To investigate whether intraoperative triggered electromyographic (T-EMG) monitoring could effectively reduce the breach rate of pedicle screws and the revision rate.Patients with posterior pedicle screw fixation from L1-S1 were enrolled between June 2015 and May 2021. The patients in whom T-EMG was utilized were placed in the T-EMG group, and the remaining patients were considered in the non-T-EMG group. Three spine surgeons evaluated the images. The two groups were divided into subgroups based on screw position  and breach degree (minor and major). Patient demographics, screw positions, and revision procedures were reviewed.A total of 713 patients (3403 screws) who underwent postoperative computed tomography (CT) scans were included. Intraobserver and interobserver reliabilities were perfect. The T-EMG and non-T-EMG groups had 374(1723 screws) and 339 (1680 screws) cases, respectively. T-EMG monitoring efficiently reduced the overall screw breach . in the subgroup analysis, the medial/inferior breach rate was higher in the T-EMG group than in the non-T-EMG group ; however, no difference was observed between the lateral and superior breaches (p = 0.064). A significant difference was observed between the minor  and major  medial or inferior screw breach rates. Six screws  underwent revision, with a significant difference between the groups .T-EMG is a valuable tool in improving the accuracy of screw placement and reducing the screw revision rate. The screw-nerve root distance is vital in causing symptomatic screw breach.The study is retrospective registered in China National Medical Research Registration and Archival information system in Nov 17th 2022. Low back pain is highly prevalent, and the main cause of years lived with disability in the adult population with an estimated lifetime prevalence of 70–85% , 2. FromHowever, pedicle screw fixation is not without risks. The literature reported that the pedicle screw breach rate ranges from 5.5–40% , 10. MosPrevious methods to decrease the pedicle screw breach rate include pedicle ball-tip probes and intraoperative X-ray fluoroscopy, but these methods have drawbacks. The reports found that the pedicle ball-tip probe was not suitable as an assessment tool alone, as it had a false negative effect when detecting the pedicle wall . MoreoveFurthermore, literatures reported several new methods to reduce the screw breach rate. The use of intraoperative O-arm navigation or intraoperative computed tomography (CT), a novel technique with increasing utility in recent years that is expensive and radiologically hazardous , 17, remIntraoperative neurophysiological monitoring, which is widely used in spinal surgery, plays an important role in reducing intraoperative nerve injury , 25. TriWe retrospectively analyzed the data on posterior lumbar pedicle screw position with  or without  intraoperative T-EMG monitoring based on postoperative CT in our hospital to investigate whether intraoperative T-EMG monitoring could effectively reduce the breach rate of pedicle screws and the revision rate of screw breaches.This study was conducted in accordance with the Declaration of Helsinki and was approved by the ethical committee of our university hospital. As the current study was retrospective in nature and data were analyzed anonymously, this study was exempt from requiring informed consent from patients.We retrospectively identified patients who underwent posterior pedicle screw fixation at our hospitals between June 2015 and May 2021. These patients were operated on by a senior attending surgeon. The inclusion criteria were as follows: (1) indications for spinal surgery were spinal fracture, lumbar spinal stenosis, lumbar disc herniation, spondylolisthesis, scoliosis, tumor, and spondylodiscitis; (2) pedicle screws were placed from L1-S1; (3) immediate postoperative CT scan of the operative lumbar spine.The exclusion criteria were as follow: (1) preoperative CT scan without an intact pedicle wall, (2) congenital dysmorphic pedicle features, (3) hollow pedicle screws, (4) patients who underwent minimally invasive surgery with percutaneous screw insertion, and (5) the vertebras for which the surgeons determined not to reinsert pedicle screws after t-EMG ≤ 15 mA.A total of 713 patients with posterior pedicle screw fixation at L1-S1 levels were selected. Since the intraoperative EMG monitoring system was available in our hospital from April 1st, 2018, these patients were divided into two groups according to intraoperative EMG monitoring utility: (1) T-EMG group, patients in whom intraoperative T-EMG was utilized , and (2) non-T-EMG group, patients without T-EMG . Patient demographics, indications for surgery, surgical information, operative level, and pedicle screw position on postoperative CT scans were reviewed. The revision cases were recorded.Pedicle diameter and length were measured preoperative on CT scans of all patients to evaluate the proper screw size. The patients were placed in the prone position on a radiolucent operating table. After completing subperiosteal dissection, the pedicle entry point was identified at the junction of the transverse process with the superior articular process of each vertebra. After the starting point was identified, an awl was used to access each pedicle, followed by a pedicle probe. After confirming four walls and a floor of the pedicle with a ball-tip sound probe, the pedicle trajectory was tapped with an undersized tap. Titanium alloy pedicle screws  were placed bilaterally in a standard fashion. Intra-operative anteroposterior and lateral images were obtained in both groups to confirm the pedicle screw positions. The intra-operative tangential view of the medial and lateral pedicle walls was obtained to confirm the breach of the pedicle wall if necessary.In the T-EMG group, each pedicle screw was individually tested by an experienced neurophysiologist. The triggering EMG technique was based on the initial report by Calancie et al . StimulaWhen the screw was stimulated at a threshold of ≤ 15 mA or had shown any breach sign of the medial pedicle wall on the intraoperative tangential view, it was identified as a suspected pedicle breach. All suspected violations identified by trigger-EMG . The spine protocol generated 0.5-mm source slices. The dosage parameters were 120 kV and software-based modulated mAs, with a maximum of 200 exposures. The raw data reconstructed sagittal and coronal sections of 0.5 mm thickness. According to the Laine  classifiPatient who suffered persistent radiated pain or neurological deterioration after surgery was first evaluated by X ray, CT, MRI, and inflammatory markers to clarify the exact reasons. Initially, patient was treated by conservative treatment if there were malposition of the pedicle screws without other reasons such as a nerve root compression by migration of the cage or grafted bone, haematoma formation, infection. A second surgery to revise the breached screw without T-EMG monitoring was performed when conservative treatment was failure. Before removing the violated screw, the breached medial pedicle wall and the adjacent nerve root were clearly exposed, the nerve root was carefully retracted to central canal to avoid being curled by backing rotated screw. After re-inserting the screw, in addition to the use of intraoperative fluoroscopy, the medial pedicle wall and the adjacent nerve root were also confirmed again to avoid directly contact. The postoperative CT scan after the second surgery was obtained.Statistical analysis was performed using the SPSS software (version 26.0) for Windows . The Student’s t-test for continuous variables which matched a normal distribution, chi-squared test, and Fisher’s exact test for categorical variables were used with a P value < 0.05 as statistically significant. All the tests were two-tailed. The intraobserver and interobserver reliability were assessed by Cohen’s kappa test, with the values ≤ 0 as indicating no agreement and 0.01–0.20 as none to slight, 0.21–0.40 as fair, 0.41– 0.60 as moderate, 0.61–0.80 as substantial, and 0.81–1.00 as almost perfect agreement .A total of 713 patients with 3403 screws from L1-S1 on postoperative CT scans were included in the study. There were 327 men and 386 women, with a mean age of 60.51 years . There were 1680 screws in the non-T-EMG group, with 189 screws penetrating the pedicle wall. The overall screw breach rate was 11.25% (189/1680). There was a significant difference in the overall screw breach rate between the 2 groups  screws violated the medial/inferior cortex , and 26 (19.40%) breached the lateral/superior pedicle cortex in the T-EMG group. The number of screw breaches per total number of screws in each segment is presented in Table 0% screwsThe minor and major medial/inferior screw breach rates were 6.21% (107/1723) and 0.06% (1/1723) in the T-EMG group, and 8.33% (140/1680) and 0.60% (10/1680) in the non-T-EMG group, respectively. There were significant differences in major and minor medial/inferior screw breach rates in the T-EMG and non-T-EMG groups (Table Six screws (6 patients) underwent revision due to persistent postoperative neurological deficits, all of which were in the non-T-EMG group. There was one for grade 1 Fig. , one forPedicle screw breach in posterior lumbar surgery is common  and seveLaine et al.  classifiIn the present study, the overall pedicle screw breach rate in the non-T-EMG group (11.25%) was significantly higher than that of the T-EMG group (7.78%). When comparing by the subgroup, the medial/inferior pedicle penetration rate between T-EMG (6.27%) and non-T-EMG (8.93%) groups significantly differed. In contrast, the lateral/superior pedicle subgroup showed no significant differences . The results suggest that applying intraoperative T-EMG during posterior screw placement could effectively reduce the overall breach rate of the pedicle screw, especially for medial/inferior breaches. However, it did not reduce the rate of lateral/superior screw breaches. This was due to the anatomical relationship between the pedicle and relevant nerve root. The exit nerve root typically runs beside the medial or inferior wall. The screw-nerve distance was very close when the pedicle screw penetrated the medial/inferior pedicle wall. It is much easier for T-EMG monitoring to trigger a positive reaction . The curPerumal et al.  comparedstudies have reported that medial/inferior breaches exceeding 4 mm usually induce symptomatic neurological deficit , 11, 37.Based on our experience, we speculate that the screw-nerve root distance determines whether a breach causes a neurological deficit. Theoretically, a breach screw should contact the relevant nerve root to cause clinical symptoms. The possibility of screw-related neurological deficits is low if the screw is at a long distance from the exit nerve root. Montes et al.  discoverNot surprisingly, patients with major breaches were at a high risk for revision in the non-T-EMG group. Interestingly, although most of the minor breaches were asymptomatic 99.19%, 245/247), two patients in the non-T-EMG group required screw revision . Several studies have found that the accuracy of screw placement cannot reach 100% using 5-15mA , 51–55 a9/1723. SThere is no consensus on the screw stimulation threshold during T-EMG monitoring. A recently published meta-analysis suggested that the stimulation threshold at 8mA had higher specificity and sensitivity, while increasing the threshold resulted in higher specificity but not sensitivity . We seleOur study had several limitations. First, this is a retrospective study, and the groups were assigned based on the availability of a neuromonitoring device. Therefore, there are huge biases to select the patient cases based on the surgeons’ preference and learning curve as well as the difficulty and type of surgery. Second, the diseases were from a broad category, and the patient cohort contained different diseases with various bone qualities, anatomy, and pedicle diameter, which might complicate the comparison difference. However, most the patients had degenerative diseases, and the disease difference between the two groups was insignificant, the pedicle diameters of two groups were similar, which could minimize bias. Third, surgeries were performed at a long-time frame , surgeon’s experience was increasing during the past years, which bias the results. However, the spinal surgeries were performed by a senior attending physicians who was well trained with rich experience, which could reduce this bias.The T-EMG technique has good clinical utility for evaluating pedicle screw accuracy. Compared to intraoperative fluoroscopy alone, it is of great value in improving the accuracy of screw placement and reducing the rate of symptomatic pedicle screw breach and the related pedicle screw revision. The screw-nerve root distance plays a vital role in causing symptomatic pedicle screw breaches. T-EMG can be used as a standard tool for assessing the accuracy of pedicle screw placement to reduce incorrect screw placement.


