Pediatric pulmonary hypertension (PH) is a serious condition with increased risk for malnutrition due to increased caloric needs and reduced energy intake. This combination of disease and dynamic elements make it particularly challenging to meet expected growth patterns. Pediatric PH patients require close monitoring and individualized nutrition interventions to best meet nutrient needs. The prevalence of malnutrition and effective nutrition interventions in pediatric PH has not been studied.Using our electronic medical record (EMR) patient care dashboard, malnutrition prevalence was assessed by reviewing the active problem list of all active PH patients at our center. A chart review compared patients with diagnosed malnutrition in the EMR to those with malnutrition identified by a registered dietitian (RD) using a standardized tool. Chart reviews also assessed outcomes of RD interventions.z-score assessment.195 patients were identified as active PH patients followed by our PH center during the study period (November 2021 to January 2023). Of these, 5% (10/195) had an ICD-10 code for malnutrition listed in their chart. However, upon further chart review of the remaining 185 patients, 22% (41/185) had malnutrition identified by a RD using Texas Children's Malnutrition Tool, totaling 51/195 (26%) malnourished patients. The PH RD saw 25/51 (49%) patients during PH clinic visits in the study period. At follow up visits , 56% (14/25) patients seen by the PH RD either improved or resolved their malnutrition status by Malnutrition is present in pediatric PH, although underappreciated and underdiagnosed. Managing malnutrition in pediatric PH requires close monitoring, multidisciplinary involvement, and individualized nutrition recommendations. This is best achieved by a dedicated PH RD who is familiar with the unique needs of this population and available to provide consistent nutritional assessments and interventions to reduce malnutrition in this population. Pediatric malnutrition is defined as an imbalance between nutrient requirements and nutrient intake that results in deficits of energy, protein, or micronutrients that may negatively affect growth, development, and long-term health outcomes , 2. EvenPatients with chronic disease are at an increased risk for developing malnutrition due to increased energy expenditure. Malnutrition in the setting of chronic illnesses can be multifactorial, requiring complex individualized nutrition interventions. In addition, poor nutritional status can be an independent predictor of morbidity and mortality . A studyChronic lung disease in general increases energy expenditure and energy needs for growth in children; the same is true for those with cardiovascular disease. Therefore, pediatric PH has increased risks of malnutrition in an already vulnerable patient population. Limited research has been done to examine the energy requirements of adults with PH, and even less research is available on energy requirements for pediatric PH patients . One stuz-scores and overall nutrition status.We therefore aimed to understand the prevalence of physician-diagnosed malnutrition in our PH cohort, compare this to rates of malnutrition identified by standardized assessment and examine nutritional outcomes of PH-dedicated Registered Dietitian (RD) assessment and intervention. We expected that increased RD involvement by a designated PH RD would improve weight gain, body mass index (BMI)/weight-for-length This was a single center, retrospective cohort study of pediatric patients with diagnosed PH from November 2021 to January 2023. The study population included all active patients with PH managed at our PH center during the study period. Identified charts were assessed for ICD-10 codes for malnutrition , which were listed in the active problem list of the EMR. Texas Children's dietitians applied the Texas Children's Pediatric Malnutrition Tool  to the r195 patients were active during the study period . Five pez-scores. Of note, severity of malnutrition approximated severity of PH. Of the 51 total patients identified with malnutrition, 27 patients (53%) were classified as mild malnutrition, 13 patients (25%) as moderate malnutrition, and 11 patients (22%) with severe malnutrition based on stratification of body mass index (BMI)/weight-for-length z-score at follow up PH clinic visits during the study period  patients who had RD involvement either improved or resolved their malnutrition status based on improvement in BMI riod see .PH has many risk factors for abnormal nutrition status. Dyspnea is a common symptom of PH. In addition to increasing energy needs, oral intake with dyspnea may be limited due to poor coordination of breathing and swallowing, with associated risk of dysphagia and aspiration. This increased effort can prevent adequate calorie consumption and may require the use of enteral nutrition support to safely meet elevated energy needs. Additionally, prolonged hospital stays decrease the ability to practice age-appropriate feeding skills and increases the incidence of oral aversions, which can limit age-appropriate foods consumed and further decrease caloric intake . Use of In our cohort, only 5% of patients had a diagnosis of malnutrition listed in the EMR. However, manual chart reviews found malnutrition was prevalent at much higher rates of 26% (51/195) using a dedicated nutritional screening tool. The Texas Children's Malnutrition Tool is a standardized tool that is used throughout the institution to screen and assess for malnutrition . Per hosOur study found that malnutrition was commonly not coded as a medical problem, resulting in decreased team awareness of malnutrition, missed opportunities for RD consultation and fewer occasions for interventions to address malnutrition. At our institution, physicians are responsible for adding and identifying appropriate malnutrition ICD-10 codes. However, with an embedded RD in clinic and commitment for yearly nutritional screening, collaboration with a PH RD will improve accurate identification of malnutrition and promote essential communication among the medical team. This is essential for improved EMR capture of the malnutrition burden of nutritional concerns in this population and allow for appropriate interventions to reduce severity. Prior to the study period, RD assessments were on a consultation basis only. These were most often for poor weight gain, weight loss, decreased intake, or adjustments of enteral nutrition support. Dietitian interventions for these consults included introducing nutrition supplements, modifying enteral nutrition feeding regimens, and educating families on high calorie foods. In addition to assessments, RDs assisted providers in ordering nutrition supplements and specialty enteral formulas to best meet patient needs. Advocating and facilitating access for patients to receive necessary supplies to meet their increased energy needs was a recognized crucial component of RD involvement to improve nutrition outcomes. Unfortunately, during this period, patients were seen on a consult-only basis and there was no structured RD follow up nor tracking of consultation. On average, staff RD were consulted on 4–6 patients per month.In November 2021, our PH center acquired a designated PH RD to have embedded support within the clinic, to increase access to consultation and intervention. The PH RD communicated with families in between PH clinic appointments on a bi-weekly to monthly basis to ensure growth patterns were following age-appropriate recommendations. Oral nutrition supplements and enteral nutrition were assessed and adjusted as indicated to meet constantly fluctuating energy needs due to changes in activity, intake, or acute illnesses. Nutrition reassessments were conducted as determined by the PH RD if growth remained below recommended for age, suboptimal intake continued, or malnutrition persisted. On average, the PH RD consults on 25–30 new and follow up patients per month. RD re-assessments in PH clinic are conducted 3–4 months after the initial RD assessment.Active interventions, close monitoring and frequent follow up by a designated PH dietitian improved or resolved malnutrition status in 56% of patients followed by the PH RD from November 2021 to January 2023. Our findings supported our expectation that increased RD involvement by a designated PH RD would be associated with improved weight gain, BMI/weight-for-length z scores and overall nutrition status.Limitations of this study include single center study with a limited sample size. Inherent to retrospective chart review analyses, there may have been missed malnutrition diagnoses by providers and missed malnutrition documentation in the absence of RD consultation. We did not have year-over-year data to offer direct comparisons prior to November 2021, although this information is now available for future tracking. Although nutritional outcomes were improved, the study period was too short to identify other nutrition-related improvements on objective markers of PH disease severity such as hemodynamics. NYHA FC was not significantly affected by nutritional intervention since most children are FC I–II, consistent with registry data .Nutritional assessment is a crucial component of comprehensive pediatric PH care. The vulnerable nature of pediatric PH requires close assessment and monitoring of energy expenditure and calorie supplementation by a trained dietitian who is familiar with nutritional requirements of children with cardiopulmonary disease. Our study found that a designated PH RD increased awareness of malnutrition and improved the nutrition status of many patients seen during a designated period at our PH center. The RD was able to identify patients at risk of developing malnutrition using a standardized screening tool and recommend appropriate nutrition interventions, which resulted in improved nutritional outcomes. Further studies to provide guidelines and dietary recommendations for this population are needed.


