To evaluate cerebrospinal fluid dynamics, using cine phase-contrast magnetic resonance imaging (cine-PC MRI), in healthy pediatric subjects, determining the normal flow values in this population, as well as identifying differences related to age, sex, and body surface area.This was a descriptive cross-sectional study involving 32 healthy children and adolescents, in whom the flow of cerebrospinal fluid through the cerebral aqueduct was evaluated quantitatively with cine-PC MRI. We used specialized software to analyze the images obtained with cine-PC MRI, drawing a region of interest on the aqueduct. A flow-time curve was obtained, as were automated measurements of the various parameters.The following normal (mean) values were obtained: net flow, 1.10 ± 0.99 mL/m; stroke volume, 12.2 ± 10.1 µL/cycle; mean velocity, 0.72 ± 1.00 cm/s; peak systolic velocity, 5.28 ± 2.30 cm/s; peak diastolic velocity, 4.51 ± 1.77 cm/s. These values were not affected by age or sex. In addition, body surface area was not found to correlate significantly with mean velocity or stroke volume.In children and adolescents, the basic cerebrospinal fluid flow parameters, as determined by cine-PC MRI, appear to be independent of age and sex. The CSF also plays an important role in the homeostasis of the interstitial fluid of the brain parenchyma, as well as in regulation of neuronal function. It is mainly produced by the epithelium of the choroid plexuses, located in the lumen of the lateral ventricles and in the fourth ventricle. After circulating through the ventricular system, subarachnoid spaces, and ependymal canal, CSF is absorbed into the venous system by the arachnoid granulations,5. Additional sites of absorption include the spinal arachnoid villi near the epidural spinal veins and the meningeal sheaths of the spinal and cranial nerves. Lymphatics located proximal to arteries and nerves are also part of the CSF absorption mechanism.Cerebrospinal fluid (CSF) is a transparent liquid, with a density of 1,003-1,008g/cm,8. In addition, these measurements are useful in the preoperative evaluation of patients with idiopathic intracranial hypertension and in the postoperative follow-up after endoscopic third ventriculostomy,10. Nevertheless, the applicability of flow measurements for the preoperative assessment of patients with normal-pressure hydrocephalus is controversial. Bradley et al. found that patients with a CSF stroke volume greater than 42 µL had a favorable response after shunting. However, in a more recent study, Kahlon et al. established that there is no clear relationship between stroke volume and clinical improvement after surgery.Evaluation of CSF flow dynamics, in adults and children, is an essential clinical tool for the assessment of various neurological diseases. Recently, cine phase-contrast magnetic resonance imaging (cine-PC MRI) has emerged as a useful technique for the measurement of multiple physiological parameters without the need for invasive maneuvers. In children, CSF flow studies are also clinically valuable, because of the high number of disorders affecting CSF kinetics, such as a Chiari I malformation and a brain tumor,14. In addition, cine-PC MRI techniques facilitate the differentiation between communicating and non-communicating hydrocephalus in the pediatric population, as well as the distinction between hydrocephalus and ex vacuo ventricular dilatation, which is a challenging issue in pediatric neuroradiology,16.Cine-PC MRI techniques have also demonstrated applicability in patients with arachnoid cysts, aiding in the differentiation of those that communicate with the ventricular system from those that do notIn order to perform appropriate evaluations of CSF flow dynamics in multiple pathological conditions, it is first essential to characterize the measurements in normal individuals. Unfortunately, most CSF characterization studies have been conducted in adults. The corresponding data for the pediatric population is scarce, worldwide and in Latin American countries. Therefore, we assessed CSF flow parameters using cine-PC MRI in healthy pediatric subjects, with the objective of characterizing the potential effects that age, sex, and body surface area (BSA) have on those parameters., using a confidence level of 80% and a probability of 0.05%, was 32 participants. The study was approved by the bioethics committee of the hospital. The parents or legal guardians of the subjects gave written informed consent.This was a descriptive cross-sectional study conducted over a three-month period in the radiology department of a tertiary care pediatric hospital. Pediatric subjects were recruited from among presumably healthy individuals undergoing cine-PC MRI of the brain during that period. The minimum sample size, calculated on the basis of the formula devised by Viechtbauer et al.All of the subjects were between 1 month and 14 years of age. Subjects with a CSF pathology, a structural brain/spinal pathology (known or suspected), or a history of head trauma were excluded, as were those in whom intra- or extra-axial lesions, ventriculomegaly, or alterations in the craniocervical junction were observed during the study. The cases were stratified by age group: 1-12 months (infants); 1-5 years (toddlers and preschool children); 5-11 years (children); and 12-14 years (adolescents).All images were obtained in a 1.5-T MRI scanner . The routine brain MRI procedure was performed, which involved acquiring the sequences required according to the indication of each study, using a standard head coil. Subsequently, a T2-weighted fluid-attenuated inversion recovery pulse sequence  was acquired in the axial plane for the evaluation of CSF characteristics (if it was not previously included), as were cine-PC MRI sequences with prospective cardiac triggering, a pulse oximeter being used for the qualitative and quantitative evaluation of CSF flow through the cerebral aqueduct. Velocity encoding was set at 20 cm/s. The images obtained were processed with the software READY View . The region of interest was drawn at the level of the cerebral aqueduct, as was an area of background subtraction in a magnified image, in the axial slices of the quantitative cine-PC MRI sequence  and 2. Ap < 0.05 were considered statistically significant.Statistical analysis was performed with the IBM SPSS Statistics software package, version 22.0 . Continuous quantitative variables are presented as means and standard deviations. Comparisons among and between age groups and sexes were made by using analysis of variance and Student’s t-tests. The correlation between BSA and stroke volume was evaluated with the Spearman correlation coefficient, whereas the Pearson coefficient was used in order to evaluate the correlation between BSA and mean velocity. Values of During the three-month study period, a total of 42 subjects underwent brain MRI including cine-PC MRI images for CSF flow evaluation. However, in seven subjects, there were artifacts that made it difficult to evaluate the images; in two, there were errors during the image processing; and one subject was found to have a brain tumor. Those subjects were excluded from the study, resulting in a final sample size of 32 subjects.2.Of the 32 subjects included, 20 (62.5%) were female and 12 (37.5%) were male. Eleven (34.0%) of the subjects were in the 5- to 11-year age group, whereas the other three age groups accounted for seven subjects (22%) each. In the sample as a whole, the mean height was 116.0 ± 31.4 cm, the mean weight was 27.3 ± 16.2 kg, and the mean BSA was 0.90 ± 0.38 mThe data obtained through cine-PC MRI revealed the following mean values: net CSF flow, 1.10 ± 0.99 mL/m; stroke volume, 12.2 ± 10.1 µL/cycle; mean velocity, 0.72 ± 1.00 cm/s; systolic peak velocity, 5.28 ± 2.30 cm/s; and diastolic peak velocity, 4.51 ± 1.77 cm/s. Those values are further dissected by age group and sex in p = 0.67). p = 0.67).No statistically significant differences were found between age groups and sexes regarding any parameter under investigation. . The advent of MRI has allowed us to improve our understanding of CSF flow dynamics significantly, in physiological and pathological conditions, in a simple, noninvasive manner. This imaging modality enables us to evaluate multiple flow parameters such as peak velocity, mean velocity, and stroke volume. This quantitative evaluation is performed by means of cine-PC MRI sequences, which use a velocity encoding gradient to generate signal contrast between moving and stationary hydrogen atoms. Cine-PC MRI is a noninvasive technique that allows the evaluation of flow without requiring the use of ionizing radiation or the injection of contrast medium, making it suitable for use in the pediatric population. The main drawback of the technique is that it requires sedation in young children.Traditionally, CSF flow dynamics have been studied through animal experiments and with mathematical models, most of which provide rough and indirect approximations, which may have limited applicability in human subjects studied 83 healthy pediatric subjects and found a peak velocity of 5.77 ± 3.18 cm/s and a mean velocity of 0.83 ± 0.73 cm/s. They also found significant differences in peak velocities between adolescents and children, as well as between males and females, although they found no significant differences in mean velocities. Conversely, we found no significant sex- or age-related differences between any of the dynamic CSF parameters evaluated. When making a comparison based on a variable such as age in pediatric studies, it should be borne in mind that the classification may vary depending on the age ranges assigned to each subgroup, which can lead to different reporting of results in comparison with the published literature.There have been few studies evaluating CSF dynamic parameters and their association with age and sex in normal pediatric subjects. Öztürk et al. evaluated CSF flow in 137 children and found no significant correlation between body mass index and CSF flow parameters. Although we hypothesized that mean velocity and stroke volume would correlate with BSA, we found no such correlations. That might be due to minimal variations in the cross-sectional area of the cerebral aqueduct in the pediatric population, which has been reported to be approximately 0.5 mm2, compared with 0.8 mm2 in adults,23. These results indicate that the CSF parameters measured are independent of biometric values.Moreover, literature is also limited in relation to the evaluation of these measurements according to biometric parameters. Demirtaş et al..We consider the present study to be of relevance because it contributes to the limited existing literature regarding MRI evaluation of CSF dynamics in healthy populations. In addition, corresponding studies from Latin America are scarce, and this study may help characterize CSF parameters in the Latin American population. Our study has some limitations. First, there were artifacts in some of the cine-PC MRI sequences, which led to their exclusion from the study and a reduction in the sample size. Second, in some cases, the placement of the region of interest was complicated by the presence of a very narrow cerebral aqueduct, which could increase the margin of error. Finally, neonates were not evaluated. These limitations have also been reported by other authorsWe determined the normal values for various parameters of CSF flow dynamics in healthy pediatric subjects, stratifying them by sex and age group, and found no significant differences. We also found that BSA did not correlate with mean velocity or stroke volume.


