Development of Highly Sensitive Chemiluminescence Enzyme Immunostaining Assay to Determine Glycyrrhizin Content Using Anti-glycyrrhizin Monoclonal Antibody.

Licorice, the root of Glycyrrhiza spp., is used in a large number of herbal medicines, such as traditional Chinese medicines, Japanese Kampo medicines, and therapeutic drugs. Since glycyrrhizin (GL) is among the main components in licorice and exhibits numerous beneficial pharmacological activities, the content of GL directly affects biological activity. The quality control based on GL content is an important factor in ensuring biological activity; however, the content of GL in licorice varies depending on plant cultivation environment, genetic factors, and species type. Previously, we prepared an anti-GL monoclonal antibody (anti-GL mAb) and employed it in various immunochemical assays for quality control of licorice and licorice-based products. In this study, we employed the anti-GL mAb in chemiluminescence enzyme immunostaining (CLEIS) to develop a very simple, rapid, specific, and sensitive quality control assay for licorice products, with a limit of detection of 3.9 ng. Furthermore, the CLEIS assay enabled semiquantitative analysis of GL in Kampo medicines. Our results showed that multiple samples can be simultaneously analyzed using CLEIS, and it is a useful tool for determining GL content, as well as ensuring chemical quality control of licorice-containing products and herbal medicines.

Kwakhurin-magnetic particles conjugates enable fast enzyme immunoassay for the detection of kwakhurin in Pueraria candollei.

INTRODUCTION: Kwakhurin (Kwa) is one of the unique isoflavonoids produced in Pueraria candollei var. mirifica (P. candollei), which has long been used as folk medicine for rejuvenation in Thailand. Recently, the use of P. candollei-derived products has widely spread among Japanese women for cosmetic purposes. Correspondingly, there has been an increase in the number of reports regarding possible health hazards caused by estrogenic activity inherent to the plant; thus, the need for a detailed evaluation of the phytoestrogen content of P. candollei-derived products has gained a sense of urgency in recent years. OBJECTIVE: This study aims to develop a rapid enzyme immunoassay that can be applied to the quantitative analysis of Kwa in P. candollei and its derived products. MATERIAL AND METHOD: A rapid and sensitive immunoassay was developed with a combination of Kwa-specific monoclonal antibody (MAb 11F) and Kwa-magnetic particles (MPs) conjugates, which increased the surface area of the solid phase, resulting in a decrease in the immunoreaction time. RESULT: This novel MPs-based enzyme immunoassay (MPs-EIA) was used to determine Kwa concentration in the range from 2.44 to 78.1 ng/mL with a limit of detection of 1.90 ng/mL. Validation analyses revealed that the proposed MPs-EIA protocol was sufficiently precise and accurate for effective quantitative analysis of Kwa in P. candollei and its derived products.

[Retrospective Study of Image Quality in Pediatric Cardiac Cine MRI].

In pediatric cardiac cine magnetic resonance imaging (MRI), it must overcome several challenges including the patient's size and higher heart rate. The aim of this study was to retrospectively evaluate imaging optimization. Cardiac cine MRI data from 24 patients was analyzed (age range: 3 months-10 years, average age: 5 years, male/female: 11/13, R-R interval: 450±4 to 819±7 ms). About 11 cases out of 24 have good image quality. For small variations in the R-R interval and higher temporal resolution improved image quality with significant difference (P<0.05, Mann-Whitney U-test). In this study, values of temporal resolution <30 ms yielded good image quality for heart rates over 100 bpm. On the other hands, the factors dependent on the patient like heart rate and ejection fraction have no significant difference. The segmentation of data acquisition is more significant than recording small fields of view or thin slices for infantile and pediatric cardiac cine MRI. Similar to adult cases, variations in heart rate affect the image quality; however, we demonstrated that using segmentation of data acquisition results in improved image quality.

[Improvement of Brain Contrast Using Spin Echo T1-weighted Image at 3 T MRI].