Lactobacillus rhamnosus GG and okara at different concentrations (1–3%). Additionally, the synbiotic ice cream was subjected to physicochemical and sensory attributes over 60 days of storage. High viability of L. rhamnosus GG (8.17 log CFU/mL) was observed during storage at 3% okara. Moreover, adding okara at 2% or higher improved viscosity, reduced overrun, and maintained probiotic viability. When compared to the control (ice cream without okara), synbiotic ice cream exhibited a higher protein content and a lower fat level. The synergistic combination of probiotics and okara in ice cream is a potentially novel approach for developing functional ice cream. The addition of okara is not only helpful in increasing the nutritional value of the ice cream but will also be a way forward to minimize agricultural waste. Synbiotic ice cream developed in this study may be considered a potential functional food rich in protein and low in fat.There is an increasing challenge in probiotic viability and stability during food product formulation, processing, and storage. However, synbiotic functional foods have promising potential to deliver the targeted benefits. This study aimed to isolate the okara from soybean residue, and obtained okara flour was further characterized using Fourier transform infrared spectroscopy (FTIR) and scanning electron microscope (SEM). Synbiotic ice cream was developed by fortification with  The synergistic combination of probiotics and soybean residue (okara) in ice cream is a potentially novel approach for developing functional ice cream. Adding okara (soybean residue) helps increase the nutritional value of the ice cream as well as a way to minimize agricultural waste. To date, the demand for functional dairy products such as ice cream is getting more attention than other dairy products. Ice cream is a popular frozen dairy product with a satisfying flavor and texture that is mostly liked by all ages  like vitamins, and trace elements, okara is regarded as a cheap source of carbohydrates and proteins and may be used as a prebiotic  as previously described by dos Santos et al. . The oka2.3.2−1 range. Co‐adding 64 images with a spectral resolution of 4 cm−1 allowed for their transmission mode recording.The FTIR spectra of okara were done following the process described by Quintana et al. . The spe2.4L. rhamnosus GG inoculum was prepared in the Man Rogosa and Sharpe (MRS) broth and incubation at 37°C overnight. The bacterial cells were then centrifuged and washed twice in sterile peptone water. 100 mL of sterilized full cream milk was combined with 1 mL of L. rhamnosus GG (6.5–7.5 log10 CFU/mL) after 25 h of incubation. The resulting mixture was mixed with different concentrations of okara 1%, 2%, and 3% while continually stirring. The probiotic blend of okara was then used to formulate functional synbiotic ice cream.1% of the 2.5T1), 2% (T2), and 3% (T3). The control sample was prepared by mixing 1 mL of L. rhamnosus GG without okara (T0). Before physicochemical analysis was carried out, all of the samples were placed into plastic containers with a volume of 250 mL. The mixture was then aged for 20 h at 4°C. For physicochemical, microbiological, and sensory investigation, all samples were stored in a refrigerator at −20°C.The ice cream base was formulated as per the procedure illustrated by Ibrahim et al.  with sli2.62.6.1Fat, ash, total dietary fibers, and protein were evaluated by AOAC  method.2.6.2L. rhamnosus GG without okara (T0), L. rhamnosus GG + 1% okara (T1), L. rhamnosus GG + 2% okara (T2), and L. rhamnosus GG + 3% okara (T3) were weighed and calculated as shown below:Based on the method stated by Rinaldi et al. , the oveWhereby: IA and IB represent the corresponding weights of the ice cream mixture and the actual ice cream, respectively.2.6.3T0, T1, T2, and T3 were placed on a steel mesh screen and allowed to melt for 90 min at room temperature. The melting rate was then calculated according to the following equation:The melting behavior of ice cream was performed by the method described by Elkot et al. . 100 g o2.6.4T0, T1, T2, and T3 at room temperature.According to the method of Halim et al. , a refra2.6.5T0, T1, T2, and T3) was determined at 25 ± 1°C.By following the method described by Baú et al.  and usin2.6.6T0, T1, T2, and T3) as described by Acu et al. . The panelists received 15 min training sessions prior to the examination to become comfortable with the attributes and scaling methods. Color, taste, texture, and general acceptability were the sensory features that the panelists were asked about and were scored on a scale of 1–8, whereby 1–2 represent “don't like at all,” 3–4 represent “slightly dislike,” 5–6 represent “like,” and 7–8 represent “extremely like.”The sensory analysis rating procedure of synbiotic ice cream was employed following the protocol proposed by Falah et al. . At the 2.9p < .05). Various okara contents (1–3%) were considered as independent variables, whereas physicochemical, microbiological, and sensory analysis data were placed as dependent variables. The analysis was conducted using the 64‐bit edition of IBM SPSS statistics. However, the statistics were reported as mean and standard deviation (SD).The two‐way analysis of variance (ANOVA) was used to compare the results of the physicochemical, sensory, and microbiological analysis for statistically significant at 95%  than in T1 (14.90%) and T3 (12.87%). There were no significant differences between the treatment groups (p > .05). The decrease in fat content could be due to the high percentage of insoluble fibers in the increased concentration of okara, which potentially promote fat absorption. A study performed by Tong et al. (The results of this study demonstrated a decreasing trend in the fat contents with an increase in okara concentration. A high amount of fat (21.78%) was observed in g et al.  reportedT2 had a slightly higher (5.62 ± 0.55%) percentage than other tested samples and the control (3.25 ± 0.63%). Okara is a possible solid fermentation substrate that can decrease or decompose several anti‐nutritional components, including saponin, lectin, and trypsin, to create microbial protein feed , the overrun for synbiotic ice cream in T1, T2, and T3 ranged from 14.50% to 24.93% , followed by the T1, T2, and T3, which had 75.54%, 19.93%, and 18.77%, respectively, shown in Table On the other hand, the rate 97.8%, follow3.4.3T3 showed the highest TSS (39.00°brix) followed by T2 (37.22°brix) and T1 (36.42°brix). The TSS results for T2 were slightly greater than T1, indicating a considerable rise in viscosity level. Contrary to our findings, numerous studies have reported a low TSS value in ice cream with a high water‐freezing potential, contributing to more ice crystals and affecting the texture of the ice cream  viscosity values as shown in Figure The results related to viscosity and TSS are shown in Figure 3.4.4p < .05) lower values for its hardness on day 1 than the control group  different from one another; however, they were significantly (p < .05) lower than the control group. On days 40 and 60 of frozen storage, ice creams with okara 1% and 2% had a slight increase in hardness. It is interesting to notice that on the last day of storage (60 days), the ice cream with 3% okara contained no significant impact on the hardness when compared to the control. The differences in hardness during frozen storage might be due to the high fiber contents in okara. In this aspect, adding 3% okara would need little force to penetrate the hardness of the ice cream. In this study, it has been shown that okara contained a high amount of fibers which might be responsible for influencing ice cream texture by increasing the viscosity and freezing point depression by decreasing ice crystals size. This scenario might be influenced by microstructure changes, including fat stability, phase volume, and sizes of ice crystals in the ice cream  had significantly  difference. After 20 days of storage, the pH level in T1 (6.62) and T2 (6.64) was higher than in T0 (6.59) and T3 (6.61) treatments. There was a slight increase in the pH values on day 40, T0 (6.53), T1 (6.64), and T3 (6.57), while T2 dropped to 6.55. At day 60, T2 had a pH level that was significantly (p > .05) higher than T1, T3, and T0 group, which was 6.57, 6.50, 6.70, and 6.32, respectively. Our findings demonstrated that T0 had the lowest pH readings throughout the storage time, ranging from 6.32 to 6.95. This was probably due to the fact that T0 did not contain okara, thus causing the pH to drop to an acidic level, protecting probiotic bacteria from acidic conditions. Since L. rhamnosus GG can tolerate a pH as low as 2–2.5, it is widely considered that probiotic bacteria are more