Background:Cutibacterium acnes is normal skin flora as well as a common culture contaminant. It can cause infections in the setting of sterile implants, although clinical presentations can be subtle. Differentiating true infection from sample contamination is challenging and has implications for patient care. We describe an investigation of a cluster of 7 hospitalized pediatric patients with C. acnes isolated from anaerobic cultures of cerebrospinal fluid (CSF) over 3 weeks at a quaternary-care children’s hospital. Methods: An outbreak response was coordinated between the infection prevention and control (IPC), microbiology, and neurosurgery teams. We defined a case as a hospitalized patient with C. acnes isolated from a CSF culture beginning in November 2020. We reviewed charts of all cases and CSF culture collection on all case units, transport to and processing at the microbiology laboratory, and the IPC team measured adherence for all processes. Results: There were 8 positive cultures in 7 cases from November 10 to 27, 2020. The median case age was 2 months . Cases occurred on 4 different units. All positive patients had at least 1 implanted neurosurgical device used for CSF drainage. There were no clear commonalities in surgeon responsible for device placement, hardware type placed, or staff collecting CSF samples. A standard protocol for CSF collection was followed for all cases. Overall, 3 patients cleared cultures without intervention, 2 received oral antibiotics, and 2 underwent surgical removal of their device. Specimen processing was unchanged, although due to supply issues, an alternative anaerobic culture media  was used for 6 weeks, during which all cases were identified. Compared to routine media, the alternative is known to enhance organism detection. The company reported no concerns for media contamination or C. acnes outbreaks. Once routine media became available, CSF culture positivity for C. acnes returned to baseline (late November or early December) (Fig. Conclusions: We identified a likely pseudo-outbreak related to temporary use of a more sensitive culture media. No direct patient harm was identified, although many had increased risk of harm by surgical intervention or prolonged length of stay. Technological advances may enhance organism identification but challenge existing paradigms of care. More studies are needed to better delineate the intersection of diagnostic advancements with patient care standards.Funding: NoneDisclosures: None


A confirmed CNS infection was significantly associated with older age  and with an increased WBC count in the CSF . Ninety-seven (97) of 99 (98.0%) children received empiric antimicrobial treatment, and 35 (35.3%) were treated with multiple antibiotics. Eighty-four (84) patients (84.9%) were discharged home, and 11 (11.1%) were transferred to the National Hospital because their condition had worsened. No deaths were recorded. Point-of-care tests, including real-time PCR assays to identify common pathogens, should be implemented for more accurate diagnosis and more appropriate antibiotic use.From July to October 2020, 99 cases of central nervous system (CNS) infections were identified in Thai Binh Pediatric Hospital, Viet Nam, representing a five-fold increase compared to the baseline incidence during the previous five years. Clinical data were retrospectively collected. Cerebrospinal fluid specimens (CSF) were secondarily tested for pathogens using viral culture and PCR assays. Patient median age was 5 years (0–12 years); 58.6% were male. Of these children, 83.8% had CSF white blood culture (WBC) counts of ≥ 10 cells/µL, including 58 of 99 (58.6%) with a WBC count ≥ 100 cells/µL. Overall, 72 (72.7%) patients had confirmed infections with a pathogen identified in the CSF, the majority of which (66) were enterovirus. Sequencing results suggested that the rise of incidence observed in 2020 was due to Echovirus 4 ( These infections, including meningitis, encephalitis, and brain abscesses, are potentially life-threatening conditions, particularly among young children . In addiRapid and accurate identification of microbial pathogens is essential in the management of CNS infections. Common methods used in microbiology laboratories are antigen or antibody detection, direct microscopic examination, and culture techniques. But these methods are limited by low sensitivity and/or specificity. Currently, with high accuracy and short execution time, molecular methods performed on cerebrospinal fluid (CSF) samples are considered the “platinum” standard, in contrast to the culture “gold” standard in the etiological diagnosis of CNS infections . HoweverSeveral infectious pathogens are known to cause CNS infection, including the broad categories of viruses, bacteria, mycobacteria, fungi, and parasites. In children, viruses are the more common pathogens responsible for CNS infections, which are mostly mild and self-limited . BacteriIn Thai Binh, Viet Nam, CNS infections, including meningitis and encephalitis, are frequent infectious diseases in children . In our This study was carried out in TBPH, a large hospital in the central province of Thai Binh. In 2019, this hospital had 565 beds distributed across 15 different medical departments. Ill children under 16 years are directly admitted from home  or referred from district hospitals in the province. Children with severe disease who do not improve are transferred to the Vietnam National Children’s Hospital in Hanoi (VNCH). The identification of microorganisms in the laboratory is very limited, relying mainly on common bacterial culture and serological tests .Between July and October 2020, all ill children admitted to the TBPH meeting the clinical case definition for CNS infections  5] were were5] wClinical data, including sociodemographic characteristics, clinical symptoms, basic laboratory investigation, treatment and outcome were retrospectively obtained from medical electronic records.n = 24). CSF was collected from all patients for standard laboratory testing , standard bacterial culture and anti-JEV  IgM detection in the TBPH hospital laboratory.Routine laboratory investigations were performed at the discretion of the receiving clinician and based on hospital guidelines. Blood specimens were collected from all patients for white blood cell (WBC) counts and C-reactive protein (CRP) detection. Blood cultures were performed on a small number of patients ) plus 0.05% sodium polyanethol sulfonate . The vented bottles were incubated at 37°C for up to seven days. Bottles were routinely sub-cultured onto solid media at 24 h and seven days, with additional subculture if turbidity was noted on daily inspection.CSF specimens (2–3 mL) were routinely submitted for red and white blood cell counts , glucose and protein estimation . IgM against Japanese encephalitis virus (JEV) were detected using the commercial kit JE MAC-ELISA  according to the manufacturers’ recommendations.In addition, CSF was submitted for Gram stain, then cultured onto 5% sheep blood agar, chocolate agar, MacConkey and Sabouraud agar plates  for up to 48 h. Bacterial isolates from blood and CSF cultures were identified using the VITEK Mass Spectrometry System .For each CSF specimen, 100 µL was passed through a 0.22-μm pore size centrifugal filter  and then were inoculated into four wells of 96-well culture microplates containing MDCK cells (ATCC CCL-34) in MEM with 10% fetal calf serum and 1% L-glutamine. After centrifugation at 4000×g, microplates were incubated at 37°C. Each sample was cultured until a cytopathic effect (CPE) was observed, usually requiring one to four passages (seven days per passage). Viral growth was detected by searching for CPE daily, using an inverted microscope, then confirmed using a multiplex PCR assay ).Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitidis, Escherichia coli K1, Streptococcus agalactiae, Listeria monocytogenes, enterovirus (EV), herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), human herpesvirus type 6 (HHV-6), cytomegalovirus (CMV), varicella zoster virus (VZV), human parechovirus (HPeV), and Cryptococcus neoformans/gattii. This test includes automated sample homogenization, nucleic acid extraction, reverse transcription, nucleic acid amplification, and results in analysis in approximately one hour per run per specimen.Molecular identification was first carried out using the BioFire FilmArray Meningitis/Encephalitis Panel (ME). Approximately 200 μL of CSF was subject to FilmArray® ME Panel testing according to the manufacturer’s instructions. The panel identifies 14 common agents of community-acquired meningoencephalitis: Klebsiella pneumoniae (phoE gene) and Staphylococcus aureus (nucA gene)), and five main Candida species  on supernatant after viral culture. These five Candida spp. were selected because unlike Cryptococcus spp. infections that usually occur in adults and exceptionally in children, Candida spp. are agents of neonatal infections . Their prevalence is probably low but possibly overlooked because they are not routinely diagnosed in Vietnam. The laboratory manipulation procedure and protocol have been described elsewhere . In a national acute encephalitis syndrome surveillance study conducted from 1998 to 2007, the annual incidence of encephalitis in Vietnam ranged from 3.0 to 1.4 cases per 100,000 population, with JEV responsible for 52% of cases, and with a decreasing trend over the 10-year period of study [JEV is one of the most frequent pathogens responsible for CNS infection in Asian tropical countries, including Vietnam ,20,21, bof study , while tOur results showed that EV was responsible for most cases in the outbreak that occurred in Thai Binh in 2020, in line with previous studies ,23-25. EThe typical clinical course of EV-associated meningitis is a biphasic fever, accompanied by neurological symptoms during the second febrile peak. Non-specific signs in patients with EV-associated meningitis, including nausea, vomiting, headache, cutaneous rash, and respiratory symptoms, were found in all age groups. In older children, neck stiffness and photophobia are frequent, while neonates often present with irritability, lethargy, and bulging fontanelles. Increased polymorphonuclear leukocytes are a typical sign at the early stages of disease in the CSF laboratory examination .Currently, there are more than 100 genotypes of EV identified, subdivided into 4 species , CV-A5, CV-A7, CV-A9, CV-A16, CV-B2-5, E-4, E-6, E-9, E-11, E-14, E-16, E-25, E-30, E-31, and EV-A71 have been demonstrated to cause meningitis . There iRegarding treatment, almost all patients were treated with antibiotics, while bacteria were detected in 2 cases in the CSF and in 4 cases in the blood. It is unlikely that antibiotherapy before admission accounted for this result, given that only 12.1% children received it and that no statistical association was evidenced. It cannot be excluded that the relative lack of capacity for microbiological testing at the hospital may have played a role. In any case, systematic antibiotic treatment in CSN infections in children is not recommended, and antibiotic overuse is an important contributor to the development of antibiotic resistance worldwide .Our study has some limitations. This is a monocentric retrospective study, so it may not represent the entire country. Moreover, most cases were related to an outbreak. Also, due to the lack of microbiological testing facilities in Thai Binh Pediatric Hospital, blood culture was performed in only a small proportion of patients. Based on these results, point-of-care tests, including real-time PCR assays to identify common pathogens in CNS infections, should be implemented for more accurate diagnosis and more appropriate antibiotic use.Click here for additional data file.


In Keivan et al.,FIGURE 1 Histopathological H&E and IHC images of testis in control , TT , and TT + exosome  groups. Red arrow shows the interstitial connective tissue containing Leydig cell, and green arrow shows basal spermatogenic cell lines. 100× magnification. N = 18 animals in 3 different groups. H&E, hematoxyline and eosin; IHC, immunohistochemistryAlso, the authors would like to revise the graphical abstract and image in online as provided below,Graphical abstract(1) In this research, adult male mice were used for testopathy modeling. (2) Through temporary torsion of spermatic cord, the laboratory model of testicular torsion was induced. (3) Thus, the pathological condition of testopathy was confirmed by laboratory documentations. (4) Exosomes were derived from mouse blood serum. (5) Blood‐derived derived exosomes were injected into the testis. (6) Finally, the pathological symptoms caused by testicular torsion decreased to a physiological status and sperm production returned to normal conditions.The Authors apologize for this error.