Brain T1-weighted images using spin echo (SE) sequence has poor contrast at 3.0 Tesla magnetic resonance imaging (3.0 T MRI) systems from the influence of crosstalk and magnetized transfer (MT) effect, and prolongation of the T1 value. Therefore, improving of scan parameters has been reported such as excitation flip angle (FA) and interleave data acquisition. The purpose of this study was to show the effects of alterations of presaturation pulse amplitude and chemical shift selective (CHESS) pulse amplitude. Gray-to-white matter contrast increased with decreasing amplitude of presaturation pulse in whole brain imaging. Presaturation and CHESS pulse consist of radio frequency pulse. Therefore, both pulses have a similar effect on MT pulse. Manual alteration of presaturation pulse amplitude for each scan lacks versatility on clinical use. However, decreasing amplitude of presaturation pulse is equal to decreasing thickness of presaturation pulse. About CHESS pulse, it requires no manual alteration for each scan. For example, switching fat suppression mode from strong to weak increase T1 contrast. Our study demonstrated that using not only low excitation FA and interleave date acquisition but also low amplitude of presaturation and CHESS pulse increase the contrast in T1 SE brain scans at 3.0 T MRI.

Perioperative Amino Acid Infusion for Preventing Hypothermia and Improving Clinical Outcomes During Surgery Under General Anesthesia: A Systematic Review and Meta-analysis.

Amino acid (AA) infusion is sometimes selected to avoid hypothermia during general anesthesia. However, the widespread clinical use of AA infusion therapy has not been established. This study aimed to clarify the evidence that AA infusion can increase patient body temperature and improve clinical outcomes using the Grading of Recommendations Assessment, Development, and Evaluation system. We searched MEDLINE (PubMed), Cochrane Central Register of Controlled Trials, and Igaku Chuo Zasshi (Japana Centra Revuo Medicina) in November 2015. Studies were reviewed by 2 independent assessors to identify randomized controlled trials (RCTs) involving AA infusion compared with placebos during surgery under general or combined general/epidural anesthesia. Study quality was assessed using the Grading of Recommendations Assessment, Development, and Evaluation system and the Cochrane methodology. The primary outcome was difference in body temperature before and after perioperative AA infusion. Shivering frequency, blood loss volume, postoperative intubation time, and hospitalization period were also assessed as clinical outcomes. We analyzed the outcome data using a random effect model. From 298 screened titles, 14 RCTs met our inclusion criteria, including 626 patients (327 in AA and 299 in placebo groups). In 626 participants from 14 RCTs, AA infusion increased body temperature by a mean difference (MD) of 0.46°C (95% confidence interval [CI], 0.31-0.62, low-quality evidence). Regarding other outcomes, AA infusion decreased shivering frequency by a risk ratio of 0.34 (95% CI, 0.12-0.94; 7 RCTs, 248 participants, very low-quality evidence), shortened postoperative intubation time by MD of -125 minutes (95% CI, -210 to -38.8; 2 RCTs, 158 participants, moderate-quality evidence), and shortened the hospitalization period by MD of -1.81 days (95% CI, -2.07 to -1.55; 3 RCTs, 230 participants, low-quality evidence) compared with placebo. There was no significant difference in the volume of blood loss between the 2 groups (standardized MD, -0.20, 95% CI, -0.44 to 0.04; low-quality evidence). There was no publication bias. AA infusion in the perioperative period increased patient body temperature and improved clinical outcomes compared with placebo. However, the evidence to support the use of AA infusion is limited, and further large-scale RCTs are required.

Simultaneous determination of glycyrrhizin and liquiritin in licorice roots and Kampo medicines by combination enzyme-linked immunosorbent assay using anti-glycyrrhizin and anti-liquiritin monoclonal antibodies.

Immunoassay systems using monoclonal antibodies (mAbs) are one of the most useful techniques in the analytical, biochemical, and clinical fields. In this study, a combination enzyme-linked immunosorbent assay (ELISA) using both anti-glycyrrhizin and anti-liquiritin mAbs (anti-GL/Liq mixture mAbs) was developed for quality control of licorice and its products. The combination ELISA demonstrated high sensitivity, reproducibility, and specificity for the total content of GL and Liq by a single assay. The developed ELISA was effective and useful as the first screening method in the selection of high-quality licorice from the Glycyrrhiza species and in confirming the quality of licorice-containing Kampo medicines.

Nano-Structural Investigation on Cellulose Highly Dissolved in Ionic Liquid: A Small Angle X-ray Scattering Study.