Diffusion magnetic resonance imaging white matter tractography, an increasingly popular preoperative planning modality used for pre-surgical planning in brain tumor patients, is employed with the goal of maximizing tumor resection while sparing postoperative neurological function. Clinical translation of white matter tractography has been limited by several shortcomings of standard diffusion tensor imaging (DTI), including poor modeling of fibers crossing through regions of peritumoral edema and low spatial resolution for typical clinical diffusion MRI (dMRI) sequences. Track density imaging (TDI) is a post-tractography technique that uses the number of tractography streamlines and their long-range continuity to map the white matter connections of the brain with enhanced image resolution relative to the acquired dMRI data, potentially offering improved white matter visualization in patients with brain tumors. The aim of this study was to assess the utility of TDI-based white matter maps in a neurosurgical planning context compared to the current clinical standard of DTI-based white matter maps.2. For each patient, two directionally encoded color (DEC) maps were produced as follows. DTI-based DEC-fractional anisotropy maps (DEC-FA) were generated on the scanner, while DEC-track density images (DEC-TDI) were generated using constrained spherical deconvolution based tractography. The potential clinical utility of each map was assessed by five practicing neurosurgeons, who rated the maps according to four clinical utility statements regarding different clinical aspects of pre-surgical planning. The neurosurgeons rated each map according to their agreement with four clinical utility statements regarding if the map 1 identified clinically relevant tracts, (2) helped establish a goal resection margin, (3) influenced a planned surgical route, and (4) was useful overall. Cumulative link mixed effect modeling and analysis of variance were performed to test the primary effect of map type (DEC-TDI vs. DEC-FA) on rater score. Pairwise comparisons using estimated marginal means were then calculated to determine the magnitude and directionality of differences in rater scores by map type.Fourteen consecutive brain tumor patients from a single institution were retrospectively selected for the study. Each patient underwent 3-Tesla dMRI scanning with 30 gradient directions and a b-value of 1000 s/mmA majority of rater responses agreed with the four clinical utility statements, indicating that neurosurgeons found both DEC maps to be useful. Across all four investigated clinical utility statements, the DEC map type significantly influenced rater score. Rater scores were significantly higher for DEC-TDI maps compared to DEC-FA maps. The largest effect size in rater scores in favor of DEC-TDI maps was observed for clinical utility statement 2, which assessed establishing a goal resection margin.We observed a significant neurosurgeon preference for DEC-TDI maps, indicating their potential utility for neurosurgical planning. In oncoTraditional DEC maps are based on the diffusion tensor imaging (DTI) model, where the major eigenvector of the tensor model represents the primary white matter fiber orientation of the tissue . In thesHowever, the DTI model, which only represents a single tract orientation per voxel, has a limited capability to resolve areas of crossing fiber tracts . AnotherTrack Density Imaging (TDI) is a technique that leverages dMRI tractography to create a white matter map with enhanced resolution relative to the acquired dMRI data . TDI mapThe main aim of this study was to assess the potential of DEC-TDI as a tool for neuro-oncology surgical planning. We compared the potential clinical utility of DEC-TDI maps versus scanner default DEC-FA maps according to neurosurgeons’ expert rating. We developed four clinical utility statements to rate the maps. Five practicing neurosurgeons participated in the expert rating process. We found rater scores were significantly higher for DEC-TDI maps than DEC-FA maps on data from fourteen consecutive brain tumor patients. Overall, we observed a general neurosurgeon preference for DEC-TDI maps, indicating their potential utility for neurosurgical planning.22.1Sixteen consecutive brain tumor patients treated by a single neurosurgeon [AJG] with clinical MRI scans collected from 8/22/2019–7/28/2020 at Brigham and Women’s Hospital in Boston, MA were identified. One patient was excluded due to excessive motion artifacts and another was excluded due to a processing error in the tumor segmentation map, leaving fourteen  for inclusion in this study . Tumor g2.22, TR = 3200 ms, TE = 58 ms, flip angle = 90°, voxel size = 2.0 mm isotropic). DEC-FA DICOM files were output directly by the MRI scanner and were computed from the DWI data using diffusion tensor modeling. T1-weighted scans  and T2-weighted scans  were acquired as clinically indicated for each patient.Preoperative structural and diffusion images were acquired using Siemens 3T scanners . Diffusion-weighted images (DWI) were acquired using an echo planar imaging (EPI) sequence  images, b = 1000 s/mm2.3Diffusion-weighted DICOM Import (DCM2niixGUI) module (https://www.slicer.org) (https://dmri.slicer.org)  (https://www.nitrc.org/projects/dtiprep)  module  in 3D Slcer.org)  via the cer.org) . Binary mrtrix3) . This stmrtrix3) . We appldtiprep)  to perfo2.4MRTrix using the second order integration over fiber orientation distributions (iFOD2) algorithm -based tractography without anatomical constraints was performed for each patient DWI dataset in lgorithm . Tractoglgorithm , as folllgorithm . The tralgorithm . The maxlgorithm . The remlgorithm  and recelgorithm , as foll2.5MRtrix at 0.5 mm isotropic grid size using each patient’s baseline b = 0 image as a template for consistent track display in diffusion space  and Transforms modules in 3D Slicer. A physician with significant surgical planning experience [PJ] segmented brain tumors in 3D Slicer with reference to preoperative T1- and/or T2-weighted brain MR images with and without gadolinium contrast. Tumor diameters were then measured in axial slices using the Annotations module in 3D Slicer. For each patient, the single axial slice with the largest tumor diameter was selected for visualization for expert rating. If a patient had multiple lesions, the diameters of the individual lesions were summed to determine the appropriate axial slice. Then screenshots of the selected axial slices were captured .2.The resolution of this DEC map is helpful in establishing a goal margin of resection.3.This DEC map would influence my planned surgical route.4.Overall, this DEC map is useful.We defined criteria for an effective pre-surgical planning white matter visualization with the input of two practicing neurosurgeons . The neurosurgeons recommended assessing the ability of each map to help demonstrate clinically relevant peritumoral white matter tracts, to establish a goal margin of resection, and to influence the planned surgical route, as well as to assess the overall usefulness of each map. Therefore, we developed the following four clinical utility statements for evaluation of DEC maps:2.8Five MDs  with extensive neurosurgical training performed expert rating to assess how well each DEC map met the four clinical utility criteria described above. To ensure that raters were blinded to map type and patient, the 28 DEC maps (two per patient) were presented in a fully randomized order. Rating was performed using a survey in Qualtrics  following similar past methodology for expert rating of visualizations . The sur2.92 tests  by assessing how map type impacted rater score for each clinical utility statement. The data were modeled using a two-way repeated ordinal regression with a cumulative link mixed effect model (CLMM)  followin2 tests . In a po2 tests . Signifi2 tests , using t2 tests .33.1DWI data acquisition time was approximately 2–3 min per subject. Average tractography runtime across included subjects was 81.1 ± 12.1 min. DEC-TDI maps were generated in less than one minute following tractography.3.23.3Example cases were selected to illustrate neurosurgeon preferences for a particular DEC map. 3.4For all four clinical utility statements, the cumulative model including patient as a random blocking variable significantly outperformed the model not including a blocking variable . TherefoThe analysis of deviance results for the variables included in the cumulative model are reported in The results of the estimated marginal means (EMM) analysis are plotted in 4In this study, we have investigated the potential application of traditional and advanced DEC maps to aid neurosurgical planning in brain tumor patients. To our knowledge, this study is the first to specifically assess the utility of DEC-TDI for neurosurgical planning in brain tumor patients. We found that five neurosurgeon expert raters broadly preferred DEC-TDI maps over traditional DTI-based DEC-FA maps in a cohort of 14 brain tumor patients. This study provides evidence that DEC-TDI maps may help neurosurgeons identify important peritumoral tracts, decide on a goal resection margin, and plan a surgical route. To assess why DEC-TDI maps were generally preferred by neurosurgeon raters, we consider the interpretation of clinical utility statements 1 and 2, which assessed specific components of neurosurgical planning. The largest effect size in rater scores in favor of DEC-TDI maps was observed for statement 2, “The resolution of this DEC map is helpful in establishing a goal margin of resection” . This re5Our study has several limitations. To make expert rating feasible for neurosurgeons, the study design restricted visualization to a single axial slice of each DEC map. However, in clinical practice, access to the full DEC map could allow for improved tract visualization in relation to the tumor, including in different orientations. A second limitation to the study design is that the DEC-TDI maps enabled visualization only within the brain mask (where tractography was performed), while the DEC-FA maps included all anatomy that was scanned, such as the brain, cerebrospinal fluid, and skull. Our goal was to preserve the default nature of the DEC-FA maps, presenting them to expert raters as they are produced by the scanner without additional processing. The corresponding difference in visual presentation of the maps may have biased raters toward the brain extracted DEC-TDI maps, while providing raters with a way to to identify the map type despite the random, blind presentation of maps. In terms of patient selection, we selected a cohort of consecutive patients for this study with the goal of studying multiple different tumor histopathologic diagnoses, locations, sizes, and degree of edema without any a priori judgment of which cases would be most suited for DEC visualization. A number of patient- and tumor-specific factors therefore impact the potential clinical utility of DEC maps. First, for tumors located near the cortical surface , there is relatively less contrast visible in the DEC maps. Similarly, diffusion anisotropy is lower in areas of peritumoral edema, which leads to reduced brightness and contrast in DEC-FA maps and can limit fiber tracking , therefo2 and over 45 gradient directions to better model crossing fibers while reducing the effect of noise. DEC-TDI maps derived from tractography that suffers from false positives or false negatives could increase the likelihood of a suboptimal surgical approach. While the DEC-FA maps were automatically generated on the scanner, in our study the average time to generate a DEC-TDI map (including tractography and map generation) was over an hour per patient on a modern Linux computer workstation. In addition, while our study employed recommended and default parameters for tractography and DEC-TDI generation, further study may be needed to optimize and standardize these parameters for generalization across different brain tumor patients and dMRI sequences.Other study limitations relate to challenges of clinical implementation of DEC-TDI. While DEC-TDI benefits from the advantages of tractography, it also