Spinal cord stimulation (SCS) is a promising treatment for patients with disorders of consciousness (DoC); however, the laboratory examinations and different electrodes  that are associated with postoperative outcomes are unclear. The study aims to study the association between the change in postoperative cerebrospinal fluid (CSF) protein level and improvement in consciousness after SCS in DoC patients and to explore whether different electrodes were associated with elevated CSF protein levels.A total of 66 DoC patients who received SCS treatment from December 2019 to December 2021 were retrospectively analyzed. Patients were grouped according to their elevated CSF protein level. The clinical characteristics of the patients and SCS stimulation parameters were compared. The preoperative sagittal diameter of the spinal canal is the distance from the midpoint of the posterior border of the vertebral body to the midpoint of the posterior wall of the spinal canal at the level of the superior border of C3. The postoperative sagittal diameter of the spinal canal is the distance from the midpoint of the posterior edge of the vertebral body to the anterior edge of the stimulation electrode. Patients with improved postoperative CRS-R scores greater than 3 or who progressed to the MCS + /eMCS were classified as the improved group and otherwise regarded as poor outcome.P = 0.001), and elevated CSF protein levels were significantly associated with a reduced sagittal diameter (P = 0.044). In DoC patients receiving SCS treatment, we found that elevated CSF protein levels (P = 0.022) and preoperative diagnosis (P = 0.003) were significantly associated with poor outcomes at 3 months. Logistic regression analysis showed that elevated CSF protein levels were significantly associated with poor outcomes .We found that more DoC patients had elevated CSF protein levels among those receiving SCS treatment with permanent electrodes than temporary percutaneous electrodes (The results suggest that reducing the effect of electrode pads on anatomical changes may help improve the outcomes of DoC patients receiving SCS treatment. CSF protein levels are associated with poor postoperative outcomes and whether they are potential biomarkers in DoC patients receiving SCS treatment remain further exploration. Disorders of consciousness (DoC) are among the most frequent complications in patients after severe brain injury and are mainly caused by trauma, stroke, or anoxia . DoCs inCerebrospinal fluid (CSF) is secreted by the choroid plexus of the ventricle, and its properties are similar to those of plasma and lymph, which act as diagnostic tools for many conditions affecting the central nervous system . The maiWe retrospectively reviewed 66 patients with DoC who were treated with SCS at the Neurosurgery Department in Beijing Tiantan Hospital and Peking University International Hospital between December 2019 and December 2021. Considering that the patients could not understand and legally consent to treatment, the SCS procedure and the possible risks were explained to the patients’ legal representatives and/or family members. Once the realistic expectations were explained, their legal representatives and/or family members signed an informed consent document compatible with our institution’s legal and ethical committee regulations, where this trial was approved (2011-0415). When their caregivers provided written informed consent for their participation, the patients were enrolled.The inclusion criteria were as follows: (1) Clinical diagnosis of DoC ; (2) DoCData were extracted from the enrolled patients’ medical records between December 2019 and December 2021. The baseline variables included age, sex, pathogenesis, duration of DoC, diagnosis, hydrocephalus, and paroxysmal sympathetic hyperactivity (PSH). After obtaining informed consent, 2–3 ml of middle CSF samples were taken during routine lumbar puncture. The samples were kept on ice and transferred to −20°C refrigerator within 10 min.We also collected CSF samples from patients preoperatively and on the 7th day postoperatively, and the total CSF protein level was determined by rate nephelometry. The detection process was conducted in accordance with the manufacturer’s instructions.39286 electrode is 56.4 mm long, 7.6 mm wide, 1.9 mm thick, 4 mm contact length, 1.5 mm contact spacing, 42.5 mm contact tip to contact tip, and 65cm electrode tip to wire tip. In total, 3,777 electrode contact is 3 mm, the spacing is 6 mm, the length from the electrode contact tip to the contact tip is 66mm, and the total length from the electrode tip to the contact tip is 75 cm. The detailed procedure for the placement of permanent electrodes  can be found in our previous study . A midliFor temporary percutaneous electrode placement , the patient was placed in a prone position, and the T7-8 intervertebral space was positioned under the C-arm as the puncture point. The electrode  was inserted at the C2 level and fixed. The day after the temporary SCS operation, electric stimulation was applied to the patient’s dorsal column at a voltage of 2.5 V and a frequency of 70 Hz with a 120 μs wave width, which is consistent with the SCS treatment using permanent electrodes. The overall stimulation lasted for 21 days, and then the electrode was removed.Before and after surgery, 64-slice spiral computed tomography (CT)  was used to obtain plain cervical CT scans, coronal scans, and sagittal scans of the enrolled patients. The axial images of the upper edge of the C3 vertebral body were obtained from the imaging system. The data were measured and processed independently by two senior clinicians using ImageJ (1.42) software, and the measurement results were averaged. Finally, the sagittal diameter of the spinal canal was obtained. The preoperative sagittal diameter of the spinal canal is the distance from the midpoint of the posterior border of the vertebral body to the midpoint of the posterior wall of the spinal canal at the level of the superior border of C3. The postoperative sagittal diameter of the spinal canal is the distance from the midpoint of the posterior edge of the vertebral body to the anterior edge of the stimulation electrode. Patients with a sagittal diameter reduction of more than 1.5 #39286 electrode pads (3 units) were classified as the reduced sagittal diameter group .The consciousness level of each patient was assessed using the CRS-R16. On the day before surgery and the 30th day after surgery, the CRS-R score was independently evaluated by 2 clinicians. For patients with an auditory score of ≤2 points, a visual score of ≤1 point, a motor score of ≤2 points, a language score of ≤2 points, a communication score of 0 points and an arousal score of ≤2 points, the diagnosis was VS. For patients with an auditory score of 3 to 4, a visual score of 2 to 5, a motor score of 3 to 5, a language score of 3, a communication score of 1 and an arousal score of ≤2, the diagnosis was MCS. Patients with improved postoperative CRS-R scores greater than 3 or who progressed to the MCS + /eMCS were classified as the improved group and otherwise regarded as poor outcome.t-test. Chi-square tests were used for categorical variables. P for trend was tested using one-way analysis of variance (ANOVA) for continuous variables and linear-by-linear association for categorical variables. Logistic regression analysis was performed to determine the factors related to outcomes with default parameters. The significance threshold was a two-sided P-value less than 0.05.SPSS 22.0 software was used to process the data. The Kolmogorov–Smirnov test was used to determine normality. Normally distributed measurement data are expressed as the mean ± standard deviation. Age was compared using an independent samples P = 0.087) or sex (0.732) between the groups. In this cohort, 27 (40.9%) cases were caused by traumatic brain injury, 11 (16.7%) cases were caused by anoxia and 28 (42.4%) cases were caused by stroke, and there was no significant difference between the groups (P = 0.998). We also found no significant difference in the duration of DoC (P = 0.839). There were significantly more patients in a MCS in the improved group (P = 0.003). There were significantly more patients with elevated CSF protein levels in the non-improved group (P = 0.022). Regarding complications, there was no significant difference in the occurrence of hydrocephalus (P = 0.752) or PSH (P = 0.752) between the groups.Between December 2019 and December 2021, SCS was performed on 66 patients, 47 males (71.2%) and 19 females (28.8%), with an average age of 46.38 ± 13.33 years. The characteristics are summarized in P = 0.201). There were also no significant differences in sex (P = 0.099), pathogenesis (P = 0.532), duration of DoC (P = 0.379) or diagnosis (P = 0.062) in the elevated CSF protein level group. Complications such as hydrocephalus (P = 0.369) and PSH (P = 0.369) were also not different.The clinical features of DoC patients receiving SCS according to elevated CSF protein levels are summarized in P = 0.032).We analyzed the potential factors associated with outcomes in DoC patients receiving SCS treatment, and the results are shown in P < 0.001) and elevated CSF protein level (P = 0.001) than in the temporary puncture electrode group (P = 0.044, To further examine whether surgery-related factors could affect CSF protein levels, we analyzed the relationship between SCS electrodes and the sagittal diameter or CSF protein level. The results showed that in the permanent electrode group, there were significantly more patients with a reduced sagittal diameter (de group . We alsoIn this cohort study, we found that in DoC patients receiving SCS treatment, an elevated CSF protein level was significantly associated with poor outcomes at 3 months. This result suggests that CSF protein levels may play a role in the process of rehabilitation and may be a potential biomarker in DoC patients receiving SCS treatment. Logistic regression analysis showed that elevated CSF protein levels were significantly associated with poor outcomes. We also found more DoC patients with elevated CSF protein levels among those receiving SCS treatment with permanent electrodes rather than temporary percutaneous electrodes and more DoC patients with a reduced sagittal diameter in the permanent electrodes group. Furthermore, we found that elevated CSF protein levels were significantly associated with a reduced sagittal diameter. The reason an elevated CSF protein level was more significant in the permanent electrode group was because of the larger size of the electrode pads.In recent decades, because the pathogenesis of DoC has not been clearly stated, developments in the treatment of DoC has stagnated and different treatments have minimal effects. In recent years, with the development of neuromodulation technology, many emerging tools have been applied to the evaluation, diagnosis, and treatment of DoC . SCS, asvia dynamic circulation, and increased CSF protein levels have been reported to play a role in neurodegenerative diseases such as Alzheimer’s disease, aging and other pathological processes, including subarachnoid hemorrhage and trauma (The possible mechanisms underlying the change in total CSF protein level after SCS treatment may be due to the disruption of the blood–brain barrier by intraoperative local procedures or the disturbance of regional CSF circulation. Traditionally, CSF protects the brain and spinal cord from mechanical injury, but the understanding of the dynamic metabolism and function of CSF has rapidly improved in recent decades . It has d trauma . Disruptd trauma . Previoud trauma . SCS eled trauma . Thus, td trauma .Furthermore, we found that elevated CSF protein levels after SCS were associated with lower patient emergence rate at 3 months. Previous studies have shown similar results: at the proteomic level, DBS, an invasive neuroregulatory procedure, can up- or downregulate the expression level of various proteins in CSF and peripheral blood and may further affect prognosis . AdditioOur study has several limitations. First, a predictive model was not generated due to the limited number of patients enrolled in this study. Second, in the current study, we tracked the change in the total CSF protein level for a short period of time and did not explore the time-course trend of the total CSF protein level, which may further clarify the effects of such factors on patient outcomes. Finally, we did not perform further proteomic analysis of CSF in this study. Future research on CSF composition may help to further elucidate this issue and explore treatment methods. Large-scale, long-term follow-up studies are still needed to validate the preliminary results obtained in this study.In this study, we found that elevated CSF protein levels were significantly associated with poor outcomes in DoC patients after SCS treatment and significantly related to a reduced sagittal diameter, which suggests that CSF protein levels may play a role in rehabilitation. The level of CSF protein may also serve as a biomarker in DoC patients receiving SCS treatment and reducing the effect of electrode pads on anatomical changes may help improve the outcomes of DoC patients receiving SCS treatment.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of Beijing Tiantan Hospital. The patients/participants provided their written informed consent to participate in this study.QH and YY designed the study and wrote the manuscript. TL, YX, XX, YD, XC, and XG collected the data and samples. JH and JZ supervised the study. All authors contributed to the article and approved the submitted version.