tolerant of acidic circumstances  affected during storage. On day 1 no significant difference among the treatment was observed. However, as the storage period increased a rapid change in the viable count was recorded. These differences ranged from 7.60 to 7.82 log10 CFU/mL. On day 20, the viability of L. rhamnosus GG in T3 is greater than in T0, T1, and T2. The viability started to rise in T2 (7.79 log10 CFU/mL) and T3 (8.02 log10 CFU/mL). In comparison, T0 showed the lowest counts of L. rhamnosus GG throughout the frozen storage time ranging from 7.60 to 7.82 log10 CFU/mL. Furthermore, the ice cream sample containing okara showed a more stable number of probiotics than the control samples.The probiotic bacteria T3 exhibited the most viable population of probiotic bacteria; this might be due to the highest protein concentration that serves as an essential source of nitrogen for L. rhamnosus GG growth. Okara is a significant source for solid‐state fermentation of microbial protein as food since it is thought to be an excellent supply of nutrients, including minerals, lipids, protein, and vitamins. Additionally, dietary fiber present in the okara may serve as a potential substrate for the growth of probiotics and safeguard the probiotics from cold damage and shear stress linked with the integration of small air bubbles during mixing and freezing shock. According to Ibrahim et al. . Our current study evaluated the sensory analysis of synbiotic ice cream after 60 days of frozen storage ; methodology ; writing – original draft . Fedrick C. Mgomi: Formal analysis ; writing – review and editing . Farhan Saeed: Data curation ; formal analysis . Aftab Ahmed: Data curation ; writing – review and editing . Aasma Asghar: Validation ; writing – review and editing . Sakhawat Riaz: Data curation ; writing – review and editing . Huda Ateeq: Formal analysis ; writing – review and editing . Yasir Abbas Shah: Data curation ; formal analysis ; writing – review and editing . Mahbubur Rahman Khan: Formal analysis ; validation . Yi Li: Supervision ; writing – review and editing . Muhammad Afzaal: Supervision ; writing – review and editing .This work was supported by the High Level Talents Support Program of Yangzhou University.The authors declare that they have no conflict of interest.This article does not contain any studies with human participants or animals performed by any of the authors.For this type of study, formal consent is not required.Corresponding and all the co‐authors are willing to participate in this manuscript.


An efficacious tuberculosis (TB) vaccine is critical to reducing the global burden of TB. TB vaccine trials require the identification of multiple sites globally that have both a high incidence of TB and the capacity to conduct a clinical trial. To expand the diversity of potential phase III TB vaccine trial sites to be considered for inclusion, we describe a novel epidemiologic method that incorporates approaches from a variety of public health practices. Our approach incorporates analytic methodology to enable quantification and validation of qualitative information from disparate data sources, and epidemiologic analysis to systematically assess site-specific TB epidemiology. The integration of robust data-driven practices, and more quantitatively focused analysis, allowed for the objective evaluation of sites, which resulted in the identification of sites and catchment areas with high TB burden that may not have been previously considered. This suggests that an integrated epidemiologic methodology, not traditionally utilized for clinical trial site evaluations, could be integrated into site feasibility assessments as it results in more rapid site identification and reduces unintended bias. Tuberculosis (TB) remains one of the greatest global health challenges with approximately 10 million cases reported to the World Health Organization (WHO) each year –4. WhileWhile several successful phase II studies of TB vaccines have paved the way for development of global registrational phase III clinical trials , the devTherefore, a phase III trial requires identification of a large number of clinical trial sites that recruit from communities with very high TB incidence to ensure sufficient enrollment of participants and adequate capacity to sustain the long follow-up time vaccine efficacy studies require. Identification of a sufficient number of quality sites is pivotal to ensure efficacy endpoints are met, however, traditional mechanisms of assessing site feasibility require a resource-intensive approach. Typically, for industry-sponsored clinical trials, timelines are tight and contract research organizations (CROs) have limited time and budget to determine the optimal strategies to target the research population. Most CROs implement a site feasibility assessment process using a questionnaire template that may or may not be customized for the specific study. To rapidly identify capable sites, there is also a tendency to revisit the same clinical research sites and seek information from familiar investigators or Key Opinion Leaders (KOLs), which limits the potential reach of studies. Additionally, the important epidemiologic risk factors of disease transmission for those sites are not evaluated in a systematic way that can inform clinical trial design and impact the incidence of efficacy endpoints . As a reWe combined methods across epidemiology, public health analysis, and data science to develop an expanded approach to site feasibility that incorporates an integrated assessment of disease epidemiology and more in-depth source evaluations and data validation which can impact the type of information that informs overall site feasibility. A combination of these methods can be applied to gather a sustained understanding or situational awareness of a specific disease, or biological event, In tandem, these analytic practices can also be utilized to identify and assess the individual factors within a given region, community, or population that impacts the degree and severity of disease transmission relevant to a location . MoreoveA study site selection for an epidemiologic study in preparation for a phase III clinical trial effort with a target population of healthy 15–34-year-olds at high risk of TB infection served as the foundation for our research. To systematically evaluate a large expanse of possible Phase III sites, we integrated epidemiologic assessments with traditional site feasibility processes to rapidly aggregate, vet, and analyze site-specific data. We also incorporated precise epidemiologic assessments to improve situational awareness of TB-related disease burden. Overall, the methodology comprised of four major components: 1) integrated epidemiologic source evaluations, 2) epidemiologic analysis, 3) indicator/attribute scoring, and 4) expert assessment . To faciIncidence, Site Capacity, Regulatory Landscape, Experience, and Accessibility  that influenced a site’s eligibility for inclusion in a clinical trial. Borrowed from traditional biosurveillance practice, which is loosely defined in epidemiologic practice as the act of surveying factors of a biological event to inform public health practice, ‘Indicators’ are the pre-defined factors that serve as the basis for extraction of relevant event-based data . As suchThis indicator, meant to access a site’s level of TB burden, was further divided into smaller attributes for evaluation. These included Reported Rate (IR1) of TB among population size 15–34-year-olds for years 2017, 2018, 2019, and 2020; Accuracy (IR2) of data as determined by study target population data, number of years of data provided, and if any additional TB prevalence data was provided ; Granularity (IR3) regarding specificity and characterization of the study target area; and Justification (IR4) of TB data based on the type of evidence a site was able to provide to validate TB frequency and incidence .The Capacity indicator was also further divided into attributes for evaluation, including Study Area Capacity (CP1), TPT Policies (CP2), and Lab Capacity (CP3) of the site. Study Area Capacity was based on reported proximity to international travel and/or shipping facilities; sites that were located in urban or peri-urban areas were more likely to have international travel and shipping capabilities nearby, and thus were awarded a higher score. TPT Capacity assessed reported current and planned updates to national TPT guidelines as site-specific TPT policies and implementation can impact the design and conduct of the clinical trial and evaluation of vaccine efficacy. Lab Capacity assessed necessary experience in collecting and processing blood and sputum samples, understanding of international lab shipping protocols, and