We investigated nano-structural changes of cellulose dissolved in 1-ethyl-3-methylimidazolium acetate-an ionic liquid (IL)-using a small angle X-ray scattering (SAXS) technique over the entire concentration range (0-100 mol %). Fibril structures of cellulose disappeared at 40 mol % of cellulose, which is a significantly higher concentration than the maximum concentration of dissolution (24-28 mol %) previously determined in this IL. This behavior is explained by the presence of the anion bridging, whereby an anion prefers to interact with multiple OH groups of different cellulose molecules at high concentrations, discovered in our recent work. Furthermore, we observed the emergence of two aggregated nano-structures in the concentration range of 30-80 mol %. The diameter of one structure was 12-20 nm, dependent on concentration, which is ascribed to cellulose chain entanglement. In contrast, the other with 4.1 nm diameter exhibited concentration independence and is reminiscent of a cellulose microfibril, reflecting the occurrence of nanofibrillation. These results contribute to an understanding of the dissolution mechanism of cellulose in ILs. Finally, we unexpectedly proposed a novel cellulose/IL composite: the cellulose/IL mixtures of 30-50 mol % that possess liquid crystallinity are sufficiently hard to be moldable.

Pulmonary interstitial emphysema due to respiratory syncytial virus infection.

Pulmonary interstitial emphysema (PIE) primarily affects premature infants on positive pressure ventilation. PIE is rarely reported in infants and children in the absence of mechanical ventilation and/or associated respiratory infection. We report a case of PIE in a 22-month-old girl who had severe respiratory distress due to respiratory syncytial virus infection. Chest computed tomography showed cystic lung lesions mimicking congenital cystic adenomatoid malformation. The cystic lesions spontaneously resolved after conservative treatment. Based on the clinical course and the chronological changes on imaging, the cystic lung lesions were diagnosed as localized persistent PIE.

Risk factors for recurrence of frozen shoulder after shoulder manipulation under ultrasound-guided cervical nerve root block.

Background: This study aimed to investigate risk factors for recurrence of frozen shoulder after shoulder manipulation under ultrasound-guided cervical nerve root block (MUC). Methods: We retrospectively reviewed 135 frozen shoulders in 121 patients who underwent MUC. We defined frozen shoulder as a limited shoulder range of motion (ROM) (passive forward flexion <120°, external rotation <30°, or internal rotation lower than L3). Patients fulfilling any one criteria were considered to have frozen shoulder. If patients continued to have severe pain and limited ROM at 3 months after MUC, we defined as recurrence of frozen shoulder and they were offered a further MUC or arthroscopic capsular release (ACR). We compared the ROM, Constant Shoulder (CS) score, and University of California, Los Angeles score before and 3 months after MUC between patients with the successful of MUC group (Success group) with those recurrence of frozen shoulder who required a further MUC or ACR group (Recurrence group). Multiple logistic regression analysis was used to identify risk factors for recurrence of frozen shoulder after MUC. Results: Patients who underwent MUC were retrospectively enrolled and divided into: the successful of MUC group (Success group, n = 112) and required a further MUC or ACR group (Recurrence group, n = 9). The Recurrence group had significantly lower external rotation and CS score before MUC than those in the Success group (P < .05). The Recurrence group showed significantly inferior all ROM and functional scores 3 months after MUC (P < .05). The levels of blood glucose and hemoglobin A1c both before and 3 months after MUC in the Recurrence group showed inferior compared with those of Success group. The difference, although not statistically significant, trended towards significance (before MUC/3 months after MUC; the glucose levels P = .06/.06, the hemoglobin A1c levels P = .07/.09, respectively). The visual analog scale pain score (at rest, during activity, at night) both before and 3 months after MUC in the Recurrence group showed significantly higher scores compared with those of Success group (P < .05). Multiple logistic regression analysis revealed that lower CS score before MUC was independent risk factor for recurrence of frozen shoulder after MUC. Conclusion: The overall incidence of recurrence of frozen shoulder after MUC was 7.4%. The lower CS score before MUC was an independent risk factor for recurrence of frozen shoulder after MUC. Moreover, patients in the Recurrence group tended to have poorly controlled diabetes and higher visual analog scale pain score both before and 3 months after MUC.

Highly sensitive indirect competitive enzyme-linked immunosorbent assay based on a monoclonal antibody against saikosaponin b2 for quality control of Kampo medicines containing Bupleuri radix.