Meningoencephalitis of unknown origin (MUO) is an inflammatory disease of the canine central nervous system (CNS) that shares several features with multiple sclerosis (MS) in humans. In approximately 95% of MS patients, ≥ two immunoglobulin G (IgG) oligoclonal bands (OCBs) are detectable exclusively in the cerebrospinal fluid (CSF).To investigate OCBs in CSF and serum in dogs affected by MUO, intervertebral disc disease (IVDD), idiopathic epilepsy (IE), intracranial neoplasia (IN), steroid-responsive meningitis-arteritis (SRMA), and diseases outside the CNS. We hypothesize that the highest prevalence of CSF-specific OCBs (≥ two OCBs uniquely in the CSF) would be found in dogs affected by MUO.Client-owned dogs (n = 121) presented to the neurology service due to neurological deficits.Prospective study. Measurement of IgG concentration in CSF and serum via a canine IgG ELISA kit. OCB detection via isoelectric focusing (IEF) and immunoblot.Presence of CSF-specific OCBs was significantly higher in dogs with MUO (57%) compared to 22% in IN, 6% in IE, 15% in SRMA, 13% in IVDD, and 0% in the non-CNS group (p < .001). Dogs with MUO were 9.9 times more likely to show CSF-specific OCBs than all other diseases together .MUO showed the highest prevalence of CSF-specific OCBs, indicating an inflammatory B cell response. Future studies are needed to evaluate the prevalence in the specific MUO subtypes and a possible similarity with human MS. Meningoencephalitis of unknown origin (MUO) encompasses idiopathic, non-infectious inflammatory diseases of the canine central nervous system (CNS), which can be further subdivided into granulomatous meningoencephalitis (GME), necrotizing meningoencephalitis (NME), and necrotizing leukoencephalitis (NLE) via histopathological examination . AlthougStudies have highlighted characteristics –10 similSimilar to MS, the pathogenesis of MUO is not fully understood. So far, a T cell-mediated delayed-type hypersensitivity with involvement of mainly T and just few B cells has been postulated in dogs with GME . The lacThe only clinical study available in veterinary medicine on OCBs in German Shepherd dogs diagnosed with degenerative myelopathy involved just a small number of diseased and healthy dogs and lacked the comparison with other neurological diseases .The aim of the present study therefore was to investigate CSF and serum for the presence of CSF-specific OCBs with IEF and immunoblot in dogs affected by MUO in comparison to dogs affected by idiopathic epilepsy, intracranial neoplasia, intervertebral disc disease, steroid-responsive meningitis-arteritis, or diseases not affecting the CNS. We hypothesized that the highest prevalence of CSF-specific OCBs would be found in dogs affected by MUO.The experimental procedures used in this study were approved by the ethical committee of the Veterinary service, Cantone of Bern (BE121/2020).Sampling of CSF and serum was done during the diagnostic work-up of each patient (no sampling was done only for research purposes), and all efforts were made to minimize stress and suffering for each patient. In addition, we have obtained the owner’s written consent for each of the dogs involved in this study. Human samples were taken during the diagnostic work-up of the patient in the University Hospital, Zurich (not taken only for the purpose of the study). Patients signed a consent that residual amount of CSF and serum not needed for the diagnostic work-up can be used for research purposes. Samples were anonymized prior to researchers having access to the samples.CSF and serum samples were prospectively collected from dogs presented to the Small Animal Clinic, Division of Clinical Neurology, Vetsuisse Faculty, University of Bern, Switzerland due to neurological impairment between April 2018 and June 2020. Patients were included in the study if all criteria were met: All dogs had a complete medical record including signalment, they received a complete diagnostic work-up with clinical and neurological examination, full blood work , MRI of the brain and/or spinal cord and CSF puncture. When possible, dogs received a histopathological and immunohistopathological examination of brain biopsies/the brain.Dogs were classified into six disease categories: meningoencephalitis of unknown origin (MUO), idiopathic epilepsy (IE), intracranial neoplasia (IN), intervertebral disc disease (IVDD), steroid-responsive meningitis-arteritis (SRMA), and non-CNS category. A clinical diagnosis of MUO was based on the following criteria: (1) dogs older than 6 months, (2) multiple, single or diffuse intra-axial hyperintensities on T2-weighted MR images, and/or (3) pleocytosis in CSF analysis with > 50% of mononuclear cells, and (4) exclusion of infectious diseases . ResponDogs were assigned to the IE category, if they presented recurrent epileptic seizures and their diagnostic work-up met the Tier 2 confidence level as defined by the International Veterinary Epilepsy Task Force (IVETF) . Dogs wePaired serum and CSF samples were collected from all dogs during the diagnostic work-up. CSF was obtained by atlanto-occipital puncture in lateral recumbency during general anesthesia and examined within 30 minutes for total protein and total nucleated cell count (TNCC) as well as cytology. Pleocytosis was defined as a TNCC > 5 cells/μL and was further characterized as mild (6–50 cells/μL), moderate (51–200 cells/μL) or marked (> 200 cells/μL) . The pleStatistical analysis was performed using NCSS . For all analyses, p < .05 was considered statistically significant. Summary descriptive statistics were calculated to describe the population of patients included as well as different CSF findings and pathology results. Concentrations were non-normal distributed. Therefore, the median was used additionally to describe total protein, TNCC, IgG in CSF and serum per disease category. A Kruskal-Wallis test was used for assessment of group differences of continuous, non-parametric data . Moreover, associations were assessed between the presence of CSF-specific OCBs and the TNCC as well as CSF cytology. Logistic regression and contingency tables were used to evaluate a possible relation between CSF-specific OCBs and single disease categories, and MUO versus all other disease categories summarized. Kappa statistics were used to assess the interobserver agreement.125 dogs were initially included in the study. Four dogs from the MUO category had to be excluded as they were diagnosed with idiopathic cerebellitis (n = 2) and eosinophilic meningoencephalitis (n = 2). Therefore, 121 dogs were enrolled in the present study comprising 28 dogs with MUO, 18 dogs with IE, 23 dogs with IN, 13 dogs with SRMA, 23 dogs with IVDD, and 16 dogs with non-CNS disease . The mosIn the MUO disease category, 15/28 dogs showed a good response to treatment and were still alive at the time of writing, 2/28 were lost to follow-up and 11/28 died or were euthanized. Of the latter, a postmortem examination was declined by the owner in 7/28 dogs. Histopathology was available in 4/28 dogs with MUO  and 16/23 dogs with IN .Total protein in CSF was available in 97/121 dogs with a median of .2 g/L and a mean of .45 g/L (5th-95th percentile .1–1.90 g/L). The TNCC was determined in 119/121 dogs with a median of two cells/μL and a mean of 200 cells/μL (5th-95th percentile .3–1234.7 cells/μL). Total protein was not determined in cases with CSF examination in the emergency night shifts, when only Pandy reaction was assessed. TNCC was not recorded in one dog with IN and one dog with IVDD. As only three dogs were found with a moderate pleocytosis, the categories moderate pleocytosis and marked pleocytosis were grouped together as marked pleocytosis to simplify the statistical analysis. The IgG concentration in CSF and serum was measured in all samples with a median of 46.93 mg/L in CSF and 22.11 g/L in serum and a mean of 191.74 mg/L (5th-95th percentile 11.78–1080.64 mg/L) in CSF and 23 g/L (5th-95th percentile 10.43–38.24 g/L) in serum. Medians as well as range of total protein, TNCC, IgG in serum and CSF per disease category can be found in An increased IgG concentration in CSF was significantly associated with the presence of CSF-specific OCBs (p < .01)  and an iPresence of CSF-specific OCBs was significantly higher in dogs diagnosed with MUO  comparedThe pathogenesis of human MS and the MUOs in canine patients are both not fully understood. Consequently, both diseases lack specific diagnostic tests for ante mortem definitive diagnosis. Instead, a range of defined clinical criteria is used to narrow down the diagnosis, one of which is the presence of CSF-specific OCBs as part of the McDonald criteria to diagnose MS. Up to 95% of human MS patients present with CSF-specific OCBs . SimilarOne potential explanation might be that the majority of dogs showed a good response to treatment and was therefore still alive at the time of writing or lost to follow-up resulting in a low number of patients with histopathological confirmation of MUO. In the majority of cases, the diagnosis of MUO was based on clinical factors, hence it cannot be excluded that neoplastic or other inflammatory diseases were included in the MUO disease category, lowering the actual prevalence of CSF-specific OCBs in the MUO category. The clinical diagnosis of MUO was based on information from clinical and neurological examination, full blood work , MRI of the brain and/or spinal cord and CSF puncture. There are currently no clear and internationally recognized diagnostic criteria defined for diagnosis of MUO. In the present study, the guidelines provided by Granger et al. presenting criteria for the diagnosis of MUO were followed , althougIn human medicine, the overall prevalence of CSF-specific OCBs in inflammatory diseases in general, but not MS specifically, is not well established. However, the prevalence range is wide, with around 95% in MS, and for example 16.4% in neuromyelitis optica , 6–17% iEach of the MUO subtypes presents with specific features, such as predilection for white matter in NLE and GME , or a geStudies in human medicine demonstrated OCBs in various non-inflammatory neurological diseases with a prevalence of 67% in glioma patients, and 8.7–14% in patients with epileptic seizures , 39. In Ante mortem differentiation between inflammatory and neoplastic lesions can be challenging and so far a definitive diagnosis can be reached only via biopsy , 40. UsiSRMA is an inflammatory disease that does not solely affect the CNS but also has a strong systemic effect due to the widespread vasculitis. Therefore, the majority of cases was expected to show either a systemic OCB pattern  or CSF-Finally, the prevalence of CSF-specific OCBs in dogs diagnosed with IE was 6%  in order to reach the best resolution possible. The loading order was from left to right, starting with dog no. 1 with the serum sample, then CSF sample etc. Dog no. 1 (study no. 82) was diagnosed with intracranial neoplasia, dog no. 2 (study no. 83) and dog no. 3 (study no. 84) with idiopathic epilepsy, dog no. 4 (study no. 85) with MUO, and dog no. 5 (study no. 86) with a disease outside the CNS. The samples from the human MS patient (study no. 23/2020) served as positive controls. CSF-specific OCBs were detected in dog no. 4 (study no. 85). (PDF)Click here for additional data file.S1 TableData used to reach the conclusions drawn in the manuscript.(DOCX)Click here for additional data file.S2 TableM = MUO, meningoencephalitis of unknown origin, E = idiopathic epilepsy, N = intracranial neoplasia, S = steroid-responsive meningitis-arteritis, D = intervertebral disc disease, C = non-CNS disease, TNCC = total nucleated cell count.(XLSX)Click here for additional data file.