suffers from limitations of tractography including false positive streamlines and challenges tracing through peritumoral edema that can lead to false negatives , as wellIn future clinical applications, DEC-TDI visualization may be useful to aid in virtual dissection and visualization of specific white matter tracts adjacent to the tumor. In conjunction with automated tractography segmentation techniques described previously by our group , clinica6Our study demonstrates that preoperative planning white matter visualization using DEC-TDI maps is preferred by expert neurosurgeon raters over the current clinical standard, DEC-FA maps. DEC-TDI may have a potential role in conjunction with currently employed imaging techniques to help neurosurgeons plan function-preserving brain tumor resections. Further study is needed for methodology validation and clinical translation.Jared J. Sullivan: Conceptualization, Investigation, Formal analysis, Visualization, Writing – original draft. Leo R. Zekelman: Formal analysis, Writing – original draft. Fan Zhang: Methodology, Investigation, Writing – review & editing. Parikshit Juvekar: Visualization, Investigation. Erickson F. Torio: Investigation. Adomas Bunevicius: Investigation. Walid I. Essayed: Investigation. Dhiego Bastos: Investigation. Jianzhong He: Data curation. Laura Rigolo: . Alexandra J. Golby: Supervision, Conceptualization, Investigation, Writing – review & editing. Lauren J. O'Donnell: Supervision, Conceptualization, Investigation, Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Paenibacillus polymyxa ZF129, was encapsulated by the spray-drying method with gum arabic as wall material, and then pak choi seeds were pelleted with the microencapsulated Paenibacillus polymyxa ZF129 (ZF129m). The morphology, storage stability, and release behavior of ZF129 microcapsules were evaluated. Compared with the naked Paenibacillus polymyxa ZF129 cells, encapsulated ZF129 cells showed higher viability during ambient storage on pak choi seeds. Moreover, ZF129m-pelleted seeds showed higher control efficacy (71.23%) against clubroot disease than that of nonencapsulated ZF129-pelleted seeds (61.64%) in pak choi. Seed pelleting with microencapsulated biocontrol Paenibacillus polymyxa ZF129 proved to be an effective and eco-friendly strategy for the control of clubroot disease in pak choi.Clubroot is one of the most serious soil-borne diseases on crucifer crops worldwide. Seed treatment with biocontrol agents is an effective and eco-friendly way to control clubroot disease. However, there is a big challenge to inoculating the seed with bacterial cells through seed pelleting due to the harsh environment on the seed surface or in the rhizosphere. In this study, a method for microbial seed pelleting was developed to protect pak choi seedlings against clubroot disease. Typically, a biocontrol bacterium,  Plasmodiophora brassicae Woronin is one of the most devastating diseases on Cruciferae crops. The roots of the infected plants by P. brassicae will grow in the form of large galls, which can disrupt water and nutrient uptake and eventually result in the wilting and stunting of plants [P. brassicae in the soil [Clubroot caused by the soil-borne obligate parasite f plants . Clubroof plants . In addithe soil . TherefoP. brassicae [P. polymyxa ZF129 was proven to be effective in the control of soil-borne diseases [P. polymyxa ZF129 into the environment may result in a poor survival rate because of their susceptibility to various environmental factors. Thus, encapsulation of bacteria using biocompatible wall materials is an effective strategy to protect sensitive bacteria from unfavorable environments. In addition, microcapsules could be designed to deliver core materials in a controlled manner by bursting, diffusion, and dissolution, depending on the properties of the wall materials. Among the microencapsulation techniques, the spray-drying method is the most widely used in biocontrol industries because of its low operation cost and high production rate. However, the activity of the biocontrol bacteria could be negatively affected by various parameters, such as the high temperatures.Naturally occurring microorganisms have the potential to reduce clubroot ,5,6. Somrassicae ,8, whilerassicae ,10 and irassicae . The micSeed inoculation has been considered a precise and cost-effective method to deliver microbial inoculants ,14, withBrassica rapa L. ssp. chinensis L.) is one of the most popular vegetables in China, with annual consumption accounting for 30–40% of the total annual vegetable consumption in eastern China. However, in recent years, clubroot has caused increasing damage to pak choi, thus affecting its production. Therefore, this research aimed to propose a biocontrol method through pak choi seed pelleting with an encapsulated P. polymyxa ZF129. The physical and biological properties of pelleted pak choi seeds were systematically investigated, including the degree of uniformity, germination rate, storage stability, and control efficacy of clubroot. The results of this study would provide a theoretical basis and technical support for the application of biological seed treatment to vegetable crops.Pak choi (P. polymyxa ZF129 strain (GenBank CP040829.1) was provided by the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences . Herein, 0.3% w/w of aqueous-carboxymethyl cellulose (CMC)  was prepared as the binding agent. Gum arabic powders were purchased from . Pak choi (Brassica rapa L. ssp. chinensis L.) seeds were obtained from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences .P. polymyxa ZF129 used for the inoculation were stored at 20 °C in Lysogeny Broth (LB) with 20% (v/v) glycerol. The P. polymyxa ZF129 strain was cultured in the LB at 30 °C with a shaking rate of 180 rpm in the dark for 48 h. Cell concentrations were determined by following the absorbance at 660 nm.Bacterial stocks of P. polymyxa ZF129 cell cultures with a cell concentration of 108 CFU/mL were mixed with 50% (w/v) of gum arabic solution in a 1:1 ratio. Then, the mixture was spray-dried in a laboratory spray dryer with an airflow and feed flow of 500 L/h and 6 mL/min, respectively. Subsequently, the inlet temperature was set independently at 120 °C, 180 °C, and 200 °C. Afterward, the sample mixture was agitated using a magnetic stirrer at room temperature during the whole process. Finally, the dried microcapsule powder was collected in a Schott glass bottle and stored at 4 °C before further analysis.The 48-h w/v) of aqueous carboxymethyl cellulose (CMC) solution was prepared as a binding agent. For P. polymyxa ZF129 microcapsule pelleted seeds (ZF129m seed), 100 g of pak choi seeds, 300 g of pelleting powder containing 10 g of ZF129 microcapsules (108 CFU/g), and 290 g of inert filler materials were placed in the rotary coater, and rotation speed was set at 90 rpm. During the pelleting, pak choi seeds were wetted with the binder, which was dropped through an atomizer. Meanwhile, for the P. polymyxa ZF129 pelleted seed (ZF129 seed), a volume of the P. polymyxa ZF129 culture containing the same number of cells with the abovementioned microcapsules was concentrated via centrifugation to pellet the cells. In the binding agent, the supernatant and cells were respectively decanted and resuspended. Then, cells were embedded in the filler matrix through wetting. The pelleting seeds were dried at 35 °C for at least 0.5 h before being used. Stiffness testing was conducted on 10 pelleted seeds per treatment using a tablet hardness meter . For each recipe, a total of 100 pelleted seeds were planted in multi-cavity trays that contained 100 g of sterilized commercial substrate soil . The germination rate of seeds was recorded daily until a constant germination rate was achieved, with untreated seeds serving as the germination control.Four types of filler materials  were chosen as inert pelleting fillers, and their combination was designed by DesignExpert v11 . Herein,P. polymyxa ZF129 microcapsules was characterized by field emission scanning electron microscopy . The samples were spread onto conductive carbon tape and then stuck on a SEM stub. Furthermore, samples were sputter-coated with gold using a JFC-1600 Auto Fine Coater  to a thickness of 10 nm. Afterward, samples were imaged at different magnifications with an acceleration voltage of 3.00 kV.The morphology of P. polymyxa ZF129 microcapsules was determined using a dynamic laser scattering (DLS) instrument . Ethanol was used as a dispersant for microcapsules [The particle size distribution of capsules . The sizP. polymyxa ZF129 microcapsule powder was added, and the dish was reweighed (b). The loaded dish was placed in the oven at 105 °C for 24 h, then cooled to room temperature in a desiccator and reweighed (c). The heating and cooling processes were repeated until the weight (c) was constant [Weighing dishes were initially dried in an oven at 105 °C to a constant weight and then cooled in a desiccator containing silica gel. Then, the weight of the empty dish was recorded (a), and approximately 3 g of a = weight of an empty dish;b = weights of dish and wet powder;c = weights of dish and dried powder.P. polymyxa ZF129 microcapsules was introduced in 9 mL of deionized distilled water. Then, the sample was serially diluted in distilled water and plated on the LB medium. After 48 h of incubation at 37 °C, cell counts were determined, the survival of encapsulated bacteria was evaluated, and the number of colony-forming units per gram (CFU/g) was measured.0 displays the free viable cells before encapsulation.Briefly, 1 g of spray-dried P. polymyxa ZF129 microcapsule powders, samples were placed in tightly sealed sterile bottles and stored for 30 days at 4 °C and 25 °C. The viability of cells at different storage times  was determined using the colony counting method.For the P. polymyxa ZF129-pelleted seeds, including ZF129 seeds and ZF129m seeds, seed samples were placed in tightly sealed sterile bottles and stored for 60 days at 25 °C. A total of 10 seeds were placed in 10 mL of sterilized water and then shaken to allow bacteria to release from the seed pelleting matrix. Then, the enumeration of P. polymyxa ZF129 within the suspension was conducted using the colony counting method.In addition, for the P. polymyxa ZF129 cell from pelleted seeds was studied based on the protocol described by Cortés-Rojas et al. [The release behavior of the s et al. . with lis et al. . Second,P. polymyxa ZF129 microcapsule pelleted seeds (ZF129m seeds), ZF129 suspension pelleted seeds (ZF129 seeds), and non-treated seeds were sown in trays (540 mm × 280 mm) containing 4000 g of Plasmodiophora brassicae Woronin (P. brassicae)-infected soil (108 spores/g soil). The P. brassicae inoculum used in this study belongs to pathotype 4. The pak choi plants were cultured in the greenhouse with the following conditions: 25 °C, 16-h photoperiod, and 70% RH. After 60 days, all plants were harvested, and the clubroot severity was rated using a slightly improved grading standard [standard , which i540 mm × 0 mm contData were subjected to analyses of variance (ANOVA) using SPSS 13.0 software . Mean comparisons were conducted using a least significant difference (LSD) test at a 0.05 probability level.P. polymyxa ZF129 microcapsules prepared at different inlet air temperatures is shown in P. polymyxa ZF129 microcapsules prepared at the inlet air temperature of 120 °C showed a spherical shape with small wrinkles on the surface. As the inlet temperature increased from 180 °C to 200 °C, the surface of the microcapsule appeared smooth; however, the shape became irregular, and more concavities were observed. These concavities were also reported in other studies using gum arabic as a wall material. Thus, it can be explained that concavities were caused by the rapid evaporation of the atomized liquid drops during spray drying [The morphology of y drying .The physical properties, such as single seed rate, abscission rate, and disintegration rate, were tested for pelleted pak choi seeds to screen the filler formula for seed pelleting, which was prepared based on the 11 filler formulas . Among tP. polymyxa ZF129 