ability to utilize nearby labs to expand capacity.Timeliness assessed the response times of potential study sites. Sites were scored higher if the turnaround time to provide their site information was 5 business days or less. Sites that took more than 10 business days to respond were differentiated from sites that did not respond at all to acknowledge the effort of these sites. Sites that were able to successfully provide the requested information, even if it was beyond the anticipated time frame, were awarded Timeliness points for taking the time to respond.Completeness evaluated the robustness of data collected from sites, including their ability to provide necessary supplemental information reflecting their epidemiological, laboratory, and implementation capacities.The Incidence-related questions were given a total weight of 60, the Site Capacity-related questions were given a weight of 20, and Timeliness and Completeness each were assigned a weight of 5, resulting in possible total score of 100. Questions were assigned to each attribute, sub-weighted, and assigned scoring options associated with those weights .We designed an initial FAT to collect, vet, and evaluate site-specific data related to Incidence and certain attributes of Site Capacity. The FAT was developed using Smartsheet’s ‘Forms’ capability, a collaboration and work management software tool that can be used to collect and assess large amounts of contextual data via a digital questionnaire . We are As data were received via a completed questionnaire, it was automatically input into the Smartsheet database for evaluation. Additional metadata collected included tracking dates sent, received, geographic locations, and points of contact for each site.We developed a standardized workflow  for asseWe conducted an assessment of Incidence based on the data provided by each site .Additional information regarding the ethical, cultural, and scientific considerations specific to inclusivity in global research is included in the Supporting Information .Promising sites were identified, and then further evaluated in a subsequent phase of the study (not described here) where Site Capacity, Regulatory Landscape, Experience, and Accessibility specific criteria would be evaluated.The initial FAT was sent to 221 sites in 25 countries across three continents. Of these, 191 completed the FAT for scoring. Some countries, such as South Africa and the Philippines were considered priority countries due to their high TB burden and previous experience in conducting TB vaccine trials. While we still assessed and scored sites in these countries (South Africa [n = 27 sites] and the Philippines [n = 8]), using our systematic method, these sites were ultimately selected for further evaluation based on their previous experience with conducting TB vaccine trials in high incidence areas, which was considered priority for the clinical trial. More than half of sites (51%) that completed the FAT required at least one follow-up that included clarification questions or requests for additional information that would allow for more accurate scoring. Using our methodology and workflow for scoring, it took us approximately 10 weeks to collect responses, score, conduct expert assessment, follow-up with sites, and finalize scores for all 191 sites.Site scores ranged from 40–92 out of a total 100 points (n = 191) . Sites tIn addition to identification of promising sites based on a systematic and quantitative evaluation, our methodology also allowed us to obtain a more granular understanding of the epidemiology of TB that was highly informative in identifying specific catchment areas. TB incidence reported at the national level may not necessarily reflect the local epidemiologic situation. We found that often local case notification or incidence calculated from site-specific data was higher than national TB incidence reported by WHO. The burden of TB has only been exacerbated by the COVID-19 pandemic due to disrupted case finding and treatment strategies, and the case numbers of TB are expected to increase substantially over the next several years , 16. TheThe level of detail and the granularity of the information we collected went well beyond a typical feasibility study. Our method enabled objective assessment of layers of data that included not only incidence data, but also epidemiological factors, demographic, and location/capacity-related information. This allowed us to conduct a deeper evaluation of TB burden at the local level not traditionally considered in site feasibility evaluations. We compared site value across disparate regions and locations in an objective, quantitative manner  that improved justification and decreased overall bias in site selection for a trial. In addition, the Score Cards could be sorted by various factors, such as by country, indicator, or attribute.The totality of a country’s site data serves as a justifiable means for referencing country-level factors that may influence clinical trial development. We found that site score comparisons at the country level were highly relevant to site selection because we could assume that similar extenuating factors contributed to incidence and reporting of TB case notification data. This helped rapidly determine which sites within a country were most promising for inclusion and therefore merited further assessment. For example, DRC was not originally considered a high priority country to include in the trial; however, there were several sites in the country with very high TB incidence. This, combined with high scores in the feasibility assessment, demonstrated that sites in DRC have both the incidence and capacity and should be considered for inclusion in the trial. In contrast, Kenya has historically been known to have high TB burden; however, the data provided showed that the sites in Kenya had lower TB incidence than expected, likely because of improvement in early detection and treatment of active TB cases.The incidence of TB is heterogeneous within a country and a beneficial outcome of this methodology was that it enhanced the equitable assessment of sites by focusing on site level data. CROs usually conduct site feasibility by reaching out to known sites, investigators, or KOLs which is not an optimal approach in identifying sites for Phase III TB vaccine clinical trials. Clinical trials often are not available or accessible to sites that work with underrepresented populations. These populations are often at the greatest risk of infectious diseases like TB and could benefit greatly from having access to novel therapeutics and interventions. Clinical trials are integral to improving and sustaining equity related to high burden of disease, yet <20% of trials are conducted in LMICs or developing countries . In addiNotably, utilization of this method for site feasibility required a multi-disciplinary team of epidemiological and clinical trial experts with relevant experience in the fields of infectious disease transmission and public health. Input from these experts, in conjunction with data analysts and public health experts, ensured the information was assessed within the appropriate epidemiologic context.Thus, we conducted this site feasibility study in an objective manner that reduces resource demands, enhanced the ability to objectively evaluate vital epidemiological data, and expands diversity of sites evaluated. Our novel approach accounts for variability across target populations, geographies, and capacities, ultimately striving for comparability in epidemiological measures reported across potential sites. Moreover, the use of standardized evaluation protocols increased confidence in the final grading and scoring of sites that was conducted in a time and cost-efficient manner.Our application of an integrated epidemiologic methodology to site feasibility allows for the operationalization of equity and accessibility to clinical trials. The rigorous assessment of indicators and attributes of a site’s epidemiology and capacity resulted in a systematic and objective evaluation of sites that reduced the influence of bias. We found that some sites had significant incidence and basic site capacity that could potentially yield robust results for a clinical trial but would have been unlikely to be considered through traditional site feasibility assessment methods. Using a systematic approach like the integrated epidemiologic methodology described here expanded the range of sites serving underrepresented populations and help provide more equitable access to life-saving therapeutics.S1 Questionnaire(DOCX)Click here for additional data file.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.S3 Appendix(XLSX)Click here for additional data file.