TP53 significantly decreased LTR5Hs transcriptional activity. Chromatin immunoprecipitation (ChIP) and CUT&Tag experiments finally confirmed the direct binding of the p53 protein with the two LTR5Hs p53 binding sites. Overall, the two p53 binding sites in the third section (−263 to 0) of LTR5Hs were revealed to play critical roles in the difference in transcriptional activity among the three subtypes.The long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) are distributed throughout the human genome and provide favorable conditions to regulate the expression of their adjacent genes. HML-2 is the most biologically active subgroup of the HERV-K family, and expression of its members has been associated with many cancer types. The LTRs of HML-2 have been classified into three subgroups  based on phylogenetic analyses. The current study aimed to explore the LTR transcriptional activity differences among the three subtypes and further explore the underlying factors. A total of 43 LTR5A elements, 62 LTR5B elements, and 194 LTR5Hs elements were selected. A phylogenetic tree showed that the LTR5Hs group was clearly separated from the LTR5A and LTR5B groups. A luciferase reporter assay indicated that LTR5Hs had the strongest promoter activity, followed by LTR5A and LTR5B. To investigate the underlying factors, LTR5Hs was divided into 4 sections, and the homologous fragments in LTR5B were replaced successively. Replacement of the third section (−263 to 0) significantly increased LTR5B activity. Subsequent mutation experiments revealed that the increased transcriptional activity was induced by the TATA box and the two p53 binding sites within the section. Further interference with IMPORTANCE Human endogenous retroviruses (HERVs) were integrated into the human genome in ancient times and have been coevolving with the host. Since the Human Genome Project, HERVs have attracted increasing attention. Many studies have focused on their characterization, evolution, and biological function. In particular, the expression of HERV-K has been associated with many diseases, such as germ cell tumors, neurotoxicity, ovarian cancer, prostate cancer, and melanoma. Indeed, two HML-2-produced proteins, Np9 and Rec, are associated with certain cancers. However, their roles in these disease associations remain unclear. The current work focused on subgroup HML-2 of HERV-K, which is recognized as the most biologically active subgroup, and aimed to explore the mechanistic basis of transcriptional activity. The results revealed that p53 deeply determined the activity of HML-2 LTR5Hs. p53 is a rather important tumor suppressor protein. It can regulate the expression of genes related to cell cycle arrest, organic processes, and apoptosis in response to cellular stress and is critical for the control of homeostasis. Previous ChIP and expression studies of individual genes suggested that p53 sites in HERV LTRs may be part of the p53 transcription program and directly regulate p53 target genes in a species-specific manner. However, the exact function of p53 in the regulation of HERV LTR expression is largely elusive. Our results clearly demonstrated the interaction between LTR5Hs of HML-2 and p53. They are of great significance for the future comprehensive study of the physiological and pathological functions of LTRs of HERVs. Human endogenous retroviruses (HERVs), which stem from exogenous retrovirus infection during the evolution of primates and subsequent integration into the genome, account for approximately 8% of the human genome –3. The v–5–9–A complete HML-2 provirus is approximately 9.5 kb in length and consists of four overlapping genes that encode structural and nonstructural proteins, with an LTR structure (5′ LTR-gag-pro-pol-env-3′ LTR) at each terminus . Each fl13–Based on phylogenetic analyses, the LTR sequences of HML-2 are classified into three subgroups: LTR5A, LTR5B, and LTR5Hs. LTR5B is the oldest ancestral type, while LTR5Hs is the most recently acquired. LTR5A and LTR5Hs were independently generated from LTR5B , 16, 17.HERV-K HML-2) LTRs exist as part of complete provirus structures or as single-LTR structures called solo LTRs . Within  LTRs exi19–Although the functions of many transcription factors have been revealed, it is unclear whether the transcriptional activities of the three HML-2 LTR subtypes are different, and the LTR TFBSs associated with HERV function are largely elusive. In this study, we investigated the transcriptional activity differences among LTR5A, LTR5B, and LTR5Hs of HML-2 and explored the key TFBSs.Based on BLAT (BLAST-Like Alignment Tool) analysis using Dfam consensus representatives, a total of 704 LTR5A sequences, 828 LTR5B sequences, and 256 LTR5Hs sequences were identified . After alignment using ClustalW in BioEdit, we screened 43 LTR5A sequences, 62 LTR5B sequences, and 194 LTR5Hs sequences, which were longer than 60% of the Dfam full-length references for each type, for further analyses. The majority of sequences in the data set covered the third fragment (−263 to 0), which was the target region of the current work. The identified sequences and reference sequences used to construct the phylogenetic trees are shown in Table S2.To characterize the phylogenetic relationships among HML-2 LTR5A, LTR5B, and LTR5Hs, the 43 LTR5A sequences, 62 LTR5B sequences, 194 LTR5Hs sequences, and reference sequences were used to construct maximum-likelihood (ML) trees. Overall, our classification was the same as those of the previous reports defining the three major subgroups , 17. Ourcis-regulatory roles of these LTR elements were predicted with the Genomic Regions Enrichment of Annotations Tool (GREAT), which can predict genes potentially regulated by these elements based on spatial proximity. In total, 263, 331, and 316 potentially regulated genes were obtained for LTR5A, LTR5B, and LTR5Hs, respectively. The gene names and distances from LTR elements are shown in Table S3. The results showed that there was little overlap among the three subtypes  were predicted to be regulated by all three subtypes . Ninety-nine genes were predicted to be regulated by both LTR5A and LTR5B, 9 genes were predicted to be regulated by both LTR5A and LTR5Hs, and 6 genes were predicted to be regulated by both LTR5B and LTR5Hs. The results are based on the potential regulatory genes predicted by GREAT according to spatial proximity. Further research is required to confirm any of the implied associations between the solo LTRs and the nearby genes.Typically, noncoding regions lack biological function annotations. To study the biological significance of LTR5A, LTR5B, and LTR5Hs in the human genome, the putative subtypes . Only 9 We further analyzed the Gene Ontology (GO) enrichment results of the genes predicted to be regulated by the 3 types (Table S4). The results showed that the genes potentially regulated by LTR5A, LTR5B, and LTR5Hs were involved in almost completely distinct biological processes (BPs) and molecular functions (MFs)  and C. MTo investigate the potential differences in the promoter activity among the 3 HML-2 LTR subtypes, we screened three representative strains  to F. ThTo investigate which region determines the significantly high activity of LTR5Hs, LTR5Hs was divided into 4 sections. The homologous fragments in LTR5B were successively replaced to generate 5Hs1-5B (−792 to −553), 5Hs2-5B (−552 to −264), 5Hs3-5B (−263 to 0), and 5Hs4-5B (1 to 177) constructs . The 4 cNF-κB, TP53, and POU2F1 motifs , TP53-1 , TP53-2 , TP53-1 with TP53-2 (87.11%), and POU2F1  motifs  to investigate how their diversity affected promoter activities using the dual-luciferase reporter system. This region contains multiple TFBSs. We analyzed the differences among the HML-2 LTR5A, LTR5B, and LTR5Hs subtypes and identified the following sites: the TATA box and the 1 motifs . A total) motifs .TP53-1, and TP53-2 binding sites of LTR5Hs3-5B exhibited significantly decreased transcriptional activity of this LTR. Importantly, the combination of the mutations in TP53-1 and TP53-2 decreased the transcriptional activity more significantly than either mutation alone . In comparison, the mutations in either NF-κB or POU2F1 sites did not significantly change the promoter activity compared with that of wild-type LTR5Hs3-5B in HEK293T cells .To further study which TFBSs affect transcriptional activity, we mutated the above-mentioned TFBSs in LTR5Hs3-5B to the match corresponding loci of LTR5B. The constructs with mutations in the TATA box, on alone  and D. ITP53 TFBSs in the promoter activity of LTR5Hs, we silenced the expression of the TP53 with specific small interfering RNAs (siRNAs). Two siRNAs, siRNA1 and siRNA2, were designed to target the TP53. According to the results of Western blot analysis of p53 protein (TP53 RNA expression levels (TP53 by siRNA reduced LTR5Hs activity as determined by luciferase reporter assays. We observed that the promoter activity significantly decreased after silencing of TP53 in both HEK293T cells (3.28-fold for LTR5Hs3-5B and 1.86-fold for LTR5Hs) and HeLa cells (2.5-fold for LTR5Hs3-5B and 2.34-fold for LTR5Hs) (To elucidate the roles of  protein  and quann levels  and C in LTR5Hs)  and G. E LTR5Hs)  and F. TTP53 probe encompassed both putative p53 binding regions   followed by qPCR (ChIP-qPCR). The negative-control (NC) probe  was located 5 kb upstream from the transcription start site (TSS) of LTR5Hs, and the 867,144) . In addi867,144)  and C, shIP follo867,144)  and E.TP53-1 and TP53-2 binding sites. Notably, there were no peaks in the NC (19q13.13), LTR5A (11p15.4), or LTR5B (12p11.1), as shown in To further confirm the binding regions between LTR5Hs and the p53 protein, we performed a CUT&Tag (cleavage under targets and tagmentation) assay. The p53 CUT&Tag profiling tracks of LTR5Hs are displayed over chromosomes, including 10q21.3, 12q13.13, and 19q13.13  to H. ThWe further verified the other two loci, 7q22.1  and 11q13.2 , using the same types of experiments. The results of ChIP-qPCR of the chromatin pulled down by p53 antibodies in HEK293T and HeLa cells are shown in Fig. S3A and B. The Cistrome Data Browser data on p53 enrichment at 7q22.1 and 11q13.2 are shown in Fig. S3C and D. The red boxes represent the p53 binding region. The ChIP-seq results on database showed that the p53 protein was enriched in 7q22.1 in H9 and MCF-7 cells and enriched in 11q13.2 in MCF-7 cells. The p53 CUT&Tag profiling tracks of LTR5Hs are displayed over chromosomes, including 7q22.1 and 11q13.2, as shown in Fig. S3E and F. The superposition of p53 mapping at the representative sites revealed the binding region between the p53 protein and LTR5Hs.TP53-1 and TP53-2 binding sites in nucleotides −263 to 0. Among these 169 sequences, 72 of these (42.60%) were identified as being enriched with the two p53 binding sites.Additionally, based on the CUT&Tag profiling track results, we did not find a p53 binding site in the homologous fragment (−263 to 0) or p53 enrichment in the 43 LTR5A and 62 LTR5B sequences, which was consistent with the BLAT results in Human Genome Assembly 38 (hg38). Among the 194 LTR5Hs