was encapsulated by the spray drying method using gum arabic as the wall material under different inlet air temperatures . The counts of viable cells before and after spray-drying treatment are shown in P. polymyxa ZF129 culture was 62%. After spray-drying treatment, the spore rate increased up to 67.35% and 83.66% because vegetative cells could be easily killed by the spray-drying process while spore cells had more tolerance [olerance . Meanwhiolerance . The resolerance .P. polymyxa ZF129 microcapsules with different inlet temperatures  is graphed in To investigate the effect of inlet temperature on the size of the ZF129 microcapsules, the size distribution of the three microcapsule samples was measured by the dynamic light scattering technique. The hydrodynamic size distribution of the The viability of encapsulated ZF129 with different inlet temperatures  was monitored during storage for 30 days at 4 °C and 25 °C . To makeP. polymyxa ZF129 microcapsule powders retained viable cell counts close to or higher than 105 CFU/g after 30-day storage  lower than those stored at 4 °C  and non-encapsulated ZF129-loaded seeds (ZF129 seeds), respectively. The cross section of the seed pelleting layer is shown in Lactobacillus acidophilus [To investigate the storage stability of ZF129 microcapsule-loaded seed, the viable ZF129 cell counts on the seed pelleting matrix were tracked for up to 60 days at 25 °C . The numdophilus .P. brassicae-incubated soil under greenhouse conditions. In the meantime, a group of bare seeds were planted in healthy soil as a healthy control. After growing for 60 days, the phenotype of pak choi root galls with different treatments was photographed  was 1.58-fold larger than that of the infected control group (23.44 cm2), possibly because the formation of clubroot would prevent the transportation of nutrients from roots to leaves.The efficacy of seed pelleting on pak choi plant growth was also investigated. Phenotype analysis revealed that ZF129m seed produced 2.35% more shoot biomass and 0.6-fold lower root biomass than bare seed . It can P. polymyxa ZF129. The P. polymyxa ZF129 microcapsule powder was prepared by an optimized spray drying method with gum arabic as wall material. The optimized seed pelleting formulas showed good performance in terms of high germination rates and uniform morphology. Pak choi seed pelleted with encapsulated P. polymyxa ZF129 (ZF129m seeds) showed better storage stability than that of non-encapsulated P. polymyxa ZF129 pelleted seeds (ZF129 seeds). Importantly, the ZF129m seeds also have better bioactivity against clubroot disease, with a control efficacy of 71.23%. Overall, this work not only provided a “seed pelleting with microbial-loaded microcapsules” strategy to control the clubroot disease but also supported the development of biological seed treatments.In this work, pak choi clubroot disease was successfully controlled through seed pelleting with encapsulated


We highlight a delicate balance among Mg2+, the intra- and intermolecular RNA-RNA interactions in modulating RNA folding and function.T-box riboswitches are unique riboregulators where gene regulation is mediated through interactions between two highly structured RNAs. Despite extensive structural insights, how RNA-RNA interactions drive the folding and structural transitions of T-box to achieve functional conformations remains unclear. Here, by combining SAXS, single-molecule FRET and computational modeling, we elaborate the folding energy landscape of a translational T-box aptamer consisting of stems I, II and IIA/B, which Mg 2+, intra- and intermolecular RNA-RNA interactions in modulating its folding and function.T-box riboswitches are RNA-based gene regulators, composed of highly structured noncoding RNAs: the T-box and a tRNA ligand. Here, the authors assess the folding of a translational T-box aptamer and dissect the role of Mg The functional diversity of RNAs is determined by their propensity to fold into distinct secondary and tertiary structures, interact with other molecules to form complex quaternary assemblies, and transit dynamically between alternative conformational states in response to specific cellular signals2. Previous studies have provided important insights into how RNA interacts with ion, metabolite or protein to guide the folding and conformational dynamics of RNAs to facilitate cellular functions6. However, our understanding of how RNA-RNA interactions occur and drive the RNA folding and conformational dynamics remains relatively limited.RNAs play essential roles in multiple cellular processes including gene expression, processing, catalysis and gene regulation7. Canonical T-box riboswitches contain highly conserved helical structures designated as stems I, II, IIA/B and III that are upstream to an anti-terminator  or anti-sequestrator  element  of mRNAs encoding aminoacyl tRNA synthetase and proteins involved in amino acid synthesis and transportent Fig. . These e20, revealing a wealth of complex RNA-RNA interactions that create high-affinity tRNA binding sites by T-boxes. Multiple contact sites have been revealed between the T-boxes and the tRNAs, which are driven and stabilized by a large set of intra- and inter-molecular RNA-RNA interactions. For example, in the crystal structure of a translational ileS T-box in complex with tRNA19, the inter-molecular base-pairing interactions between the tRNAIle anticodon and the Specifier are laterally reinforced by the intra-molecular interactions between stem I containing a K-turn and stem II bearing an S-turn, which are arranged perpendicularly and joined at their bases near the stem IIA/B pseudoknot. The pseudoknot is coaxially stacked with stem II to extend the dsRNA trajectory and anchors the base of the stem I to dock with the middle section of stem II, creating an extended binding groove for the tRNA binding  and single-molecule fluorescence resonance energy transfer (smFRET) have been applied to investigate the structural dynamics and kinetics of a transcriptional ileS T-box riboswitch from Nocardia farcinica  system, which allows for a comprehensive labeling scheme including 7 pairs of fluorophore labels across the T99. smFRET analysis reveals that Mg2+ has a minimal impact on the folding of individual stems I and II, but is required for the formation of stem IIA/B. Consequently, the Mg2+-facilitated inter-stem preorganization between stems I and II to form a competent tRNA binding conformation is dynamically coupled with the formation of stem IIA/B pseudoknot and its coaxial stacking with stem II. Subsequent binding of the cognate tRNAIle anticodon by stem I Specifier induces the docking of stem I on the S-turn and surrounding regions of stem II, and concomitantly, moving stem IIA/B away, which further stabilizes the Specifier-anticodon interaction. The essentiality of stem IIA/B and stem II S-turn regions in such processes is further demonstrated by dynamic analysis and 3D structural visualization of several point mutants and transcriptional intermediates of T99. Taken together, our results delineate a conformational energy landscape for a translational T-box riboswitch in which cooperative RNA-RNA interactions drive RNA folding and promote high-affinity tRNA recognition.In this study, we have combined isothermal titration calorimetry (ITC), SAXS with smFRET techniques to probe the folding and conformational dynamics of the aptamer domain (T99) of a translational ica Fig. . Our res2+ ions are known to be important for the folding and ligand binding of many riboswitches25. To understand how Mg2+ affects T99 RNA, we first probe its global folding and conformational changes through SAXS 2I(q)/I0versus qRg, reflects the degree of flexibility and compactness of the molecule in solution. At low Mg2+, T99 displays a significant enrichment in the high scattering angles, characteristic of an unfolded conformation. As Mg2+ increases, there are significant changes in the plots resulting in transitions from a bimodal peak to a more bell-shaped curve indicative of a folding event  and the maximum particle dimension (Dmax) were plotted as a function of Mg2+  in 2 mM Mg2+ is significantly larger than that in higher Mg2+, indicating that the binding properties for T99 in high Mg2+ may differ from that in low Mg2+ (discussed later). Given that Mg2+-induced folding is essential for high-affinity tRNA binding and tRNA binding induces further structural changes in T99 RNA, these two processes are cooperatively coupled.ITC measurements were also performed for the T99 in the presence of varying Mgg2+ Fig. . While n26. Recently, we have achieved site-specific fluorescent labeling of large flaviviral RNAs up to 719 nts using the NaM-TPT3 UBP system28  which can be incorporated into RNA orthogonally with amine-modified rTPT3TP (rTPT3A). Using the similar strategy29, DNA templates containing an upstream T7 promoter, a dNaM and a dTPT3 modifications at specific sites are prepared by overlapping PCR first. Then the rNaMCO and rTPT3A are incorporated into specific sites of the RNA by in vitro transcription. The rNaMCO- and rTPT3A-modified transcripts are subsequently conjugated with azide-modified Cy5 and NHS ester-modified Cy3 dyes through click chemistry and amine-NHS ester reactions, respectively  and transits into middle-FRET occasionally. In 20 mM Mg2+, the population occupying the high-FRET state substantially increased to 74%, however, further addition of tRNA slightly decreased the population at the high-FRET state (~65%). These findings are consistent with previous studies that the K-turn is largely formed even in the presence of only monovalent salt which can be further stabilized by Mg2+ 30. However, tRNA binding doesn’t cause significant structural rearrangements in stem I.Stem I harbors a conserved kink-turn (K-turn) structural motif that bends the duplex RNA trajectory by ~120°, acting as a geometry tool to facilitate the docking between stems I and II Fig. . To probRNA Fig. . The peaRNA Fig. . The dis2+, however, no further change was observed upon the addition of tRNA . Under all three conditions, T99/35-54 displayed predominantly a ~0.4 middle-FRET state. The FRET efficiency increased slightly in high MgRNA Fig. .2+ promotes and stabilizes the formation of pseudoknot32. In accord with this, the population of the high-FRET state in T99/82-99 increases substantially from ~42% in the absence of Mg2+ to 57% in 20 mM Mg2+. Further addition of tRNA only causes a modest increase (~3%) in the occupancy of high-FRET state. The Mg2+ dependence of stem IIA/B pseudoknot formation was further verified in a homologous translational T-box riboswitch from Mycobacterium tuberculosis. Similar to T99/82-99, T87/71-3’end interconverts between a middle- (~0.5) and a high-FRET (~1.0) states under all three conditions  state was observed, suggesting that the inter-dye distance in T99/6-54 is too far . However, the distance between the labeling sites estimated from the crystal structure is about 31 Å, which is expected to give a high-FRET signal. Thus, stems I and II are likely to coaxially stack and adopt an undocked conformation at low Mg2+. At high Mg2+ which the T99 RNA is presumed to be folded and competent for high-affinity tRNA binding, the low-FRET state was shifted to a low-to-middle-FRET (~0.32) state, indicating that stem I is moving close to stem II. Upon addition of tRNA, a middle-FRET (0.57) state emerged and was highly populated (65%), presumably corresponding to the tRNA-bound conformation since T99/6-54 alone mainly stays in the low-to-middle-FRET state and rarely samples the middle-FRET state. These data indicate that while high Mg2+ promotes the preorganization of stems I and II into a competent conformation, subsequent tRNA binding further induces their docking, resulting in a significant compact and more closed conformation between stems I and II. We thus defined the low-to-middle-FRET in high Mg2+ and the middle-FRET in the presence of tRNA as predocked and docked states, respectively. To substantiate these findings, we also performed smFRET measurements for another labeling construct (T99/14-54). Similar phenomena were observed for T99/14-54 