Saikosaponins are naturally occurring oleanane-type triterpenoids that are found in Bupleuri radix (root of Bupleurum falcatum) and exhibit a broad biological activity spectrum. Saikosaponin b2 (SSb2) is the main saikosaponin in Kampo medicine extracts and is a designated quality control marker for the same in the Japanese Pharmacopeia. Although some monoclonal antibodies (mAbs) against saikosaponins have been produced to evaluate the quality of Bupleuri radix and related products, anti-SSb2 mAbs have not been used to quantify SSb2 in Kampo medicines. To address this knowledge gap, we herein established a new hybridoma cell line secreting a highly specific anti-SSb2 mAb and developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) based on this mAb for the detection of SSb2 in Bupleuri radix-containing Kampo medicines. The generated SSb2-recognized mAb exhibited high specificity to SSb2 in icELISA. The developed assay featured high sensitivity (linearity range = 1.95-125 ng/ml), accuracy, precision and reproducibility (coefficient of variation < 5%), and the thus determined SSb2 contents were strongly correlated with those obtained using liquid chromatograph-mass spectrometer. These results suggest that the anti-SSb2 mAb-based icELISA method can be used for the quality control and standardization of Kampo medicines containing Bupleuri radix.

Intercalation of a surfactant with a long polyfluoroalkyl chain into a clay mineral: unique orientation of polyfluoroalkyl groups in clay layers.

Eight novel polyfluorinated surfactants (C(n)F(2n+1)CONH(CH2)2 N(+)(CH3)2C16H33 Br(-); abbreviated as CnF-S, where n = 1, 2, 3, 4, 5, 6, 8, 10) were synthesized and their intercalation into cation-exchangeable clay was investigated. All of the polyfluorinated surfactants intercalated in amounts exceeding the cation exchange capacity (CEC) of the clay. The C4F-S and C5F-S surfactants exhibited intercalation up to 480% of the CEC as a saturated adsorption limit. On the basis of X-ray analysis, CnF-S surfactants intercalated between clay nanosheets to form a bilayer structure in which the surfactant molecules tilt at an angle of ∼60° with respect to the clay surface. The saturated adsorption limits and layer distances differed between surfactants with short (n = 1, 2) and long (n = 3-10) perfluoroalkyl chains. For long-chain surfactants, their saturated adsorption limits were independent of the perfluoroalkyl chain length and the layer distances systematically increased with increasing perfluoroalkyl chain length. These results suggest that the microscopic orientation differed between the short and long chains. X-ray analysis showed that the long-chain surfactants orient the perfluoroalkyl chains at a tilt of 41 ± 5° with respect to the clay layer. This value was in good agreement with polarized IR measurements of 42 ± 2° for this angle.

Development of an indirect competitive enzyme-linked immunosorbent assay for formononetin and its application in a cell-based assay using MC3T3-E1 cells.

Formononetin (FMN) is a methoxy isoflavone found abundantly in leguminous plants and associated foods. Several analytical methods have been developed to detect FMN. However, they are costly, complicated, and time-consuming. This study describes an indirect competitive enzyme-linked immunosorbent assay (icELISA) to determine FMN content in food samples using a monoclonal antibody (mAb) against FMN produced by a newly established hybridoma cell line. Validation studies were conducted, and this assay was found to be sufficiently reliable, with an analytical measurement range of 19.53-1250 ng/mL and a detection limit of 17.42 ng/mL. Furthermore, icELISA was successfully applied for a cell-based assay in which the amount of FMN and ononin uptake was quantified in MC3T3-E1 cells. Hence, icELISA is a simple and reliable method for the detection and quantification of FMN, as well as elucidation of its functions and underlying mechanisms of action.

The Association between the First Cry and Clinical Outcomes in CDH Neonates: A Retrospective Study.