Hydrogen peroxide is a primary atmospheric oxidant significantin terminating gas-phase chemistry and sulfate formation in the condensedphase. Laboratory experiments have shown an unexpected oxidation accelerationby hydrogen peroxide in grain boundaries. While grain boundaries arefrequent in natural snow and ice and are known to host impurities,it remains unclear how and to which extent hydrogen peroxide entersthis reservoir. We present the first experimental evidence for thediffusive uptake of hydrogen peroxide into grain boundaries directlyfrom the gas phase. We have machined a novel flow reactor system featuringa drilled ice flow tube that allows us to discern the effect of theice grain boundary content on the uptake. Further, adsorption to theice surface for temperatures from 235 to 258 K was quantified. Disentanglingthe contribution of these two uptake processes shows that the transferof hydrogen peroxide from the atmosphere to snow at temperatures relevantto polar environments is considerably more pronounced than previouslythought. Further, diffusive uptake to grain boundaries appears tobe a novel mechanism for non-acidic trace gases to fill the highlyreactive impurity reservoirs in snow’s grain boundaries. This work shows that hydrogen peroxidereadily enters grainboundaries in ice and snow directly from the atmosphere. Acceleratedreaction rates in these reservoirs have been described with an impacton air quality and snow composition. Its presencethere might help better assess the environmental impact of the previousfindings on accelerated reactivity.Hydrogen peroxide . We use two distinctice morphologies for the DIFT: monocrystalline (MC) ice, which ischaracterized by large crystals domains with a reduced grain boundarynetwork, and polycrystalline (PC) ice, which has relatively more andsmaller crystal domains and a more extensive grain boundary networkwithin a comparable volume.To discernthe effect of the ice microstructure on the uptake ofH2O2 sourceto provide a steady gas-phase concentration of H2O2 in humidified N2 carrier gas, a flow tube wherethe H2O2 is passed over an ice sample that canbe by-passed, and an H2O2 detector to monitorchanges to the gas-phase H2O2 concentrationin the carrier gas downstream of the flow tube. The flow tube wasa DIFT, where a hole is bored through an ice block or an ice-coated-wallflow tube. A schematic of the experimental setup is shown in the SupportingInformation .The experiment consists of anHwww.icefactory.ch). The purchased ice block is not treated before use. Polycrystallineice (PC) is grown by freezing ultraclean water  at 253 K. From each MC and PC sample, one drilled iceflow tube (DIFT) is made by cutting the respective ice into blocksof 10 cm lengths and drilling a 6 mm borehole longitudinally throughthe middle of each block. At each end, the boreholes were widenedto approximately 9 mm to attach fittings by screwing in pre-heatedthreads , which temporarily melts the ice and, uponrefreezing, creates gas-tight connections. The connections also leavethe inner borehole diameter flush with the inner fitting diameterto allow for smooth flow transitions. Additional water is frozen aroundthe fitting external to the connector to increase durability. TheDIFT boreholes are 80 mm long. Four experiments were done with oneDIFT tube each on 2 days. Experiments started with the lowest H2O2 concentration, which then increased from experimentto experiment. DIFT tubes were sealed and stored at −20 °Covernight. Before the first experiment each day, the tubes were purgedwith humidified carrier gas for 30 min. Between experiments on thesame day, desorption experiments served to purge the tubes  has been purchased from the ICE Factoryof Switzerland (ubes see .33Thin DIFT cross-sections(250 μm) are prepared after drilling and imaged using a compoundoptical microscope with a nadir polarized light source as describedearlier.34 Briefly, the quartz tube is internally etched with a solutionof 5% hydrofluoric acid in water and subsequently is rinsed severaltimes with purified water . Afterrinsing, 8–10 mL of ultra-pure water  or laboratory Milli-Q water are pipetted into the quartz tube.Excess water is removed by orienting the quartz tube vertically for1 min and draining. An ice film is formed at 258 K by slowly rotatingthe coated flow tube in a snuggly fitted double-mantled cooling jacket.This procedure results in thin ice films with an average thicknessof 10 μm, as determined by mass. Quartz tubes of either 80 or45 cm in length are used in this work. Data obtained with ice madefrom ultra-pure and Milli-Q water are in excellent agreement. Foreach experiment, a new coating was prepared.Coated wall flow tube experimentsare done in quartz glass tubes with 8 mm inner diameters. The innersurface of the quartz tube is covered with ice.2O2 is introduced to the experiment by addinga small stream of N2 carrier gas over a frozen solutionof 30% H2O2  at248 K. For coated wall flow tube experiments, gas-phase H2O2 is dosed from a custom-built permeation tube. For this,a liquid solution of 30% H2O2  fills a 17.4 cm long, thin-walled fluorinated ethylenepropylene (FEP) tube sealed at both ends. The permeation tube is placedin a commercial permeation oven  at 323 K. Aflow of pure nitrogen (Carbagas 99.9995% purity) through the permeationoven transports the gas phase H2O2 into theexperimental setup.For the DIFT experiments,H2O2 is measured using a commercial H2O2 analyzer with a detection limit of 100 ppt, correspondingto 2.5 × 109 molecules per cm3 at 298 Kand 1 atm . The instrument relies ona fluorometric method, by which H2O2 is strippedfrom the gas phase in a glass coil and subsequently reacts with p-hydroxyphenyl acetic acid and peroxidase forming a fluorescencedimer. This method detects all peroxides in the solution. The reagentsused for the analyzer were: Potassium phthalate , EDTA , NaOH , formaldehyde , p-hydroxyphenyl acetic acid ,and peroxidase . The reagentsare dissolved in double distilled water (3478.2 Roth). The instrumentis calibrated with diluted liquid solutions of H2O2 , which are titrated againsta permanganate solution  ofcertified concentration.Gas-phase H2O2 analyzerand set to either 2000 mL min–1 STP (standard temperatureand pressure: 273.15 K and 1 bar) or 500 mL min–1 STP, respectively. The flow through the permeation source is constantat 1500 mL min–1 STP. For the drilled flow tubeexperiments, the flow via the H2O2 source isset to 20 mL min–1 and mixed with a humidified flowof 215–230 mL min–1 N2. Afterpassing the drilled flow tube or by-pass, the gas flow was dilutedwith 1900–2000 mL min–1 to quickly transportthe H2O2 to the analyzer operated with a flowof 1500 mL min–1.The tubing of the flow system consistedof perfluoro-alkoxy copolymer (PFA). The flow through the coated wallflow tube is controlled by the H35 The PC-DIFT sample . The desorption curves probe the releaseof H2O2 from the ice samples. They show similarshapes with rapid increases of the gas-phase H2O2 concentration when the carrier gas flow through the DIFTs is initiatedat t = 0 min. A slow decaying release follows forboth the MC-DIFT and the PC-DIFT. The shape similarity between suchuptake and desorption curves has previously been noted.38 Generally, the magnitude of H2O2 release observed during the desorption experiments scaleswith the gas-phase concentration of H2O2 thatthe DIFTs were exposed to during the uptake experiments. The highestrelease of H2O2 to the gas phase of 3.0 ×1011 molecule s–1 cm–2 (light red) and 3.3 × 1011 molecule s–1 cm–2 (light blue), yielded gas-phase maximum concentrationsof 11 × 1011 molecule cm–3 (lightred) and 12 × 1011 molecule cm–3 (light blue) in the desorption experiments for the MC- and PC-DIFT,respectively. These were observed in runs that followed uptake experimentswith the highest gas-phase concentrations . A qualitative explanation for thelonger residence time during the recovery experiments might be thatin this mode, the concentration gradient drives diffusion back towardthe air–ice interface, where the carrier gas passes the ice,and deeper into the ice. During the uptake experiments, the concentrationgradients drive the net diffusion of H2O2 exclusivelyfrom the air–ice into the bulk ice interior. From the surface,H2O2 readily desorbs and is carried away bythe gas stream once the desorption experiment begins. This leads tochanges to the concentration gradients that drive bulk diffusion anda diffusive flux in the reverse direction toward the carrier-gas–iceinterface establishes. However, because the H2O2 concentrations remain low in the interior of the DIFT tube bulkice, diffusion away from the air–ice interface also continues.The partial irreversibility on the time scales of the uptake agreeswith Conklin et al.,38 who found between33 and 47% recovery. That their recovery is lower than that foundin our PC-DIFT experiments might be due to a different grain boundarydensity in their samples and their longer experimental time scalesthat would allow for more extensive diffusion. Also, differences inpressure and flow tube geometry between individual flow tube studies,where the transport and retention of trace gases involve multiplere-adsorption steps, might hamper direct comparison. Nevertheless,Conklin et al.38 used sintered ice spheresthat likely provide a significant but unknown grain boundary density.33 Interestingly, a different behavior was observedfor the acidic HCl. McNeill et al.39 concludedthat HCl is reversibly adsorbed to grain boundaries and observed fullrecovery within experimental time scales of up to 50 min at −60°C.We further explore the impactof grain boundaries on the reversibility of H2O2 motivates quantification by comparing theobserved uptake curves with theoretical predictions. Such comparisonshave been used to identify the uptake mechanism at a wide range ofexperimental settings and time scales, tremendously impacting