under the same conditions  state in the absence of Mg2+ to a low-to-middle-FRET (~0.34) state, the addition of tRNA at high Mg2+ results in the emergence of a pronounced middle-FRET (~0.63) state coexisting with the low-to-middle-FRET state.The labeling construct T99/6-54 was used to characterize the relative inter-stem motions between stems I and II. smFRET data for T99/6-54 were obtained under the same three conditions as above and summarized in Fig. ons Fig. . While h2+ exhibited a major but broad FRET population, which is likely due to the intrinsic dynamics of stem IIA/B pseudoknot. Increasing Mg2+ to 20 mM shifted the peak of FRET distribution to a middle value (~0.49), indicating that stems I and IIA/B are moving towards each other. Interestingly, upon the addition of tRNA, a sharp and highly populated low-FRET ( ~ 0.26) state emerges and coexists with the middle-FRET state, suggesting that tRNA binding drives stem I moving away from stem IIA/B to dock with stem II. smFRET measurements were also performed for another construct (T99/14-99) which C14 and G99 were labeled with Cy3 and Cy5, respectively. Similar phenomena were observed for T99/14-99 as that for T99/6-99  state in the absence of Mg2+ to a middle-FRET (~0.45) state, further addition of saturating tRNA at high Mg2+ results in the emergence of a pronounced low-FRET (~0.25) state.To characterize the relative inter-stem motions between stems I and IIA/B, we synthesized the T99/6-99 construct. The inter-site distance estimated from the crystal structure is ~56 Å, which is expected to give a low-FRET signal. smFRET data for T99/6-99 under all three conditions were obtained and summarized in Fig. -99 Fig. . While h2+ facilitates the folding of stem IIA/B pseudoknot and its stacking on stem II for preorganization of stems I and II to form a competent tRNA binding conformation, and subsequent tRNA binding drives further docking of stem I on stem II, and concomitantly, moving away from stem IIA/B  alone in 7.5 mM Mg2+ present a major low-FRET population with the same peak value (~ 0.08) as that of T99/6-54 in the absence of Mg2+, implying that stems I and II are undocked and locate far away from each other, thus complete disruption of the pseudoknot abolishes Mg2+-induced pre-docking of stems I and II.To experimentally delineate the roles of the structural integrity of stem IIA/B in pre-docking of stems I and II, two single-point mutants (G85C/6-54 and U90C/6-54) that impair the IIA/B pseudoknot by disrupting two conserved base triple interactions and two deletion mutants that remove IIB (T89) or the complete IIA/B pseudoknot (T77) of T99/6-54 were constructed Fig. . The docted Fig. . Under s2+ and tRNA were also investigated using smFRET  that disrupt these interactions were constructed. SAXS data indicated that these single-point S-turn motif mutations cause little effects on the global folding of T99 RNA  in 20 mM Mg2+, suggesting that high Mg2+ attenuates the mutational effects of G70U. It’s likely that the initial contact between the tRNA anticodon and Specifier in stem I drives the formation of a helical, stacked conformation for the specifier and then induces the docking of stem I towards stem II via backbone interactions with the S-turn region, which in turn reinforces the Specifier-anticodon interactions. Thus, the S-turn region is critical to stabilize the tRNA-induced docking of stems I and II.The docking dynamics of stems I and II in such mutants were characterized by smFRET , T89/6-54 and T77/6-54 (2–50 mM) in the absence or presence of tRNA  is determined as 3.9 ± 0.2 mM using the Hill-equation fit, indicating that near-physiological concentration of Mg2+ is sufficient for high-affinity binding between tRNA and T99  increase significantly as Mg2+ increases. In contrast, the reverse transition rates (kundock) are less sensitive to changes of Mg2+ concentrations, especially above 2 mM Mg2+, which maintains at about 0.19-0.26 s-1. The transitions between low- and middle-FRET states are likely caused by the tRNA binding and dissociation events since such transitions were rare for apo T-box. The free energy landscapes of T99 at different Mg2+ concentrations in the presence of tRNA were estimated from transition rates between the undocked and docked states, revealing that Mg2+ stabilized the docked state and significantly reduced the energy barrier from undocked to docked states  was comparable to the affinity reported for unlabeled T99. In addition, the transition rate kdock increases significantly while kundock changes slightly when tRNA concentration increases from 10 nM to 2 μM, suggesting that tRNA recognition by T99 favors an induced-fit mechanism.To further explore the tRNA recognition mechanism by T99, we also performed smFRET experiments for T99/6-54 with varying concentrations of tRNA (1 nM-2 μM). As tRNA concentration increases, the occupancy of low-FRET decreases and middle-FRET increases , of which the peak value (~ 0.08) is insensitive to the increasing Mg2+ and the same as that of T99/6-54 in the absence of Mg2+  as Mg2+ increases. These data suggest that high Mg2+ attenuates the detrimental effects of stem IIA/B disruption and promotes the docking of stems I and II in both T89/6-54 and T77/6-54 in the presence of tRNA. Further transition kinetic analysis shows an obvious increase of the kdock and decrease of the kundock in response to increasing Mg2+, suggesting that Mg2+ not only promotes the formation of the docked state, but also increases its stability by reducing the undocking rate  compared with T99. Thus, we speculated that the destabilization or deletion of stem IIA/B mainly increases the accessibility of stem I in the conformational space of T99 and finally results in their lower docking rate with tRNA. This is also consistent with previous ITC data on the stem IIA/B mutants, in which they exhibited a larger entropic loss while the enthalpy change remained almost the same compared with T9919. Free energy landscapes for the stem IIA/B mutants and the T99 in 7.5 mM Mg2+ were calculated, which showed that destabilization or removal of stem IIA/B significantly reduced the stability of the docked state, accompanied with an increase of the transition energy barrier from undocked to docked state by about 0.33-1.37 kcal/mol  for apo T-box. tRNA mostly binds the open and the semi-open conformations to induce folding and formation of tRNA-bound state, which also suggest a predominant induced-fit mechanism. This differs from the Mg2+-promoted conformational selection mechanism reported in small-molecule sensing riboswitches such as preQ1 and fluoride riboswitch39.Our smFRET-based analysis of the intra- and inter-stem dynamics of T99 unveils the molecular mechanism to sculpt its high-affinity tRNA binding site. Riboswitches might recognize their cognate ligands through lock and key, induced-fit or conformational selection mechanisms, which can be regulated by Mg2+, the intra- and intermolecular RNA-RNA interactions in modulating the cooperativity in T99 to regulate its folding and function. Mg2+ is essential for neutralizing the negative charge of phosphate moieties in the RNA backbone, which induces structural changes that bring together two RNA regions41, thus allowing for long-range tertiary interactions and quaternary interactions to form44. Our study suggests strong cooperativity among the tertiary interaction networks involved in stem I Specifier region, stem II S-turn region, stem IIA/B pseudoknot and Mg2+ binding in T99, in which stem IIA/B plays an essential role. At low Mg2+, the stem IIA/B pseudoknot is not formed, and stems I and II are coaxially stacked, preventing the pre-docking of stems I and II to form a competent conformation for efficient tRNA binding. Increasing Mg2+ stabilizes the stem IIA/B pseudoknot formation, which drives the coaxial stacking of stem IIA/B on stem II, resulting in pre-docking of stem I on II into a competent conformation for tRNA binding. Subsequent tRNA anticodon binding to the specifier region induces further docking of stem I with stem II S-turn region. The cruciality of stem IIA/B and stem II S-turn in the pre-docking and docking of stems I and II for high-affinity tRNA binding, respectively, was substantiated by mutational analysis. However, high Mg2+ can compensate for the destabilizing effects of stem IIA/B and stem II S-turn mutations which are destructive to the docking of stems I and II at low Mg2+. The intricate interplay among these interactions is directly visualized by computational modeling of the transcriptional intermediates of T99, showing how different structural modules cooperate with each other to promote RNA folding into functional conformations for efficient and fast tRNA recognition  at 12 ID-B. The setups were adjusted to achieve scattering q values of 0.005 < q < 0.89 Å-1, where q = (4π/λ)·sin(θ), and 2θ is the scattering angle. Thirty-two-dimensional images were recorded and reduced for each buffer or sample and no radiation damage was observed. Scattering profiles of the RNAs were calculated by subtracting the background buffer contribution from the sample-buffer profile using the program PRIMUS3.2 following standard procedures50. Guinier analysis was performed to calculate the forward scattering intensity I(0) and the radius of gyration (Rg), which were also estimated from the scattering profile with a broader q range of 0.006–0.30 Å-1 using the indirect Fourier transform method implemented in the program GNOM4.651, along with the pair distance distribution function (PDDF), p(r), and the maximum dimension of the RNA, Dmax. Low-resolution 3D shape envelopes were ab initio reconstructed using the scattering data within the q range of 0.006–0.30 Å-1 with the program DAMMIN52, which generate models represented by an ensemble of densely packed beads. The theoretical scattering intensity of the atomic structure model was calculated and fitted to the experimental scattering intensity using CRYSOL53.All the experimental procedures and instrument parameters for data collection and software employed for data analysis are similar as described before2+. ITC measurement was performed in triplicates at 25 °C, with ~20 μM T99 in the cell and ~200 μM tRNA in the syringe, using a MicroCal PEAQ-ITC microcalorimeter. The raw ITC data were analyzed using the Origin7 software package provided by the manufacturer to obtain the dissociation constant and thermodynamic parameters summarized in supplementary Supplementary Table RNA samples used for Isothermal Titration Calorimetry experiments were prepared as described above and stored in a buffer containing 20 mM Tris (pH 7.4), 100 mM KCl supplemented with different concentrations of Mg2+ in 37 °C for 30 min. Then the mixtures were loaded on 8% native polyacrylamide gel containing 10 mM Mg2+ and the gel was run at room temperature for ~ 60 min. The gel was then stained by Gelsafe dye and imaged using gel imager. EMSA for different labeling constructs were conducted on independent biological triplicates.RNA samples used for EMSA were prepared as described above and stored in a buffer containing 50 mM HEPES (pH 7.5), 100 mM KCl. tRNA (2.4 μM) was incubated with varying concentrations of dye-labeled or unlabeled T-box (0.24–7.2 μM) supplemented with 20 mM MgCO) by following the procedures in the Supplementary Methods. The deoxyribonucleotide phosphoramidites , the triphosphorylated deoxyribonucleotides  and ribonucleotides , were custom synthesized as described29. Reversed primers containing unnatural nucleotides were synthesized and used for overlapping PCR to introduce the dNaMTP and dTPT3TP into the DNA template. Then the PCR products containing dNaM and dTPT3 at specific sites were used as the DNA templates for in vitro transcription and purified as described above. The purified T99 products modified with rNaMCO and rTPT3A at specific sites were precipitated with cold ethanol (2.5 volumes) in the presence of sodium acetate (0.3 M) at – 80 °C for at least 0.5 h. After centrifuging at 4 °C, the ethanol was removed and the pellet was washed with ethanol (75%) three times, then the pellet was dried for 10 min. Finally, the product was resuspended in