sequences, 169 of these (87.11%) had HERV LTRs are often categorized as transposable elements and harbor principal core promoter elements as well as important transcriptional enhancers. Indeed, there are many examples of HERV-K (HML-2) LTRs that feature novel gene promoters or TFBSs , 27.GGAAGGGAAA and GATAGGAAA) that overlap active and intact NF-κB and NFAT-1 binding sites. The nuclear translocation of IRF1 and NF-κB results in upregulation of HERV-K in ALS (HERV-K (HML-2) LTR5Hs contains two conserved interferon-stimulation response elements , a ubiquitous transcription factor that binds to the 5′ termini of the U3 regions of HERV-K (HML-2) LTR5A, LTR5B, and LTR5Hs, has been shown to activate HERV-K expression in many cell lines, including HepG2, HeLa, GH, and Tera2 cells. Mutation of this YY1 binding site causes a 50% reduction in the activity of the HERV-K LTR (RV-K LTR .MITF-M) is an oncogene in melanoma. MITF-M  activates the responsive sequences (E boxes) in the partial sequences of HERV-K (HML-2) LTR5A, LTR5B, and LTR5Hs, leading to high-level expression in malignant melanoma  suppresses the proliferation and migration of various cancer cells by specifically controlling cancer-related splicing. Its reduced expression is related to poor overall survival in lung, breast, ovarian, and gastric cancers (RBM4 has been identified to be negatively correlated with the expression of HERV-K (HML-2) and to bind HML-2 LTR5A, LTR5B, and LTR5Hs at CGG consensus elements. RBM4 knockout leads to HML-2 transcript upregulation and increases Env protein expression in the chronic myelogenous leukemia (CML) cell line HAP1 (The splicing factor RNA-binding motif 4 ( cancers . RBM4 haine HAP1 .OCT4 motif (ATGCAAA/ATGCCAA) at base pairs 693 to 699 of LTR5Hs is conserved across diverse LTR5Hs sequences but is not present in LTR5A or LTR5B. This binding site on HML-2 LTR5Hs can promote HERV-K transcription in HEK293T cells  of LTR5Hs significantly increased LTR5B promoter activity. The results suggested that the fragment from −263 to 0 containing the enhancer/core promoter sequence determines the difference in LTR activity of LTR5Hs.NF-κB, TP53-1, TP53-2, and POU2F1 binding sites. As these elements were replaced by homologous fragments of LTR5B, the luciferase reporter assay results showed that the TATA box, TP53-1, and TP53-2 binding sites specifically influenced the promoter activity of the third section (LTR5Hs3-5B) of LTR5Hs. TATA-binding protein (TBP) is a transcription factor that specifically binds to the TATA box. For this TFBS, the TATAA variant TGTAA may affect the binding efficiency of the protein. This situation is similar to that in human immunodeficiency virus (HIV): the subtype CRF01_AE contains a different TBP binding site (with the sequence TAAAA), whereas all other subtypes have the TATAA sequence. This single-nucleotide variation reduces the assembly efficiency of the TBP-TFIIB-TATA complex for the recruitment of RNA polymerase II , we did not conduct in-depth research on its mechanism.We focused further on the third section and screened 5 different TFBSs: the TATA box, erase II . Considep53, an important tumor suppressor protein, is a specific transcription factor that regulates the expression of genes related to cell cycle arrest, organic processes, and apoptosis in response to cellular stress and is critical for the control of homeostasis –40. Prev–TP53. Furthermore, the ChIP-qPCR assay and CUT&Tag assay proved the direct binding of the p53 protein with LTR5Hs sequences. Interestingly, CUT&Tag showed that there was no direct binding of p53 with LTR5A (11p15.4) or LTR5B (12p11.1) sequences, making up for the shortcomings of ChIP-qPCR. One advantage of CUT&Tag is that the entire reaction from antibody binding to adaptor integration occurs within intact cells. Antibodies are easier to detect than with other approaches, which makes the CUT&Tag method more sensitive  were identified to be enriched with the two p53 binding sites.In the current work, we focused on the 3 LTR subtypes of HERV-K (HML-2), which is the most biologically active HERV subgroup, and directly revealed that the two unique p53 binding sites located in the −263 to 0 region of the LTR5Hs subtype pose crucial roles to promote LTR5Hs transcriptional activity. The activity of LTR5Hs was decreased by silencing of ensitive , 43. We TP53 mutation has profound effects on tumor cell genomic structure, gene expression, and clinical outlook. Mutations in TP53 most commonly occur as single-nucleotide variants at positions that cluster in the DNA-binding domain (TP53 in many tumor types (TP53 hot spot mutation was located in the locus of our p53 binding site (GCTGCCC). Further analysis of The Cancer Genome Atlas (TCGA) database revealed that there were 5 single-nucleotide polymorphism (SNP) sites located in p53 binding sites, including rs587783062, rs587781504, rs28934578, rs786202962, and rs147002414. The cancer types and SNP loci and proportions are shown in Table S5.g domain , 45. Rs2or types . This TPTaken together, our findings characterize the promoter activity differences among LTR5A, LTR5B, and LTR5Hs and reveal the critical role of the two p53 binding sites in LTR5Hs. p53 has a critical tumor suppressor function. Therefore, our findings suggest that the most integrated and most active LTR of HERV-K (HML-2), LTR5Hs, probably participates in the tumor suppressor function of p53. Notably, more than 30% of all p53 binding sites in the whole human genome are located in HERV LTRs . Thus, ohttps://www.dfam.org/home), including LTR5A_DF0000556.4, LTR5B_DF0000557.4 and LTR5Hs_DF0000558.4 , DF0000556.4_LTR5A, DF0000557.4_LTR5B, and DF0000558.4_LTR5Hs, were used as representatives (https://asia.ensembl.org/index.html) was performed on hg38 using the Dfam-derived HML-2 LTR5A, LTR5B, and LTR5Hs references , to create custom Venn diagrams for the potential genes regulated by the three subtypes.To predict the regulatory roles of LTR5A, LTR5B, and LTR5Hs in the human genome, the nearby genes were annotated with the tool GREAT . The asshttp://david.ncifcrf.gov; version 6.8) is an online biological information database that integrates biological data and analysis tools and provides a comprehensive set of functional annotation information for genes and proteins, from which users can extract biological information . HEK293T, HeLa, A549, and HepG2 cells were grown in Dulbecco’s modified Eagle’s medium ; Beas-2B cells were grown in minimum essential medium ; and L02, MT-2, and Jurkat T cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) and 1% penicillin-streptomycin (Thermo Fisher Scientific). All cell lines were maintained at 37°C in a humidified incubator containing 5% COThe sequences and reverse sequences of HML-2 LTR5A , HML-2 LTR5B , and HML-2 LTR5Hs  were cloned into the KpnI and NheI restriction sites of pGL4.17  to perform luciferase reporter assays. The pRenilla-luc-TK luciferase plasmid pGL4.74 was obtained from Promega. LTR5Hs was divided into four sections, which were used to replace the corresponding positions of LTR5B with a seamless cloning kit . All primer sequences are given in the 5′-to-3′ direction. The primers used to construct the four sections of LTR5Hs to generate the LTR5B luciferase reporters  are shown in Table S7.NF-κB, TP53-1, TP53-2, and POU2F1 binding sites in LTR5Hs3-5B-Luc vector were mutated to the corresponding sequences of LTR5B with a Q5 site-directed mutagenesis kit . The TATA box was mutated from 5′-TATAA-3′ to 5′-TGTAA-3′, the NF-κB binding site was mutated from 5′-AGGGAAAAACCG-3′ to 5′-AGGGGAGAATGG-3′, the TP53-1 binding site was mutated from 5′-GGGCTGG-3′ to 5′-TGGCTGG-3′, the TP53-2 binding site was mutated from 5′-GGGCAGC-3′ to 5′-TGGCAGC-3′, and the POU2F1 binding site was mutated from 5′-TGTATGCATAT-3′ to 5′-TAGAGTCAAACATAAATCTGGCCTATGTGC-3′. The primers used to create the mutant constructs are shown in Table S7.The TATA box, Renilla luciferase as an internal control (pRenilla-luc-TK) to assess for variation in transfection efficiency. The amounts of plasmids used for transfections in 96-well cell culture plates were as follows: 200 ng of luciferase reporter plasmid (HERV-K [HML-2] LTR) or empty luciferase vector pGL4.17, with 5 ng pRenilla-luc-TK. At 6 h posttransfection, the medium containing a mixture of plasmids and transfection reagent was replaced with fresh medium supplemented with 10% FBS. At 24 h posttransfection, the cells were treated with TNF-α (50 ng/mL) or PMA (20 nM)/ionomycin (1.5 μM). At 48 h posttransfection, the transfected cells were assayed for luciferase activity using a dual-luciferase assay kit (Promega) according to the manufacturer’s instructions.HEK293T, HeLa, Beas-2B, L02, A549, and HepG2 cells were seeded in 96-well cell culture plates for 24 h before transfection. Transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. All transfections included Renilla luminescence] experimental luciferase units in a specific stimulus)/. The experiment under each condition was performed with at least three independent biological replicates.To display the difference of promoter activity between different constructs in the same stimulation, as well as the difference of promoter activity of the same construct in different stimulation (cell culture medium and PMA-ionomycin or TNF-α), the normalized fold change were calculated with the following formula:  by using Lipofectamine 2000 (Invitrogen). The NC siRNA was a random sequence (50 nM). At 24 h after transfection, the treated cells were harvested for Western blot analysis and real-time RT-PCR. The siRNA sequences are shown in Table S7.A260/A280 value of 1.95 to 2.05. A PrimeScript RT reagent kit with gDNA Eraser (TaKaRa) was used to synthesize cDNA from the extracted RNA according to the manufacturer’s instructions.Total cellular RNA was isolated from cells using a MiniBEST universal RNA extraction kit (TaKaRa). The RNA concentration was measured with a NanoDrop spectrophotometer. The acceptable RNA purity was an TP53 RNA expression levels after silencing with siRNA. The primers used are shown in Table S7. qPCR was performed on technical duplicates or triplicates of each sample. Each condition was performed with three independent biological replicates.qPCR was performed using the SYBR green detection method  in a LightCycler 480  to measure g for 15 min at 4°C. The protein concentrations were measured by the bicinchoninic acid (BCA) (TaKaRa) method to determine the protein content of each sample. The cell lysates were prepared for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), heated at 95°C for 10 min, and then transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in a 5% skim milk solution for 1 h and then probed with primary antibodies against p53  and GAPDH  overnight at 4°C. After washing with PBS with Tween 20 (PBST) for 1 h, the membranes were incubated with secondary antibodies . Then, the membranes were imaged using chemiluminescent horseradish peroxidase (HRP) substrate  after washing with PBST for 1 h.Cells were washed with ice-cold phosphate-buffered saline (PBS) twice and then incubated with lysis