washing, sectioned were incubated with Horseradish peroxidase (HRP) polymer conjugate  and DAB (Boster Bio). The sections were counterstained with hematoxylin (Thermo Fisher scientific). Images were captured with All-In-One Fluorescent (Keyence) microscope using a ×2 and ×20 objective. To assess the positive cells for each proliferation marker Ki-67 and pHH3 in the dorsal midbrain regions of each embryo, the images were digitized and analyzed with Fiji (NIH), with image analysis. For quantification, a total of 4–6 fields per dorsal midbrain regions per each embryo were captured at ×20 magnification. Each field was divided into 100 equal squares and subjected to color deconvolution. For each of the images, the Shanbhag threshold (Fetuses were harvested at either E13.5 or E15.5 from timed mating between hreshold  was appl® 800CW goat anti-rabbit IgG and 1RDye® 680CW goat anti-mouse IgG secondary antibodies (LI-COR). The images were captured by Odyssey® (LI-COR).The midbrain tissues were dissected from E13.5 mouse fetuses as described . The disThe dissected midbrain tissues from E13.5 mouse fetuses were lysed with Trizol and total RNA was purified with Direct-zol RNA kit (Zymo research). For quantitative RT-PCR, 500 ng-1 µg of RNA was used to synthesize cDNA using iScript reverse transcription Supermix kit (Bio-Rad). The quantitative RT-PCR was performed using SsoAdvanced™ Universal SYBp < 0.05 genes with <1 count per million (cpm). Any gene with a p-value greater than FDR, after Benjamini-Hochberg correction for multi-testing, was deemed significantly differentially expressed under the test condition as compared to the control. The dataset was analyzed by the Gene Ontology (GO) enrichment analysis.The tissues were harvested as described in Midbrain dissection and total RNAs were extracted with Direct-zol RNA kit (Zymo Research). The quantity and integrity of RNAs were analyzed by Nanodrop (Thermo Fisher) and Bioanalyzer (Agilent Technologies). The library was prepared with NEBNext Ultra RNA with Poly-A selection and (NEB) was sequenced on an Illumina Hi-Seq 4000 . The differential gene expression was determined with fold change >1.5 and 2O and 90% ethanol) for 3 days at 37°C. After washing, samples were kept in 1% KOH for 48 h. For long term storage, specimens were transferred into 20, 50 and 80% glycerol solutions and were ultimately maintained in 100% glycerol. The images were captured by a Leica stereomicroscope with a Nikon digital camera.Skeletal staining was performed using a modified Alcian Blue/Alizarin Red staining procedure . BrieflyThe E17.5 fetuses were fixed with 10% formalin followed by 70% ethanol. Specimens were scanned at the University of Texas High-Resolution X-ray CT Facility using the flat panel detector on a Zeiss Xradia 620 Versa. The X-ray source was set to 70 kV and 8.5 W with no filter. A total of 2001 0.1s projections were acquired over ±180 degrees of rotation with no frame averaging. A source-object distance of 18.0 mm and a detector-object distance of 251.7 mm resulted in 9.98-micron resolution. The resulting data were segmented in Avizo software v.2020.2.The experiments were done in triplicate Unless specifically stated otherwise and the data was analyzed by the Standard Deviation (SD) with student t-test for comparing groups. For IHC data, the statistical analysis was performed with 2-way ANOVA, followed by Tukey’s test for multiple comparison (GraphPad Prism9).