DEPC-treated water and subjected to Cy5 fluorescent labeling. RNAs  were mixed with 33.5 μL 1.5 X Click chemistry buffer, 5 μL Sulfo-cyanine5 azide (20 mM in DMSO), 1 μL of sodium ascorbate (50 mM). The resulting mixture was incubated at room temperature overnight. Then the labeled T99 are precipitated by ethanol as described above and resuspended in 0.1 M NaHCO3 (pH 8.0), then the Cy5-labeled T99s were mixed with 5 μL NHS-Cy3 (20 mM in DMSO) and incubated at room temperature for 4–6 h. Then Cy5-Cy3 double labeled T99s were precipitated by ethanol as described above and resuspended in buffer containing 50 mM HEPES (pH 7.4) and 100 mM KCl, stored at –80 °C for further smFRET experiments. The labeling efficiency was calculated by measuring the absorption of RNAs at 260 nm, 546 nm and 650 nm, respectively. The labeling efficiencies for Cy3 and Cy5 are ~70% and ~90%, respectively.To develop a site-specific dual fluorophore labeling scheme for large RNAs using the NaM-TPT3 unnatural base pair (UBP) system, we synthesize an alkyne-modified rNaMTP derivative . This was followed by the addition of N-(3-Dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride (EDC) along with ethylene diamine and 0.1 M imidazole solution (pH 6.0). The mix was incubated at 37 °C for 3 h and followed by ethanol precipitation for at least three times to remove residual EDC. Then the EDC-treated T99 was resuspended in 0.1 M NaHCO3 (pH 8.0) and mixed with 30 folds NHS-Cy3 dye, incubated at room temperature for 4–6 h. Then Cy5-Cy3 double labeled T99s were precipitated by ethanol as described above and resuspended in buffer containing 50 mM HEPES (pH 7.4) and 100 mM KCl, stored at -80 °C for further smFRET experiments.5’-end fluorophore labeling of RNAs was performed by following the protocols developed in previous studies with a minor improvement2+ to the final concentration of 20 mM, and finally equilibrated at 37 °C for 20 min. Samples were diluted 1200 times in the buffer containing 50 mM HEPES, 100 mM KCl with different concentrations of Mg2+ and immobilized on the slides by biotin-streptavidin interactions. Then the samples were incubated with different concentrations of Mg2+ or tRNA for 5 min on the slide before flowing the imaging buffer containing 3 mg/mL glucose, 100 μg/mL glucose oxidase, 40 μg/mL catalase, 1 mM cyclooctatetraene (COT), 1 mM 4-nitrobenzylalcohol (NBA) and 1.5 mM 6-hydroxy-2,5,7,8-tetramethyl-chromane-2-carboxylic acid (Trolox). smFRET experiments were conducted at 25 °C by using a home-built objective-type TIRF microscope. The time resolution of each movie was 100 ms/frame and we collected 800 frames for each movie. In each field of view, about 50-60% molecules were selected for further analysis. 3-4 movies were collected for each conditions. All of the experiments were repeated at least three times, from which experimental errors were estimated.For single-molecule experiments, 450 nM double labeled T99s were annealed with 300 nM biotin-modified DNA in 50 mM HEPES (pH 7.5), 100 mM KCl by incubating the mixture at 95 °C for 2 min, then fast cooling on the ice and add Mghttp://rsb.info.nih.gov/ij). Fluorescence spots were fitted by a 2D Gaussian function within a 9-pixel by 9-pixel area, matching the donor and acceptor spots using a variant of the Hough transform55. The background-subtracted total volume of the 2D Gaussian peak was used as raw fluorescence intensity I. FRET trajectories containing donor and acceptor and displayed anticorrelation behaviors were picked and analyzed. Histograms of FRET efficiency for T-boxes at different concentrations of Mg2+ or tRNAs were built by using the frames of all traces before photobleaching . The histograms were divided by the total number of FRET frames and the frequencies were labeled as ‘probability’ in the y-axis.smFRET data were analyzed by the custom-made software program. Single-molecule movies were collected by Cell Vision software (Beijing Coolight Technology) and then analyzed by a custom-made software program developed as an ImageJ 1.43 u plugin  and middle-FRET (docked state). FRET populations for two states derived from HMM analysis were fitted to Gaussian using Origin, and the relative fraction of each FRET state was calculated as the ratio of each FRET state to the total population. The dwell times of different FRET states were calculated from idealized traces and the dwell time distribution was fitted to a two-exponential function to calculate the transition rates. Relative free energies were calculated through the equation kB is the Boltzmann constant, T is the temperature, ka→b and kb→a are the transition rates from the state a to b and from the state b to a, respectively. L state was set as the ground state (∆GLow = 0). For illustration, the energy barrier from the state a to b was calculated as -kBT·ln(ka→b) + 1.8kBT.smFRET traces were further analyzed by the Hidden Markov Model (HMM)-based software to extract the kinetics informationq range 0.006–0.3 Å–1 with a pace of 0.002 Å–1 was used in our all calculations and fittings, and the other parameters were default. The best-matching model  was selected.We exploited all-atom explicit solvent MD simulations to generate a reasonable conformational pool for post-simulation screening the model satisfying SAXS data on T99, T89 and T77 RNA. The details regarding the preparation of initial structural models for MD simulation (Supplementary Data Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Movie 1Supplementary Movie 2Reporting SummarySource Data


According to CEOWORLD Magazine‘s 2019 “Health Care Indicators” rating of 89 countries in the world, Taiwan ranks first in the world. The coverage of the National Health Insurance covers all necessary medical treatment, including outpatient, hospitalization, and prescription drugs etc. The psychiatrist was wondering which item with the highest service cost–performance (CP) ratio of the psychiatrist performance in a general hospital and used proportion of PPF as performance indicator. He used allocation to distribute the hours across job activities.The purpose of this study is to investigate items allocation proportion for outpatient and inpatient ward in a specific month, examining the distribution of performance and figure out an appropriate model to optimal medical service.Table 1. Items related to physiological examination has been excluded. The performance is calculated by combining outpatient and inpatient wards.Demographic data were collected through PPF projects included 15 outpatient items and 19 inpatient ward items from the third-month of the psychiatrist’s employment in the general hospital, as shown in . The inpatient ward performance was 2 times the outpatient performance. This result showed that most performance came from inpatient ward. Among all items of the proportion of PPF unit, the highest two for inpatient ward items were general hospital bed inpatient consultation (32.58%) and special treatment for psychiatric inpatients (14.35%), and for outpatient, the highest was psychiatric outpatient consultation - more than two (11.31%) (Table 1).Demographic data analysis found that proportion of inpatient ward PPF (67.01%) was significantly greater than proportion of outpatient PPF (32.99%) Image:Image 2:National Health Insurance is an important system to assist in epidemic prevention during the COVID-19 period. The most PPF of psychiatrist service in a general hospital came from the service of inpatient ward items, including the general hospital bed inpatient consultation with the highest CP ratio. These findings may suggest that the inpatient service could offer psychiatrists fair PPF, and the hospital environment could be favorable for recruitment.None Declared


The guttural pouch of the horse is a diverticulum of the auditory tube and has a complex anatomical structure. Disease of the guttural pouch can lead to neurological signs and hemorrhage due to its close relation to major vessels and some cranial nerves. Endoscopy allows direct visualization of the pouch and is considered to be the gold standard for its evaluation. Nevertheless, diagnostic imaging can bring useful additional information and this review article describes the value of each technique and the main imaging findings to be expected in the diagnosis of guttural pouch disease.The most common diseases of the guttural pouch are empyema, tympany, mycosis and temporohyoid osteoarthropathy. The challenge in diagnosis of guttural pouch diseases lies in the complex anatomy of the guttural pouch and adjacent associated structures. Diagnostic imaging is a good complement to endoscopy for the diagnosis of some guttural pouch diseases, especially to make a full assessment of the lesions involving the pouch and surrounding structures. This review article describes the value of each diagnostic imaging technique in the diagnosis of guttural pouch disease and the corresponding imaging findings. Radiography is generally used as the first line to complement endoscopic findings, and can give useful additional information although it is limited by superimposition. Ultrasonographic examination of the guttural pouch is of limited value due to the presence of gas in the guttural pouch but can eventually be used to detect fluid within the pouch or can help to evaluate the soft tissues located lateral and ventral to the guttural pouch. Cross-sectional imaging, especially CT, is increasingly available and appears to be the best technique to fully assess the surrounding soft tissues and to precisely identify lesions of the temporohyoid apparatus, temporal bone and skull base that are associated with guttural pouch disease. The guttural pouch is a diverticulum of the auditory tube that connects the pharynx to the middle ear bilaterally. This diverticulum has a complex anatomical structure, consisting of a cavity divided by the stylohyoid bone into a small lateral recess and a larger medial recess . The gutRadiography is generally the first-line imaging modality to be used to complement endoscopy for the diagnosis of guttural pouch diseases . Air-filUltrasonography of the region of the guttural pouches is of limited value due to the presence of air in the guttural pouch and the vertical ramus of the mandible, and the consequent inability to image through them . Due to Cross-sectional imaging, especially CT, is becoming increasingly available for imaging horse heads, particularly with the development of standing CT, allowing the elimination of general anesthesia and reduced costs . The majEmpyema is the most common disease of the guttural pouch and can be caused by upper airway infection or drainage of abscesses of the retropharyngeal lymph nodes into the ipsilateral pouch ,3,8,10. Guttural pouch tympany is a condition of young horses that is a result of trapped air within the guttural pouch due to an excessive amount of tissue at the pharyngeal orifice that allows air to enter the guttural pouch during deglutition but prevents it from exiting . It causBleeding within the guttural pouches is most frequently due to guttural pouch mycosis or injury of the rectus capitis and longus capitis muscles that attach to the skull base . Other rBasilar skull trauma with injury and avulsion of the rectus capitis and longus capitis muscles occurs when the horse falls over backward ,15,16. STemporohyoid osteoarthropathy is a disorder characterized by osseous proliferation of the temporohyoid joint that can lead to neurological disorders, especially facial nerve deficit ,18,19. TMRI may also be useful in select cases to diagnose temporohyoid osteoarthropathy ,9,17, alThe most common cause of a mass effect involving the guttural pouch is adenomegaly of the retropharyngeal lymph nodes secondary to guttural pouch empyema . HematomEven if endoscopy of the guttural pouch remains the gold standard for identifying most guttural pouch diseases, diagnostic imaging, especially radiographic and CT examinations, and occasionally ultrasound, can be very useful to complement clinical examination and endoscopic evaluation for the diagnosis of some diseases of the guttural pouch in order to precisely determine the prognosis and adapt the treatment.