theshape of uptake curves.25 In 2O2 number density exitingthe DIFT (nDIFT(t)) normalizedto the initial concentration (n0) forthe coupled adsorption-diffusion model as detailed in the Supporting Information .42 Here, the length ofthe DIFT is denoted by x, the surface area of theDIFT borehole by A, the volume of DIFT borehole is V, ν̅ is the mean thermal velocity of the gasmolecules, u is the linear gas velocity, and t the time. The mass accommodation coefficient αc describes the fraction of molecules entering the condensedphase relative to those colliding with the interface. The time constantof Langmuir adsorption is denoted by λ. The free parameter wedetermine by fitting in this work is the product H√D, where H is the dimensionlessHenry’s solubility law constant and D is theH2O2 diffusion constant in the solid phase.The solubility H is defined as the ratio betweencondensed-phase concentration and gas-phase concentration of a species.43Our observationthat grain boundaries in polycrystalline ice provide a sink for gas-phaseH2O2 air–iceinteraction can be understood as a combination of adsorption to thesurface and the diffusive loss from the surface acting on longer timescales. We derive 3 and 7 cm s–1/2 for H√D for the MC- and PC-DIFT uptake curvesfrom the fitting. That the value of H√D is larger for the PC-DIFT compared to the MC-DIFT by afactor of about two clearly shows the importance of grain boundariesfor long-term, diffusive uptake of H2O2 intothe ice matrix. In this analysis, the derived H√D must be understood as a combination of a grain-boundary-related H√D and the H√D in crystalline ice. Taken that the solubility and thediffusivity of H2O2 in the ice crystal and thegrain boundary are identical for both DIFT tubes, we assign the differencein the H√D term to varyinggrain boundary numbers in both samples. This implies that both theloss rate of H2O2 and the apparent solubilityare enhanced in environmental ice and snow as a function of grainboundary content. In the following, we estimate H√D values for the diffusive loss of H2O2 into single crystalline ice to support the hypothesisthat this long-term loss process cannot explain the derived H√D alone. Neither the solubilitynor the diffusivity of H2O2 in single crystallineice is known. Supposing H2O2 has a similar solubilityand diffusivity in ice as HCl, H√D of 0.2–2.8 cm s–1/2 can be calculated at−25 °C based on published solubility and diffusivity data.44 The scatter reflects experimental uncertainty.The similarity of the observed H√D in MC-DIFT with this estimate indicates that diffusion into thecrystalline ice phase could explain a significant fraction of thelong-term trend observed in MC-DIFT experiments but not that in PC-DIFT.However, the solubility of HCl in ice is exceptionally high comparedto HNO3, another trace gas of atmospheric relevance. Solubilitiesin single crystalline ice (solid solution) of 0.1 mol/L for HCl versus0.001 mol/L for HNO3 have been found at partial pressuresof 0.2 × 10–3 and 0.7 × 10–3 Pa and temperatures of −25 °C.45 Just as H2O2, HNO3 is highly solublein water43 and, therefore, we estimatethe solubility of H2O2 in ice in the followingbased on HNO3 solid solution data. For HNO3, H√D values of 0.008–0.02can be derived, which are significantly smaller than the results evenfor MC-DIFT. If we assume that the solubility of H2O2 in crystalline ice is closer to HNO3 than to HCl,the contribution of the solid-state diffusion to the long-term uptakein our experiments is negligible, and consequently, diffusion intograin boundaries would dominate the long-term uptake of H2O2 to ice in both samples.2O2 for grain boundaries in environmentalsnow and ice might result in significant variations in the local concentrationof H2O2. This is relevant as such non-uniformchemical morphologies and highly concentrated patches of reactantsat grain boundaries and elsewhere in the ice have recently been shownto impact chemical reactivity in frozen systems strongly.46 Grain boundaries in natural snowpacksoccur at the contact face of individual snow grains.47 As grain size, shape, and arrangement change with timein snowpacks (metamorphism),48 detailedstudies observing the impact of grain boundaries at different stagesof metamorphism are timely to elucidate the mechanism and the environmentalimpact more extensively.The direct comparisonof the uptake to samples that differ in grain-boundarydensity and that the diffusive loss is larger than expected for diffusioninto the ice crystal strongly suggests that the observed long-termDIFT uptake is driven by diffusion into grain boundaries. The preferenceof H2O2 partitioningequilibrium between the ice surface and the gas phase . 18 The partitioning coefficient, KLinC, is defined as the surface concentrationof the adsorbed H2O2 in molecules cm–2, divided by the gas-phase concentration in molecules cm–3. The ice–air interface has become more disordered in thistemperature range.14 The consistent negativetemperature trend with extrapolated results from an earlier low-pressurecoated wall flow tube study between 203 and 233 K18 indicates that the presence of this disordered interface does notchange the uptake behavior of H2O2 up to 260K.Given thatthe uptake curves are well described by a combination of long-termdiffusion and short-term adsorption 3, numeri2O2 one of the stickier atmospheric trace gasescompared to volatile alcohols, aldehydes, and ketones. Its partitioningcoefficient is 2–3 orders of magnitude larger than that ofmethanol or acetone with 100 and 10–1 cm, respectively, at 240 K and even larger than that of acetic acidfor which a partitioning coefficient of 101 cm has beenfound at 240 K.49 For comparison, strongacids such as HCl have partitioning coefficients of 104 cm at 240 K.50 The reported partitioningimplies that a large fraction of H2O2 is foundadsorbed to surface snow and not in the gas phase in this temperaturerange typical for the Arctic. A surface area of 59 cm2 percm3 has been reported for freshly fallen snow,51 which yields 2 × 1012 moleculesof H2O2 adsorbed per cm3 of snowat 260 K and 7 × 1012 molecules per cm3 at 240 K with the partitioning coefficients reported in this work;while the air above snow holds 109 molecules cm–3. Because the air–ice interface of coated wall ice films issmooth on a molecular level,16 and thatof the DIFTs can be expected to be so because any initial surfaceroughness and microstructures are generally smoothed by sinteringwith time,52 the adsorption process andthe partitioning coefficients are invariant between ice and snow.Such gas-phase concentration of H2O2 used inthis estimate is typical in the Arctic.53 For technical reasons, experiments have been done with higher H2O2 concentrations of 1011 moleculesper cm3 in the coated wall flow tube experiments. Becausesurface coverages of H2O2 were low, in the 1013 molecules cm–2 range, extrapolation tothe even lower environmental gas phase and surface concentrationsis feasible.17 H2O2 and other polar trace gases adsorption saturate at a so-called monolayercoverage of ∼2 to 4 × 1014 molecules cm–2.18 In summary, the increase of H2O2 adsorbed to the snow by a factor of 3 when temperaturesdecrease by 20 K and the fast time response of the adsorbed fraction,as evident in 2O2 in the polar regions.7 Jacobiet al.7 described the complex interplayof physical partitioning and snowpack chemistry in polar H2O2 observations. This study shows strong partitioningof H2O2 to the snowpack at high temperatureswhere it is subsequently H2O2 available forchemistry. The impact of surface adsorption can be expected to decreasewith snow age as snow surface area reduces with time during metamorphism.54 The data in this work now give the basis toaddress the impact of various snow types and stages of metamorphism.The reported partitioning coefficients between258 and 235 K makeH2O2 shows the same equilibrium adsorptionbehavior as the three types of ice used in this study. Uncertaintymight come from the higher gas-phase and resulting surface concentrationsof 1.3× 1014 to 2.6 × 1014 moleculescm–2 present during the DIFT experiments . At these surface coverages approaching a monolayercoverage,18 adsorbate–adsorbateinteractions might start to alter the adsorption process.17 Additionally, diffusion into the sparse airpores .For this snowpack, an H2O2 adsorption of 2 ×1012 molecules per cm3 of snow has been estimatedabove. The flux to the grain boundaries exceeds the number of surfaceadsorbed molecules by a factor of 10 each hour. Interestingly, inexperiments with artificial snow Kerbrat et al.23 also found a 10 times increase compared to the surfaceadsorbed HONO each hour at 260 K. Apparently, the slower diffusiveHONO loss of 1.6 cm s–1/2 for H√D reported by Kerbrat et al.23 for these snow samples and the lower surfacepartitioning23 result in the same relativeincrease. This back-of-the-envelope comparison illustrates the unmatchedstrong affinity of H2O2 for ice surfaces andthe grain boundary network of snow. Similarly, a H√D of 0.02 cm s–1/2 forSO2, a weakly adsorbing trace gas,19 was found to explain the uptake to artificial snow with negligiblesurface adsorption. Kinetic modeling results revealed diffusive lossof HNO3 at 239 K and HCl at 213 K, corresponding to 10times and 0.7 times the adsorptive surface coverage per hour, respectively.39 Detailed investigation of the relative importance of adsorptionand grain boundary diffusion of these species is timely, with focuson the quantification of the grain boundary content in artificialand natural snow and wide variations of temperature and partial pressure.Our results put the relative importance of H2O2 diffusive loss on the level with the acidic trace gases, for whichthe observation of diffusive tailing in uptake curves has kicked offthis line of research.22The uncertainty range of both DIFT data points overlap, indicatingthat Hirpores 1 mightc PC-DIFT 2B emphas