buffer on ice for 20 min. The cell lysates were centrifuged at 12,000 × The primary antibodies and HRP-conjugated secondary antibodies used were as follows: anti-p53  and anti-GAPDH  primary antibodies and goat anti-mouse or anti-rabbit IgG secondary antibodies .6 cells were harvested for ChIP. Briefly, the cells were cross-linked with 1% formaldehyde for 10 min at room temperature. Glycine was added to stop the cross-linking reaction, and the cells were washed with ice-cold PBS. Following solution removal, the tubes were chilled on ice, and the cells were lysed using ice-cold cell lysis buffer containing protease inhibitors and phenylmethylsulfonyl fluoride (PMSF). The chromatin was fragmented to 200 to 500 bp with a Bioruptor Plus  at 4°C. After centrifugation, the chromatin supernatants were diluted with cold dilution buffer. Subsequently, a human p53 antibody  was added to the chromatin, and the mixture was incubated at 4°C overnight. The antibody-chromatin samples were pelleted and incubated with protein A beads. After incubation, the antibody-chromatin/beads were washed 4 times, and DNA was purified with the DNA-purifying slurry included in the ChIP kit. Finally, the purified DNA was used for qPCR analysis using the SYBR green detection method. The ChIP-qPCR primers used are shown in Table S7.ChIP was performed with a ChIP kit  according to the manufacturer’s instructions. Approximately 3 × 10in situ ChIP library preparation kit for Illumina . In brief, the cells were incubated with 10 μL of prewashed ConA beads in a low-binding tube. Then, we added 50 μL of antibody buffer with 0.5 μg of antibody and cultured the cells for 2 h at room temperature. After washing twice, 50 μL of digitonin (Dig) wash buffer with 0.5 μg of secondary antibody was added, and the samples were incubated at room temperature for 30 min. After washing twice, we added 0.58 μL of Hyperactive pG-Tn5/pA-Tn5 transposon with 100 μL of Dig-wash buffer and incubated the samples at room temperature for 1 h. After washing twice, 300 μL of tagmentation buffer was added, and the samples were incubated at 37°C for 1 h. After DNA extraction with the phenol-chloroform and ethanol precipitation method, PCR was performed to amplify the libraries. All libraries were sequenced with an Illumina NovaSeq 6000 instrument. The CUT&Tag data were visualized with Integrative Genomics Viewer (IGV) software (CUT&Tag was performed with a Hyperactive software .


Meloidogyne incognita or Rotylenchulus reniformis. Nematode toxicity and recovery experiments were conducted in water solutions of tioxazafen, while root infection assays were conducted on tomato. Nematode paralysis was observed after 24 hr of exposure at 27.0 µg/ml tioxazafen for both the nematode species. Based on an assay of nematode motility, 24-hr EC50 values of 57.69 µg/ml and 59.64 µg/ml tioxazafen were calculated for M. incognita and R. reniformis, respectively. Tioxazafen rates of 2.7 µg/ml and 27.0 µg/ml reduced the nematode hatch after 3 d of exposure for both the nematode species. There was no recovery in nematode motility after the 24-hr exposure of M. incognita and R. reniformis to their corresponding 48-hr EC50 values of 47.15 µg/ml and 47.25 µg/ml tioxazafen, respectively. Mortality of M. incognita continued to increase after 24 hr exposure, whereas R. reniformis mortality remain unchanged after nematodes were rinsed and removed for 48 hr from the tioxazafen solution. A 24-hr exposure to low concentrations of 0.38 to 47.15 µg/ml for M. incognita and 47.25 µg/ml for R. reniformis reduced the infectivity of each nematode species on tomato roots. The toxicity of tioxazafen was similar between nematode species; however, a greater rate of tioxazafen was needed to suppress R. reniformis infection of tomato than for M. incognita.Tioxazafen is a seed-applied nematicide used in row crops. Currently, there are no data on nematode toxicity, nematode recovery, or effects of low concentrations of tioxazafen on nematode infection of a host root for  Meloidogyne incognita, and the reniform nematode, Rotylenchulus reniformis, are two of the most important plant-parasitic nematodes that affect row crops in the United States (M. incognita ranked first in lint yield loss in cotton (Gossypium hirsutum) and ranked second in grain yield losses in soybean (Glycine max) in 2019 , soybean, and cotton. The mode of action of tioxazafen involves a disruption of ribosomal activity in nematodes  by Bayer CropScience  for use in corn  determine if the effects of tioxazafen on each nematode species are reversible, and (iii) determine the effect of low concentrations of tioxazafen on the infectivity of each nematode species.The objectives of this study were to: (i) characterize the toxicity of tioxazafen to M. incognita and R. reniformis were isolated originally from cotton and maintained on tomato (Solanum lycopersicum ‘Rutgers’) and cotton (‘DP 1646 B2XF’), respectively. Eggs of M. incognita and R. reniformis were collected from roots with 0.5% NaOCl and only freshly collected eggs were used  was used in this study. These experiments were performed in 24-well Falcon tissue culture plates ; each well received 500 µl of a ×2 concentration of the test solution, which contained 30 to 40 nematodes in 500 µl of distilled water. Incubation temperatures of 28°C for M. incognita and R. reniformis were maintained throughout these experiments. Motile and immotile nematodes were determined visually during each sample time with an inverted compound microscope . Each treatment was replicated four times in three experiments for each nematode species, and the percentage of immotile nematodes was calculated for each species to determine the effective concentration response.M. incognita and R. reniformis eggs were treated for 3 d with water solutions of tioxazafen at the same rates and with the same materials used in the motility assay. The number of J2 that hatched from eggs was recorded daily and the percent hatch per day was determined. Each treatment was replicated four times, and the experiment was conducted twice for each nematode species.Freshly collected M. incognita or R. reniformis were treated for 24 hr with water solutions respective to their 48-hr EC50 values of tioxazafen, poured over a 25-μm-pore sieve, and rinsed twice with distilled water. Rinsed nematodes were transferred to a 24-well tissue culture plate containing distilled water. Nematodes treated with distilled water served as the negative control and nematodes treated for 48 hr to their corresponding 48-hr EC50 values of tioxazafen served as the positive control. Motile vs immotile nematodes were determined visually at 24 hr and 48 hr after rinse using an inverted compound microscope. Each treatment was replicated four times in the three experiments and the percentage of immotile nematodes was determined for each species.A large population  of M. incognita and R. reniformis on tomato roots was evaluated in a greenhouse experiment. A large population  of each nematode species was treated for 24 hr with water solutions of tioxazafen corresponding to their 48-hr EC50 (1:5 dilutions). Nematodes were inoculated onto the 2-wk-old tomato seedlings growing in sand in an 84-cm3 celled seedling tray. Each seedling received 2 ml of the tioxazafen solution containing 500 nematodes. The inoculum was distributed through three holes around the seedlings created by pushing a 1-ml pipette tip 3 cm into the root zone. Nematodes treated with distilled water served as the positive control. The experimental design was a randomized complete block design, each treatment was replicated six times, and the experiment was conducted twice per nematode species. Tomato plants were incubated at 28°C to 30°C on a greenhouse bench and sampled 3 wk after inoculation to determine infectivity. Galls per root system was used to determine infectivity by M. incognita. Females of R. reniformis were stained with acid fuchsin (The impact of tioxazafen on the infectivity of P > 0.05) and combined for final analysis. For these preliminary analyses, experiment repetitions were modeled as a random variable. Data from the nematode motility experiments were subjected to probit analysis using SPSS 27.0  to determine the effective concentration response . Data from hatch rate experiments were analyzed using a factorial ANOVA in the general linear mixed (GLM) model procedure with treatment replications modeled as a random variable, and nematicide rates and sample time were modeled as fixed factors using SPSS 27.0. Data from infectivity and recovery experiments were subject to ANOVA in the general linear model procedure. Data from hatch, recovery, and infectivity assays were log (x + 1) transformed when appropriate to normalize for analysis, and non-transformed data are reported. Means were separated according to Fisher’s least significant difference (LSD) procedure (α = 0.05).Data from the repeated experiments were similar  for both nematode species. A greater (P = 0.05) hatch suppression was observed at 2.7 µg/ml and 27.0 µg/ml after 3 d of continuous exposure compared to the water control for both the nematode species. Hatch was not affected by tioxazafen at concentrations <2.7 µg/ml for either nematode species.Tioxazafen was somewhat effective in suppressing tch rate . There wM. incognita and R. reniformis when removed from tioxazafen after a 24 hr treatment to their corresponding 48-hr EC50 concentration (P = 0.05) in the percent of M. incognita that were immotile 48 hr after being removed from the tioxazafen compared to the positive control, 48 hr of continuous exposure. A similar trend was observed with R. reniformis. Nematode posture for both species remained rigid when removed from the tioxazafen.No recovery in nematode motility was observed for ntration . There wM. incognita but less effective at reducing R. reniformis infection of tomato roots. Rates of tioxazafen from 0.38 µg/ml to 47.15 µg/ml reduced (P = 0.05) root galling by M. incognita compared to the water control (P = 0.05) the number of R. reniformis females observed per root system compared to the water control (Low concentrations of tioxazafen were effective at reducing  control . Only 47 control .M. incognita and R. reniformis, which was affected by the concentration and duration of exposure to the nematicide. The 24-hr EC50 value was 47.15 µg/ml tioxazafen for M. incognita, which is less toxic than other chemical nematicides used in cotton and soybean. For example, the 24-hr EC50 value for M. incognita was 1.2 µg/ml fluopyram and 0.42 µg/ml abamectin (50 value was 47.25 µg/ml for R. reniformis, which is less toxic than that reported for fluopyram, which is 1.97 µg/ml and for abamectin, which is 3.49 µg/ml (M. incognita and R. reniformis is lower than that of fluopyram and abamectin, which are other seed-applied nematicides used for cotton and soybean.Tioxazafen had a negative effect on the motility of bamectin . Similar49 µg/ml . These fM. incognita and R. reniformis was suppressed after 3 d of exposure at concentrations of 2.7 µg/ml and 27.0 µg/ml tioxazafen. Fluopyram was reported to suppress the hatch of M. incognita but not Caenorhabditis elegans at 25 µg/ml (Meloidogyne arenaria at 10 µg/ml (M. incognita (R. reniformis.Second-stage juvenile hatch for 25 µg/ml . AbamectM. incognita and R. reniformis. Abamectin was reported to have irreversible effects on nematode paralysis, whereas fluopyram effects were reversible (M. incognita infection and yield protection in cotton and soybean (Nematode paralysis was irreversible 48 hr after removal from the tioxazafen solution for both versible . The lab soybean .M. incognita than that by R. reniformis. Sublethal concentrations of abamectin and fluopyram inhibited tomato root infection by M. incognita and R. reniformis (M. incognita and R. reniformis on tomato, and this indicates that suppression of nematode infection may differ among plant-parasitic nematodes in response to tioxazafen.Low concentrations of tioxazafen were more effective at inhibiting infection of tomato roots by niformis . There wM. incognita and R. reniformis are irreversible. Since the 2020 cropping season, tioxazafen was voluntarily removed from commercial use by the manufacturer. Thus, these data serve as a reference for the toxicity of tioxazafen to two important yield-limiting plant-parasitic nematodes if it returns as a commercially available nematicide.The toxicity of tioxazafen is less than those of abamectin and fluopyram in vitro, and like abamectin, its effects on