The hCA I was inhibitedby compound 29 with a Ki valueof 3.0 nM, whereas hCA II was inhibited by compound 32 with a Ki value of 4.4 nM. The tumor-associatedhCA IX isoform was inhibited by compound 30 effectivelywith an Ki value of 43 nM, whereas theactivity of another cancer-related isoform, hCA XII, was significantlyinhibited by 29 and 31 with a Ki value of 5 nM. Molecular modeling showed that drug molecule 30 participates in significant hydrophobic and hydrogen bondinteractions with the active site of the investigated hCAs and bindsto zinc through the deprotonated sulfonamide group.To discover novelcarbonic anhydrase  inhibitorsfor cancer treatment, a series of 4-{4-[(hydroxyimino)methyl]piperazin-1-yl}benzenesulfonamideswere designed and synthesized using SLC-0111 as the lead molecule.The developed novel compounds  The human carbonic anhydrases (hCAs)pertain to the α-family of CAs and are associated with severalpathophysiological processes, such as electrolyte secretion, diversebiosynthetic reactions, pH regulation, CO2 homeostasis,and tumorigenesis.2 Fifteen isoforms of this classof enzymes have been discovered in humans, and they have distinctstructure, function, and kinetic properties, localization, and catalyticbehavior.3 The overexpression of CAs isassociated with a broad spectrum of diseases, including glaucoma,neuropathic pain, edema, obesity, and tumors. Therefore, at presentCA inhibitors are used for treatment of epilepsy, edema, glaucoma,obesity, and several cancers.4 Althoughscientists have discovered various hCA isoforms as potential and validatedtherapeutic targets for various diseases, further research is in progressto find the involvement of the CAs with other pathological disorders.5Carbonic anhydrases are ubiquitous metalloenzymes that are involved in the reversiblecatalytic hydration of carbon dioxide in cells to HCO6 Under normal conditionshCA IX is expressed in some cells, but it gets overexpressedin several cancers, such as colorectal, breast, brain, etc., in hypoxiadue to sturdy transcriptional activation induced by hypoxia-induciblefactor HIF-1.7 The overexpression of hCA IXin hypoxic tumors causes a decrease in the pH of the extracellularmatrix and thereby enhances the survival and progression of cancer.More importantly, hCA IX overexpression enhances the chemoresistanceof anticancer drugs that are weakly alkaline in nature.8 Consequently, hCA IX is one of the attractivetargets for the design and development of anticancer drugs for bothearly-stage and metastatic hypoxic cancers.9 The effect of hCA XII overexpression is mediated by hypoxiaand estrogen receptors in hypoxic tumors. It has been documented thathCA XII expression is up-regulated under hypoxic conditions likethat of the hCA IX isoform.10 hCA XIIis the cancer-associated isoform and is overexpressed in many formsof human cancer, including renal, pancreatic, gut, oral, brain, lung,and ovarian cancers. Therefore, hCA XII is also one of the remarkablebiomarkers for the inhibition of various hypoxic tumors at primaryand metastases stages.11The CA isoforms hCA IX, hCA XII,and hCA II areinvolved in several metabolic processes and pH regulation in hypoxictumors in which, due to the insufficient availability of molecularoxygen, the oxidative phosphorylation process of glucose is reducedand leads to a decrease in ATP production. Under the conditions whenthere is a lack of ATP production in the tricarboxylic acid cycledue to hypoxia, the cells meet the ATP production by adopting thealternative glycolytic pathway, in which lactic acid is produced asone of the bioproducts.2+ thatis tetrahedrally coordinated to three histidine aminoacid residues and one water molecule/hydroxide ion. The nitrogen atomof the imidazole ring of these residues is linked to the Zn2+ in the active-site region, and this site is present at the bottomhalf of the hydrophobic and hydrophilic cleft. Compounds of medicinalvalue can inhibit the functioning of CAs by interfering with thisactive site and avert the associated function.12 Ostensibly, several primary sulfonamides have been usedfor the treatment of glaucoma and epilepsy and as diuretics for decades,and this class of compounds is most investigated as carbonic anhydraseinhibitors (CAIs).13 For example, acetazolamide(AAZ) and dorzolamide are primary sulfonamides and are classifiedas first- and second-generation drugs used as CAIs.14 In addition, there are several compounds containing theSO2NH2 group that have shown significant inhibitionof CAs, including brinzolamide, ethoxzolamide, indapamide, and celecoxib3 benzenesulfonamide(SLC-0111) that is in phase II clinical trials for the treatment ofsolid hypoxic metastatic tumors.19 Therefore,in the present investigation, SLC-0111 was taken as the lead compoundto develop a novel series of CAIs. Molecules containing the -NOH group,such as psammaplin C, have recently been reported as potent inhibitorsof CAIs.21 Psammaplin C, a natural product, has shownsignificant inhibition of various CA isoforms, with Ki values in the nanomolar range, and contains an -NOHgroup in addition to an SO2NH2 group. PsammaplinC was investigated against the panel of hCA isoforms, and it inhibitedhCA I, hCA II, hCA IX, and hCA XII with Ki values of 48.1 nM, 88.0 nM, 12.3 nM, and 0.79nM, respectively.21Author: The benzenesulfonamide scaffold is widelyused in the developmentof CAIs. This fragment is a significant zinc-binding group (ZBG) andbinds to the zinc central metal atom of the CA through a coordinatebond to cause the inhibition.23 Scaffold hoppingis one of the modern strategies for the discovery of novel drug candidatesand is used in the invention of isofunctional chemotypes with molecularstructures significantly distinct from those of lead molecules.25 Inspired by this strategy, in the present research work, a novelseries of small-molecule 4-{4-[(hydroxyimino)methyl]piperazin-1-yl}benzenesulfonamideswere developed by combining the benzenesulfonamide core of SLC-0111with the >C=NOH group present in Psammaplin C, linked throughthe significantly bioactive piperazine heterocyclic scaffold  with hydroxylamine under reflux in a basic medium.26 The carboximidoyl chlorides 17–24 were prepared by chlorinating the intermediates 9–16 with N-chlorosuccinimide.27 The 4-fluorobenzenesulfonamide 25 was reacted with piperazine under reflux to obtain thecompound 26. In the next step the carboximidoylchlorides 17–24 were reacted withcompound 26 to get the target hydroxyimine-derived benzenesulfonamides 27–34. The structures of all the targetcompounds were established with the help of 13C NMR, 1H NMR, and ESI-MS, and the purity was elucidated by CHNS elementalanalysis. 1H NMR showed that the -OH group resonates inthe range of δ 10.54 to 9.42 ppm as a singlet in compounds 24–34. However, the singlet obtained inthe range of δ 163.41 to 152.98 ppm in the 13C NMRof compounds 24–34 corresponds tocarbon atom of the >C=N- group. The two hydrogen atoms ofthe>SO2NH2 group showed signals in the rangeofδ 7.09 to 7.03 ppm in the 1H NMR for compounds 24–34. All the hydrogen atoms of the piperazinering showed signals in the aliphatic region of the 1H NMRspectra of the target compounds. Similarly, the signals obtained inthe aliphatic region of the 13C NMR correspond to the carbonatoms of the piperazine heterocycle.The synthetic strategy employed for the synthesisof target compoundsis depicted in 27–34 synthesized in this study were investigated bystopped-flow CO2 hydrase assay and AAZ was taken as thereference drug.28 The compounds were screenedagainst a panel of hCA isoforms including hCA I, hCA II,hCA IX, and hCA XII. Cytosolic hCA II and transmembranehCA XII isoforms are upregulated in glaucoma and are thereforeprominent targets for intraocular pressure-maintaining drugs.30 Isoforms hCA IX and hCA XII are overexpressed in cancerpatients and are therefore validated targets for cancer treatment.31 In contrast, the cytosolic hCA I isoformis off target and is inhibited by sulfonamides effectively.32 The assessed results for the inhibition of screenedisoforms and the selectivity index (SI) values of designed drug molecules 27–34 compared with the standard AAZ arementioned in The hCA inhibition profiles of benzenesulfonamides 29, having an unsubstituted phenyl ring attached to the >C=NOHgroup, showed an inhibition constant Ki value of 3.0 nM for the hCA I isoform, and substitution ofa chlorine atom at the para position of this phenylring in compound 30 led to the molecule having a Ki value of 3.5 nM for the inhibition of hCA I.Replacement of the -Cl substituent with a -OCH3 group incompound 31 led to the discovery of a chemotype thatinhibited the hCA I activity with a Ki value of 4.0 nM. Incorporation of an electron-withdrawing group,-NO2, in compound 32 led to a novel compoundhaving a Ki value of 4.1 nM. However,the compound having a -CH3 substituent at the para position showed the inhibition of hCA I with a Ki value of 45 nM. Introduction of a pyridine ring insteadof a phenyl ring in compound 27 caused a decrease inpotency, and the observed Ki value forhCA I was 35.0 nM. However, the introduction of -F and -OH groupsin compounds 