C. elegans. This finding advances our understanding of cellular survival mechanisms under mechanical stress.Meng et al. report that upon plasma membrane damage in a cell, the Golgi apparatus accumulates at the wound site and facilitates membrane repair by converting PtdIns4P to PtdInsP2 in  C. elegans epidermal cell wounding model, we discovered phosphatidylinositol 4-phosphate (PtdIns4P) accumulation and local phosphatidylinositol 4,5-bisphosphate [PtdInsP2] generation at the wound site. We found that PtdInsP2 generation depends on the delivery of PtdIns4P, PI4K, and PI4P 5-kinase PPK-1. In addition, we show that wounding triggers enrichment of the Golgi membrane to the wound site, and that is required for membrane repair. Moreover, genetic and pharmacological inhibitor experiments support that the Golgi membrane provides the PtdIns4P for PtdInsP2 generation at the wounds. Our findings demonstrate how the Golgi apparatus facilitates membrane repair in response to wounding and offers a valuable perspective on cellular survival mechanisms upon mechanical stress in a physiological context.The maintenance of plasma membrane integrity and a capacity for efficiently repairing damaged membranes are essential for cell survival. Large-scale wounding depletes various membrane components at the wound sites, including phosphatidylinositols, yet little is known about how phosphatidylinositols are generated after depletion. Here, working with our in vivo   It mainll shape . Disruptll shape . PtdInsP2 generation at the wounded membrane in Caenorhabditis elegans epidermal cell. PtdInsP2 generation at the wound site depends on PtdIns4P, PI4K, and PI4P5K PPK-1. We further discovered that wounding triggers the enrichment of the Golgi membrane to the wound site to provide PtdIns4P, which serves as a substrate for PtdInsP2 generation during the membrane repair.Here we report that wounding triggers PtdIns4P2 using our previously described in vivo model for repairing large membrane wounds e wounds . For thie wounds  to labelpromoter . Spinninpromoter . We thenpromoter . PH::GFPpromoter , consistpromoter . The PH:promoter . The PH:promoter . Moreovepromoter , indicat PH::GFP . TogetheP2 at the wound site, we performed improved highly intelligent and sensitive-structured illumination microscopy HIS-SIM;  on animaHIS-SIM; . After nHIS-SIM; . Over tiHIS-SIM; . NotablyHIS-SIM; , reinforP2 is converted from phosphatidylinositol 4-phosphate (PtdIns4P), which is synthesized directly from phosphatidylinositol (PtdIns) by phosphorylation with PI4-kinase P2 generation in membrane repair. To visualize PtdIns4P in C. elegans hyp7, we labeled PtdIns4P by fusing the P4M domain of Legionella pneumophila SidM with the red fluorescent protein mKate2 P2 at the wound site was not due to the lateral diffusion from the unwounded area.PtdInsse PI4K; . We thenn mKate2 . The mKan mKate2 . Followin mKate2 . Unlike n mKate2 . To assen mKate2 , indicatP contributes to the generation of PtdInsP2 in membrane repair, we treated animals with Phenylarsine oxide (PAO) and KDU691, inhibitors of PI4K P2 generation depends on the PI4K activity and the accumulation of PtdIns4P at the wound site.To determine whether the PtdIns4 of PI4K , to redu PI4K P2 on the plasma membrane requires the specific enzyme PtdIns4P 5-kinase , in which two leucines (L) at the 218 and 226 positions were mutated into isoleucine  mutant resulted in a reduced accumulation of PH::GFP at the wound sites  of the Golgi apparatus transport pathway . We usedrab-1 resulted in no TGN-38::tagBFP and GFP::MANS-2 accumulation at the wound site (rab-1 increased the ratio of TryB-positive staining (rab-6.2 RNAi knockdown or utilized its loss-of-function of rab-6.2(ok2254) animals, the accumulation of the TGN-38::tagBFP was inhibited at the wound site P2 generation by expressing TGN-38::FRB and FKBP::SAC-1 in the epidermal cell to specifically deplete PtdIns4P on the Golgi membrane P2 generation at the wounded membrane.Notably, the wounding-triggered accumulation of the mKate2::P4M was colocalized with TGN-38::tagBFP, suggesting PtdIns4membrane . Before membrane . We thusund site . Moreoveund site . Similarund site . These rmembrane . We obsemembrane , suggestP2 generation after GSK-A1 treatment P2 generation during membrane repair.Although GSK-A1 treatment did not affect TGN-38::tagBFP accumulation at the wound site, it significantly inhibited the accumulation of mKate2::P4M , which ireatment . GSK-A1 S-2::GFP , suggestund site . TogetheP is used as a substrate for the generation of PtdInsP2 at the wounded site  and injected with a plasmid mix composed of 10 ng/μl gene of interest codon plasmid, 50 ng/μl co-injection marker, 50 ng/μl empty vector (blueprint plasmid) for an extrachromosomal array, 50 ng/μl SCI (single-copy-insertion) template plasmid, 50 ng/μl Cas9 with sgRNA coding plasmid, and with 2.5–5 ng/μl co-injection markers for SCI strains  to ensure efficient translation.The live imaging experiments were performed using young adult stage worms that were synchronized and grown. Prior to imaging, worms were picked and immobilized on 4% agarose pads using a small drop of 12 mM levamisole. Worms are mounted to Nikon Eclipse Ni and Z-stack images were acquired with a 100× magnification using a Nikon Plan Apo 100 X/1.40 oil microscope, with a 0.5 μm interval between each image.The HIS-SIM was performed according to the method previously described . The comrde-1(ne219); Pcol-19-RDE-1(juls346)) were placed onto HT115 Escherichia coli seeded NGM containing 1 mM IPTG and Carbenicillin. The worms were then incubated at 20°C until they reached the young adult stage to be subjected to further experiments.Adult epidermal-specific RNAi was performed as previously described . BrieflyDrugs were diluted in DMSO to make 10-mM master solution. The working solution was produced by diluting the master solution with M9 buffer seeded with OP50 to reach the working concentration, as indicated in each experiment. 100 μl aliquot of the working solution was placed on 96-well plates. Young adult worms that had been wounded were picked and placed into the drug solution and incubated at 20°C until the required time points for imaging. The DMSO group was taken as the control group.Microinjection needles were pulled from the capillary tube using a Narishige PC-10 Micropipette puller. For needle wounding, young adult stage worms were synchronized and placed under a dissection microscope. Two stab wounds were punctured using micro-injection needles for each worm as described previously . For lasWounded young adult stage worms were incubated until the respective time points for observation time points. Afterward, the worms were immersed in a 0.6% (weight/volume) solution of trypan blue  for 60 min. Observations were carried out under an upright microscope (Nikon Eclipse Ni). Number of TryB stained worms/total number of wounded worms × 100% were calculated for each experimental repeats to obtain the percentage of TryB positive staining percentage at a time point after wounding.The synchronized young adult stage worms were picked into 96-well plates containing a solution of the Golgi tracker-red  that was diluted 1:100 in BSA. The staining process was carried out according to the manufacturer’s instructions by submerging the worm in the staining solution for 20 min. The worms were then washed 3 × with M9 solution and subjected to wounding and imaging under the confocal microscope.http://rsb.info.nih.gov/ij/). For each worm, boundaries were drawn around the recruited fluorescence signal to quantify the absolute intensity (Iw). Two regions of the unwounded/wounded region were selected, and their average intensities were calculated to measure the intensity of the unwounded/wounded region (Iuw/Iw). The background intensity of each image was also quantified (Ibg). For each wound, Iw−Iuw/Iuw−Ibg was calculated to obtain the recruitment fluorescence signal (ΔFw/Fuw). Intensities measured were analyzed using GraphPad Prism 9 to generate a line graph.The fluorescence signals were quantified using ImageJ software  as described . Brieflyt), the region before photobleaching (Fu), and the region of background (Fb) were measured. The m) was calculated to evaluate the FRAP efficiency by the equation: 20 is the relative fluorescence intensity 20 min after photobleaching.For each image, the punctate signals were bleached using 100% laser transmission with an argon laser power is 30 mW, with 50% (15 mW) 405 nm laser. The fluorescence intensity was then imaged at 10-s intervals for 20 min and analyzed using ImageJ software. The relative fluorescence intensity of the photobleached region at each time point (Ft test (two-tailed unpaired) or Mann–Whitney test were used. For multiple comparisons, a one-way ANOVA test was used. Data distribution was assumed to be normal but was not formally tested. The results were considered statistically significant if P < 0.05. Statistically significant results were indicated with asterisks, where * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001. At least 10 different wound sites were quantified in each experiment, and each experiment was repeated at least twice.All experiments were conducted a minimum of three times to ensure accuracy and reproducibility. Statistical analyses were performed using GraphPad Prism 9. The average of the individual data was represented on the Y-axis of the graph, with the SD represented as an error bar. For two-way comparisons, Student’s C. elegans strains used in this study. C. elegans epidermal cell before wounding. C. elegans epidermal cell. Table S1lists reagents used in this study.Click here for additional data file.Table S2C. elegans strains used in this study.lists Click here for additional data file.Table S3lists plasmids used in this study.Click here for additional data file.Table S4lists primers used in this study.Click here for additional data file.