Congenital diaphragmatic hernia (CDH) is a life-threatening condition characterized by the herniation of abdominal organs into the thorax, resulting in hypoplastic lungs and pulmonary hypertension. The impact of the first cry, a crucial event for lung transition during birth, on CDH patients remains unclear. This study investigated the impact of the first cry during birth on CDH patient survival, along with other prognosis factors. A multi-institutional retrospective study assessed CDH patient characteristics and survival rates by analyzing factors including the first cry, disease severity, birth weight, Apgar scores, oxygenation index (OI) and surgical closure. Among the CDH patients in the study, a positive first cry was linked to 100% survival, regardless of disease severity (p < 0.001). Notably, the presence of a positive first cry did not significantly affect survival rates in patients with worse prognostic factors, such as low birth weight (<2500 g), high CDH severity, low Apgar scores (1 min ≤ 4), high best OI within 24 h after birth (≥8), or those who underwent patch closure. Furthermore, no significant association was found between the first cry and the use of inhaled nitric oxide (iNO) or extracorporeal membrane oxygenation (ECMO). In conclusion, this study suggests that the first cry may not have a negative impact on the prognosis of CDH patients and could potentially have a positive effect.

Nicotine- and tar-free cigarette smoke induces cell damage through reactive oxygen species newly generated by PKC-dependent activation of NADPH oxidase.

We examined cytotoxic effects of nicotine/tar-free cigarette smoke extract (CSE) on C6 glioma cells. The CSE induced plasma membrane damage (determined by lactate dehydrogenase leakage and propidium iodide uptake) and cell apoptosis {determined by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] reduction activity and DNA fragmentation}. The cytotoxic activity decayed with a half-life of approximately 2 h at 37°C, and it was abolished by N-acetyl-L-cysteine and reduced glutathione. The membrane damage was prevented by catalase and edaravone (a scavenger of (•)OH) but not by superoxide dismutase, indicating involvement of (•)OH. In contrast, the CSE-induced cell apoptosis was resistant to edaravone and induced by authentic H(2)O(2) or O(2)(-) generated by the xanthine/xanthine oxidase system, indicating involvement of H(2)O(2) or O(2)(-) in cell apoptosis. Diphenyleneiodonium [NADPH oxidase (NOX) inhibitor] and bisindolylmaleimide I [BIS I, protein kinase C (PKC) inhibitor] abolished membrane damage, whereas they partially inhibited apoptosis. These results demonstrate that 1) a stable component(s) in the CSE activates PKC, which stimulates NOX to generate reactive oxygen species (ROS), causing membrane damage and apoptosis; 2) different ROS are responsible for membrane damage and apoptosis; and 3) part of the apoptosis is caused by oxidants independently of PKC and NOX.

Flavin Adenine Dinucleotide-Related Adverse Event Necessitating Cardiopulmonary Resuscitation in an Infant.

Mitochondrial rescue drugs, such as vitamin B2, are a treatment modality which can be considered in unexplained cases of cardiac arrest or impaired consciousness in which mitochondrial diseases are considered in the differential. Although case reports exist of children developing a drop in their blood pressure following administration of intravenous flavin adenine dinucleotide (FAD) sodium as vitamin B2, we present the first reported case of a child requiring cardiopulmonary resuscitation (CPR) following FAD sodium infusion for severe bradycardia and hypotension. Intravenous infusion of FAD sodium should be administered as slowly as possible, with careful monitoring of blood pressure and heart rate.

Bioimprinting as a Receptor for Detection of Kwakhurin.

Bioimprinting was performed against ovalbumin (OVA) to confer its binding cavities for kwakhurin (Kwa), an isoflavonoid, produced solely by Pueraria candollei var. mirifica (P. candollei). The characterization of bioimprinted-OVA (biOVA), evaluated by an enzyme-linked immunosorbent assay (ELISA), revealed that it functioned as a specific receptor for Kwa. Using biOVA, two systems, i.e., an indirect competitive ELISA (icELISA) and the even simpler and more rapid competitive enzyme-linked bioimprinted-protein assay (cELBIA), were developed as novel techniques for the quantitative analysis of Kwa in P. candollei and its related products. The two analysis methods were found to have limits of detection (LOD) of 4.0 and 2.5 µg/mL, respectively. The high reliability of the developed icELISA and cELBIA using biOVA was also demonstrated by various validation analyses. Subsequently, bioimprinting was performed using eight other proteins to investigate them as candidate scaffolds for the generation of binding cavities for Kwa. Interestingly, two bioimprinted-IgG monoclonal antibodies (biMAbs) recognized Kwa, but their original binding affinity to hapten was lost. That is, the MAbs obtained a new binding ability to Kwa in exchange for their original binding affinity, raising the possibility that biMAb could be alternatively used as a probe for the quantitative analysis of Kwa as well as biOVA. This is the first report of small molecules recognition by MAbs used as proteins for bioimprinting.