28 and 33 caused sharp declinesin potency. Although all the compounds of the series appeared to bemore potent than AAZ, compound 29 showed higher potencyand the compound having the hydroxyl group showed the minimum potency.The overall potency followed the pattern as H > Cl ≫ OMe>NO2 > Me > Py > F > OH, and the SAR is substituentdependentfor the hCA I isoform.To determine the structure–activity relationship(SAR) trends,the CA inhibitory activities of the investigated series of compoundswere deeply analyzed. It was found that all the compounds of the seriesinhibited the activity of hCA I in the nanomolar range. Compound 32, containingthe NO2 group, appeared to be a potent inhibitor of thisscreened isoform, and the inhibition constant Ki was observed to be 4 nM. The compound containing a pyridinering showed significant inhibition of hCA II, and the recorded Ki value was found to be 5.0 nM. However, compound 34, containing the -CH3 substituent at the para position of the aryl ring linked directly with >C=NOH,showed an inhibition constant Ki valueof 6.0 nM for hCA II, while compound 33 containingthe F substituent showed an inhibition constant of 6.6 nM. Incorporationof a -OCH3 substituent led to the discovery of a compoundthat showed an inhibition constant of 7.6 nM. Compound 29 containing the unsubstituted phenyl ring showed a Ki value of 9.0 nM, while the introduction of a chlorineatom at the para position of the aryl ring led tocompound 30, having a Ki valueof 9.4 nM for hCA II. Compound 28, having the -OHgroup, appeared to be least active for the inhibition of the hCA IIisoform; its observed Ki value was 80.7nM. The overall pattern of the inhibition of hCA II is NO2 > Py > -CH3 > F > OCH3 >phenyl > Cl> OH.Further, all the compounds of thisseries inhibited hCA IIin the 4.4 to 80.7 nM range. Compound 30 having a -Cl group at the para positionof thering adjacent to >C=NOH, showed the significant inhibitionand an observed Ki value of 43 nM. Thepyridine derivative 27 inhibited the hCA IX activitywith an inhibitor constant value of 110 nM. However, compound 29, having a phenyl group linked to >C=NOH, inhibitedthe hCA IX activity with a Ki valueof 121 nM. Compound 31, having a -OCH3 group,inhibited hCA IX with a Ki valueof 739.1 nM. Compound 32, containing an electron-withdrawinggroup, inhibited the activity of hCA IX with a Ki value of 911.5 nM, and compound 28, havingan -OH group, inhibited the activity of this isoform with a Ki value of 1179.4 nM, while compound 33, having a fluoro substituent, inhibited the activity of hCA IXwith a Ki value of 1339.0 nM. Compound 34, containing a methyl substituent, appeared to be leastactive for the inhibition of hCA IX, with an observed Ki value of >10000 nM. The pattern of inhibitionis Cl > Py > phenyl > OCH3 > NO2 > OH > F >CH3 for hCA IX.In the case of the hCA IX isoform, compound 29 and 31, having phenyl and -OCH3 substituents directly linked to the >C=NOH core, inhibitedthe hCA XII activity in the nanomolar range, the observed Ki value for both these compounds being 5.5 nM.Compound 28, having an -OH substituent at the para position of the ring adjacent to >C=NOH,inhibitedthe hCA XII activity with a Ki valueof 7.7 nM. Similarly, compound 30, having a chlorineatom at the para position of the ring, inhibitedhCA XII with a Ki value of 8.2 nM.The pyridine derivative 27 inhibited the activity ofhCA XII with an inhibition constant value of 52.3 nM, whereascompound 34, having a -CH3 group, inhibitedthe activity of hCA XII with a Ki value of 93.4 nM. Compound 33, having a fluoro atomat the para position of the aryl ring, inhibitedthe hCA XII activity with an inhibition constant value of 106.2nM. However, compound 32, having a NO2 groupat the para position of the aryl ring adjacent to>C=NOH, inhibited the hCA XII activity weakly, andthe Ki value was >10000 nM. The overallactivitypattern is phenyl = OMe > OH > Cl > py > CH3 > F > NO2. These SAR trends can be determined fromthe data given in All the compounds of the presentseries inhibited hCA XIIsignificantly, and most of these compounds inhibited the activityof this tumor-associated enzyme in the nanomolar range. Compounds 30, themostpotent of the synthesized series, interacts with the human off-targetcarbonic anhydrase I and II isozymes and the tumor-related isoformsIX and XII, a computational procedure was applied based on dockingand molecular dynamics (MD) simulations carried out on both isomers(Z) and (E). According to the literature,36 all docking solutions provided poses with the benzenesulfonamidegroup deeply bound to the zinc ion through the deprotonated nitrogenatom (SO2NH–). Stabilization of the ligandwithin the active site was also supported by hydrophobic contactsof the aromatic ring with residues L198, V121 (A121 in CA I isoform),V143, and W205 and by the formation of two H-bonds between the sulfonamideNH– and S=O with the side chain OH and backboneNH of T199, respectively.In order to rationalize how compound 30 was analyzedwithout considering the first 10 ns of the MD trajectories (blackdashed line in Z)-30 and (E)-30 assumetwo and three conformations, respectively. The most stable conformerof the (Z) isomer is maintained for about 66% ofthe entire dynamic duration, also thanks to the persistence (83%)of the interaction between the hydroxamic group and Q92 to which thegroup binds via a water-bridge bond (E) isomer undergoes rapid interconversion between two mainconformers having the -NOH group oriented toward the exit of the activesite, in water-bridged H-bond distance (17%) with the side chain NH2 of Q92 -30 within CA II is characterized by thefrequent switchingbetween two conformations (1 and 2 in E) isomer (57%)engages a water-bridged H-bond with the Q92 (11%) and π–πstacking interactions with the peculiar F131 (39%)  6B. Never31 (39%) 4B.Z)-30 and (E)-30 to move freely and switching between three and two main conformations,respectively. In all those the 4-chlorobenzene ring undergoes hydrophobicinteractions with the lipophilic cleft lined by the residues V131,V153, P201, and P202 (Z)-30 conformation is involved in awater-bridged H-bond between the hydroxyimine OH and the sidechain NH2 of Q92 (38%), while the conformer (E)-30 lodges the nitrogen atom of the -NOH group in water-bridgedH-bond distance with the NH2 side chains of Q92 and Q67(51%) (The F131/V131(CA II/CA IX) and F91/I91 (CA I/CA IX) mutationsmake the binding area of the CA IX roomier, thus allowing (and P202 3–6C. The67(51%) 6C.Z)-30 -30  conformation of (Z)-30 1, 3D at theZ)-30 1, 5D, leadiZ)-30 1, 5D. The mE)-30 1, 4D, is stE)-30 1, 6D.Z) and (E) isomersof ligand 30 withinthe four carbonic anhydrase isoforms provides convincing support tothe experimentally detected enzymatic inhibition data motivating thebest inhibitory profile toward the isoforms I, II, and XII of theenzyme within which the compound is held more stably by hydrophobicand H-bond interactions.The analysis of the binding modesof both the  molarmass < 500, (2) number of hydrogen bond acceptors < 10, (3)total number of hydrogen bond donors < 5, and (4) MlogP < 5.39 Compounds 27–34 complied with the Lipinski rule and exhibited considerable oralbioavailability scores, exerting appreciable pharmacokinetic profilesand significant drug-likeness properties. Moreover, the predictiveresults manifested that these compounds could not penetrate blood–brainbarrier (BBB). All the compounds showed bioavailability scores thesame as that of AAZ which determines the desirable bioavailabilityof the compounds and enviable pharmacokinetic properties. In addition,compounds 27–34 follow the Lipinskirule for the hydrogen bond donors and acceptors that demonstrate thedrug-likeness properties of these screened compounds. The physicochemicalproperties, lipophilicity, and other drug-likeness properties of thetarget compounds 27–34 are indicatedin Next, we examined the drug-likenessproperties and other physicochemicalproperties of the synthesized compounds 27–34 bind to the zinc ion of the hCA through the -NH– group of >SO2, as all other primary sulfonamides andas validated by molecular docking studies. Compound 30 emerged as one drug candidate that inhibits the activity of tumor-associatedhCA IX and hCA XII with inhibition constant Ki values of 43 nM and 8.2 nM, demonstrating the potentialanticancer activity of the chemotype. A substantial number compoundsof the screened series in this study inhibited the activity of thehuman carbonic anhydrases hCA I, hCA II, hCA IX, andhCA XII at nanomolar concentrations, demonstrating that suchcompounds could be used as potential hCA inhibitors and treated aslead candidates. The results assessed for the pharmacological effectdemonstrated that such small-molecule benzenesulfonamides couldbe presumed as drug candidates for the treatment of multiple malignanciesthrough tumor-associated CA inhibition.In conclusion, this study led to the discovery ofsmall-molecule4-{4-[(hydroxyimino)methyl]piperazin-1-yl}benzenesulfonamidesas a novel class of compounds that showed significant CA inhibitoryactivity and could be used as anticancer and antiglaucoma agents inthe future. All developed inhibitors