There was no statistically significant difference in terms of blood loss . These results suggest that orthopaedic surgery robot for the treatment of longitudinal sacrum fracture is safer and provides less irradiation than the traditional freehand methods.Pelvic fractures mostly result from high-energy injuries in life; the longitudinal fracture of the sacrum is the most common type of sacrum fracture. This study was designed to evaluate the accuracy, safety, and efficacy of percutaneous sacroiliac joint screw placement in the treatment of longitudinal sacrum fractures with the assistance of unobstructed orthopaedic surgery robots. According to different surgical methods, 32 patients were divided into robot group and free hand group, with 16 patients in each group. The operation time, intra-operative blood loss, intra-operative fluoroscopy times, screw placement angle deviation were collected. There were statistically significant differences in terms of angle deviation of screw placement (1.96 ± 0.75° vs. 2.87 ± 1.03°;  SacraHowever, due to the complexity of the anatomy around the sacrum, the placement of sacroiliac joint screws is prone to place the screw at an unsatisfactory angle and length during the operation and to break the screw during the operation if the screw is placed many times . The tec2.2.1.A retrospective analysis of the clinical data of 32 patients with sacrum fractures were admitted to the Affiliated Hospital of Inner Mongolia Medical University from September 2018 to December 2020. This study was approved by the Ethics Committee of Inner Mongolia Medical University Affiliated Hospital, and obtained the informed consent of all the subjects.2.2.Patients were included according to the following criteria: (1) Closed and unstable sacral fractures, with or without fracture displacement, can be treated with closed reduction (or limited open reduction), after which the sacroiliac joint has space for cannulated screw placement; and (2) An open, fixable sacrum fracture can be reduced to a closed fracture after initial treatment and can be reduced by reduction and screw placement.2.3.Exclusion criteria were: (1) Severe open injuries or rupture of abdominal cavity and organs with wound contamination; (2) The unavoidable blood vessels and nerves and other tissues in the trajectory planned by the robot assistant system, or patients whose sacroiliac joint do not have space for the hollow screw placement after reduction, or the patients who are unable to use screws to fix sacral fractures effectively; and (3) Patients with systemic diseases, such as severe haemorrhagic diseases, severe heart, and respiratory diseases, or peripheral skin or soft tissue infections.2.4.Thirty-two patients with longitudinal sacral fracture were divided into two groups: the robot-assisted group  and the freehand group. The robot-assisted group was treated with transverse sacroiliac joint screw implantation through the orthopaedic surgery robot assistant system, while the freehand group was treated with sacroiliac joint screw implantation by orthopaedic surgeons.2.5.Both operations were performed by the same group of senior doctors.2.5.1.All patients undergoing surgery underwent routine admission tests after admission. Temporary pelvic external fixation was used for patients with hemodynamic instability. Patients with vertical instability of pelvic fracture were treated with tibial tubercle traction of lower extremities. All patients underwent CT scanning. On the premise of ensuring these patients’ hemodynamic stability, patients with vertical instability of pelvic fractures were examined regularly by X-ray to observe the reduction effect of their lower limbs.2.5.1.1.AIOOR is a robot assistant system based on digital and minimally invasive surgery concept. The main body comprises a 6-degree-of-freedom manipulator, the main control trolley, and an optical tracking system .2.5.2.The patients were placed in a supine position. For patients with pubic ramus fracture, open reduction and internal fixation were performed on the pubic ramus after sacral reduction.2.5.2.1.An entry point that was determined on lateral sacral and antero-posterior views during intrao-perative fluoroscopic X-rays , 17. Wit2.5.2.2.According to the pelvic images obtained by C-arm fluoroscopy and the planned position of the screw pathway, a 2.5 mm Kirschner wire was inserted into the pelvic bone. Then, a 7.3 mm cannulated screw was inserted and fixed along the Kirschner wire.2.5.3.The patients received pelvic X-ray, CT scan, and 3D imaging reconstruction to confirm the precision of the position of the ilio-sacral screw. The deviation between the actual and the planned optimal screw position was measured. The measurement method is as follows: The pelvis was scanned using a spiral CT scanner  at 120 kV with a slice thickness of 0.625 mm and a matrix of 512 × 512 pixels. The generated images were converted into DICOM format and were further processed using medical imaging software  to obtain the STL format files for the 3D reconstruction of the pelvises. Visualization software  was subsequently used to produce multi-slice sagittal views (thickness of 1.0 mm) of the 3D reconstruction images. First, we defined the geometric boundary of the safe zone on each sagittal view for the first sacral vertebra. Thus, the inscribed ellipse of the boundary was obtained from each view. Subsequently, the X, Y, and Z coordinates of the center in each inscribed ellipse  were calculated. Finally, the least-squares methods were used to fit the optimal axis that pass through the centers of the inscribed ellipses . This axis was defined as the optimal and safe pathway for the ilio-sacral screw. Also, the CT scan data obtained after surgery (the same parameters as before) were imported into the Mimics Innovation Suite for 3D reconstruction, and the actual screw position was compared with the optimal pathway in the software to measure the angle deviation of screws.All patients received regular postoperative ambulatory follow-ups.2.5.4.According to postoperative imaging data and follow-up results, the overall clinical efficacy of sacroiliac joint screw placement in the treatment of longitudinal sacrum fracture was evaluated and scored according to the Majeed function system . The fol2.6.t test, and the comparison of measurement data at different time points in the same group was conducted by repeated measurement ANOVA. LSD t test was used for multiple comparisons, and non-parametric test was used for inter-group or intra-group comparisons for measurement data that did not meet the normal distribution. p < 0.05 was considered statistically significant.All the data were statistically analyzed by SPSS statistical software . The Shapiro–Wilk test was used to determine whether the data were normally distributed, and the measurement data were expressed as mean ± standard deviation. For the measurement data meeting the normality, the comparison between the two groups of patients was conducted by two-independent sample 3.n = 5), rib fracture (n = 9), spleen rupture (n = 4), urethral rupture (n = 2) and limb fracture (n = 2) (p = 0.0145), deviation of the guide needle , intra-operative fluoroscopy time , insertion time of each sacroiliac joint screw . There was no statistically significant difference in terms of blood loss . According to the Majeed function system, 22 patients achieved excellent scores, 7 patients got good scores, and 3 patients received acceptable scores  . There wture n = , urethrae scores . The 16 4.Sacroiliac joint screw placement technique fixes sacrum fractures by placing screws laterally in the sacroiliac joint to fix the S1 vertebral body. This is a central fixation method, which is more conducive to the stability of internal fixation in terms of biomechanics  and alloAIOOR had a dual fluoroscopic imaging system. The guide needle sleeve used in the AIOOR is made of entirely roentgenolucent PEEK material. When the X-ray image of the position is taken, there is no occlusion to the position of the screw, so that the surgeon could accurately understand the position of the pilot sleeve, which achieving completely unscreened and improving the accuracy of screw placement. During the operation, it could also realize the fine adjustment of the front-end sleeve device to further ensure the sacroiliac joint screw placement’s accuracy and safety. Our results showed that although the overall surgical time of the robot group was longer than that of the freehand group due to equipment placement, the actual screw placement time of the robot group was shorter than that of the free hand group, and the angle deviation of the screw was significantly different between the two groups.The orthopaedic surgery robot assistant system inevitably has some limitations. Firstly, surgeons need to pay constant attention to the position of the optical tracker, and the photosensitive ball fixed on the patient and the robotic arm to avoid occlusion and affect the operation of the robotic arm. Secondly, the appilication of the orthopaedic surgery robot assistance system will increase surgical costs, such as the machine’s cost. However, it has the advantages of accuracy of screw placement, less radiographic exposure times to orthopaedic surgeons and patients, less blood loss and safety compared with the traditional freehand screw placement by orthopaedic surgeons. It is a new and more effective operation method, and it has outstanding application value and prospect.Common fixation methods for sacral fractures are C-clamp, anterior sacral plate fixation, posterior sacral bolt fixation, sacral plate fixation, and sacral lag screw fixation. The selection of screws can be divided into partially threaded hollow screws, full-threaded hollow sacroiliac screws and solid screws, while solid screws are rarely used because they are difficult to close percutaneous placement and cannot be closed percutaneous removal. Postoperative loosening and nailing make the sacroiliac screw less stable in the vertical direction, which may lead to internal fixation failure, especially in osteoporosis. Our experience is that for senile sacral fractures, if full screw placement is not possible, a partially threaded compression screw is used, with a pad at the end of the screw to prevent the end of the screw from penetrating the iliac cortex.Our next steps are to expand the number of cases in the study, along with long-term follow-up of the cases, to study the long-term benefits of the surgery, and to conduct a more comprehensive and accurate evaluation of the surgical effectiveness of robot-assisted screw fixation in the treatment of pelvic fractures.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving humans were approved by Ethics Committee of Inner Mongolia Medical University Affiliated Hospital. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.Y-zZ for design and writing. X-dH for clinical evaluation. S-bW participated in the implementation of the robot. GL for clinical application. All authors contributed to the article and approved the submitted version.