Severe encephalopathy associated with SARS-CoV-2 Omicron BA.1 variant infection in a neonate.

INTRODUCTION: The coronavirus disease 2019 (COVID-19), including the Omicron variant, is less severe in children than in adults. To date, there has been no detailed description of COVID-19-associated severe encephalopathy due to the Omicron variant during the neonatal and early infantile periods. CASE PRESENTATION: During the outbreak of the Omicron variant, a 29-day-old male presented with a pale and ill appearance. The patient was intubated for mechanical ventilation owing to recurrent apnea, which subsequently turned out to be a breath-holding that may have been caused by seizure. In addition, nonconvulsive status epilepticus was observed. Total duration of repetitive seizure activities was approximately 30 min per hour when seizures were most severe. Brain magnetic resonance imaging (MRI) on day 14 revealed extensive hyperintensity in the T2 sequence, hypointensity in the fluid-attenuated inversion recovery (FLAIR) sequence in the deep and subcortical white matter, and diffusion restriction in the corpus callosum. The Omicron BA.1 variant of the severe acute respiratory syndrome coronavirus 2 was detected in his respiratory sample. Follow-up MRI on day 45 revealed multiple cystic cavitations. CONCLUSION: Although COVID-19 is not severe in most children, life-threatening conditions such as COVID-19-associated severe encephalopathy can occur during the neonatal and early infantile periods.

Infantile hepatic hemangioma and hepatic mesenchymal hamartoma in an infant associated with placental mesenchymal dysplasia: a case report.

BACKGROUND: Although infantile hepatic hemangioma and hepatic mesenchymal hamartoma are relatively common in benign pediatric liver tumors, coexistence of the two tumors is rare. Placental mesenchymal dysplasia is also a rare disorder. We report the case of a baby girl born after a pregnancy complicated by placental mesenchymal dysplasia, who developed both infantile hepatic hemangioma and hepatic mesenchymal hamartoma. CASE PRESENTATION: The patient was born at 32 weeks and 5 days of gestation for impending placental abruption, weighing 1450 g. Liver tumors, composed of both hypervascular solid and large cystic lesions, were detected after birth and markedly increased to create abdominal distention within 9 months. Diagnostic imaging suspected the coexistence of infantile hepatic hemangioma and cystic hepatic mesenchymal hamartoma. Following propranolol therapy for infantile hepatic hemangioma and needle puncture of a large cyst, the cystic lesions and adjacent hypervascular lesions were partially resected via laparotomy. Pathological findings confirmed the coexistence of hepatic mesenchymal hamartoma and infantile hepatic hemangioma, which had no association with androgenetic/biparental mosaicism. The postoperative course was uneventful, and the tumor had not regrown after 3 years. CONCLUSIONS: Although the coexistence of infantile hepatic hemangioma and hepatic mesenchymal hamartoma associated with placental mesenchymal dysplasia is extremely rare, the pathological and pathogenetic similarities between these disorders suggest that they could have derived from similar embryologic origins rather than being a mere coincidence. Further follow-up is required, with careful attention to the potential for malignant hepatic mesenchymal hamartoma transformation.

Immunological Separation of Bioactive Natural Compounds from Crude Drug Extract and Its Application for Cell-Based Studies.

In this study, we present a review on a useful approach, namely, immunoaffinity column coupled with monoclonal antibodies (MAbs), to separate natural compounds and its application for cell-based studies. The immunoaffinity column aids in separating the specific target compound from the crude extract. The column capacity was stable even after more than 10 purification cycles of use under the same conditions. After applying the crude extract to the column, the column was washed with washing buffer and eluted with elution buffer. The elution fraction contained the target compound bound to MAb, whereas the washing fraction was the crude extract, which contained all compounds except a group of target compounds; therefore, the washing fraction was referred to as a knockout (KO) crude extract. Cell-based studies using the KO extract revealed the actual effects of the natural compounds in the crude extract. One-step separation of natural compounds using the immunoaffinity column coupled with MAbs may help in determining the potential functions of natural compounds in crude extracts.