Diabetic kidney disease (DKD) is the major cause of end stage renal disease in patients with type 2 diabetes mellitus (T2DM). The subtle metabolic changes in plasma and cerebrospinal fluid (CSF) might precede the development of DKD by years. In this longitudinal study, CSF and plasma samples were collected from 28 patients with T2DM and 25 controls, during spinal anesthesia for elective surgery in 2017. These samples were analyzed using liquid chromatography-mass spectrometry (LC-MS) in 2017, and the results were correlated with current DKD in 2017, and the development of new-onset DKD, in 2021. Comparing patients with T2DM having new-onset DKD with those without DKD, revealed significantly increased CSF tryptophan and plasma uric acid levels, whereas phosphatidylcholine 36:4 was lower. The altered metabolites in the current DKD cases were uric acid and paraxanthine in the CSF and uric acid, L-acetylcarnitine, bilirubin, and phosphatidylethanolamine 38:4 in the plasma. These metabolic alterations suggest the defective mitochondrial fatty acid oxidation and purine and phospholipid metabolism in patients with DKD. A correlation analysis found CSF uric acid had an independent positive association with the urine albumin-to-creatinine ratio. In conclusion, these identified CSF and plasma biomarkers of DKD in diabetic patients, might be valuable for monitoring the DKD progression. Type 2 diabetes mellitus (T2DM) is characterized by chronic hyperglycemia, due to insulin resistance. T2DM is a pandemic that has affected more than 10.5% of the adult population (536.6 million) worldwide, in 2021, according to the International Diabetes Foundation’s Diabetes Atlas estimation . The cliThe kidneys are metabolically active during the modulation of the circulating metabolites and waste removal via filtration, reabsorption, and secretion. Chronic hyperglycemia and inflammation, shift the oxidative balance toward a pro-oxidative state, accelerating kidney damage and causing the DKD progression from the glomerular hyperfiltration to the declining glomerular filtration, the progressive albuminuria, and ultimately ESRD ,7. HenceCerebrospinal fluid (CSF) exchanges systemic metabolites with the central nervous system (CNS), via a limited transcellular transport constituted by the specialized tight junction of the blood–brain barrier (BBB). The impaired BBB integrity in patients with T2DM has been recognized in MRI studies of human cohorts, which is ascribed to the tight junction disruption and enhanced paracellular permeability, due to chronic hyperglycemia . Thus, tIn this study, we explored the metabolomic signatures of current DKD and new-onset DKD, in the CSF and plasma samples of patients with T2DM, using LC-MS metabolomics. The study protocol was registered in the ClinicalTrials.gov database (Identifier: NCT03725709) and the study was approved by the Ethical Review Board of Chang Gung Medical Foundation, Taiwan . The study protocol was clearly explained to each participant and informed consent was obtained before enrollment.2 within 3 months before the sampling [This longitudinal follow-up study included 60 participants aged 20–70 years. The sample collection was performed between 1 January 2017 and 31 July 2017, while the participants underwent spinal anesthesia for elective surgeries, at Linkou Chang Gung Memorial Hospital, Taiwan. Thirty participants had a history of T2DM, while the other 30 control participants did not. The patients with a history of T2DM have a fasting glucose level of >126 mg/dL  and were taking oral hypoglycemic agents. The other 30 participants had a fasting blood glucose <126 mg/dL and formed the control group . The CSFsampling . These eThe sample collection procedures were largely the same as those used in our previous NMR metabolomic study of patients with T2DM . In thisThe biochemical parameter analyzed in the 500 μL of plasma and CSF samples, included plasma glucose, plasma glycated hemoglobin A1c (HbA1c), plasma insulin, CSF glucose, and CSF insulin levels. The plasma and CSF glucose levels were determined using the glucose oxidase assay provided by Cell Biolabs, San Diego, CA, USA. Insulin and HbA1c levels were quantified using enzyme-linked immunosorbent assay (ELISA) kits from Mercodia  and Cloud-Clone Corporation , respectively. The degree of insulin resistance in patients with T2DM was compared using the homeostasis model assessment of the insulin resistance index (HOMA-IR), calculated as fasting glucose (mg/dL) × fasting insulin (mU/L) divided by 405 .g for 30 min, the supernatant was transferred. The pellet was resuspended in 400 μL of 50% methanol with 0.1% formic acid. Following the centrifugation at 12,000× g for 30 min, the two supernatants were mixed and dried under nitrogen gas. The residue was suspended in 100 μL of 50% acetonitrile with 0.1% formic acid for LC-MS analysis.The CSF samples (50 μL) were mixed with cooled acetonitrile (400 μL) and 0.1% formic acid to precipitate proteins. Following the centrifugation at 12,000× 2; Waters, Milford, MA, USA) using an Acquity TM Ultra Performance Liquid Chromatography (UPLC) system (Waters Corporation). The column was maintained at 45 °C and the flow rate was 0.5 mL/min. The mobile phase consisted of 0.1% formic acid in water (Phase A) and acetonitrile, containing 0.1% formic acid (Phase B). The mass spectrometry was performed using a Waters Q TOF-MS  operated in ESI positive and negative ion modes. The scan range was 20–990 m/z. The desolvation gas flow rate was 700 L/h at 300 °C. The source-cone voltage was set to 35 V. The capillary voltage were 2.7 kV in the positive mode and 2 kV in the negative mode. The lock mass was leucine encephalin (m/z: 120.0813 and 556.2771 for the positive mode and m/z: 236.1035 and 554.2615 for the negative mode). The LC-MS multivariate data was statistically analyzed with the mean centering and the Pareto scaling using soft independent modeling of class analogy software .The liquid chromatographic separation was conducted on an Acquity UPLC BEH C8 column  . The idet-test or chi-squared test and the analysis of variance (ANOVA), for the comparisons among multiple groups. In this study, correlating the CSF and plasma biomarkers with current DKD in 2017, was our expected primary outcome, and correlating the biomarkers with new-onset DKD in 2021, was the expected secondary outcome. First, we identified significant metabolites in the CSF and plasma samples that could discriminate between patients with DKD, without DKD, and the control participants. We then selected significant metabolites in the CSF and plasma samples to construct the metabolite combinations. We selected the target panels with the highest AUC values as the final result. Since diabetic patients had a significantly higher age, BMI, male percentage, and chronic diseases, such as hypertension and hyperlipidemia, compared with that of the control participants, these confounding factors were adjusted in further comparisons. In addition, the correlation of these metabolites with standard renal function measurements, such as UACR and eGFR, was calculated using a regression model to compare their association. All statistical analyses were executed using the SAS software , and a two-sided p value < 0.05 was dictated as statistically significant.These collected data are presented as means ± SD for the continuous variables (such as body weight and the serum creatinine levels) and as a percentage for the qualitative variables (such as sex and chronic diseases). The statistical analyses were based on the orthogonal partial least-squares discriminant analysis (OPLS-DA) coefficients in the LC-MS signals. Comparisons between the two groups were performed using the student’s Our final cohort included 28 patients with T2DM  and 25 control participants. The analysis protocol is illustrated in The OPLS-DA score plots of the LC-MS signal integrations in the CSF and plasma samples from patients with DKD, versus the control participants, are compared in p < 0.05). The plasma samples from patients with current DKD had significantly higher L-acetylcarnitine and uric acid and lower phosphatidylethanolamine (PE) 38:4, phosphatidylcholine (PC) 36:4, and bilirubin levels than in the samples from patients without DKD. As for the new-onset DKD, these patients had significantly higher CSF tryptophan levels than the patients without DKD. The plasma samples of patients with new-onset DKD, showed significantly higher levels of uric acid and lower levels of lysophosphatidylcholine (LysoPC) 18:2, PC 38:6, and PC 36:4.The comparison between the altered metabolites in the CSF samples from the diabetic patients with current or new-onset DKD and the patients without DKD or the control participants is shown in To identify the biomarkers correlating with our DKD outcomes, we constructed metabolite combinations using CSF and plasma metabolites with a significant discrimination between patients with or without DKD. We then compared the AIC values, AUC values, and adjusted the ORs for discriminating the current DKD or new-onset DKD, from the patients without DKD and the control participants, using ANOVA and a multivariate analysis  and kynureine (95%) pathway . DownstrCaffeine, the most extensively consumed psychoactive drug with adenosine antagonist properties, has neuroprotective effects in some neurological disorders, such as Parkinson’s disease (PD) . A largeAcylcarnitine is generated by the fatty acid oxidation inside the mitochondria, and its accumulation is associated with insulin resistance and mitochondrial dysfunction . L-AcetyBilirubin, a powerful endogenous antioxidant with anti-inflammatory properties, can act on cellular pathways to halt the progression of DKD . Owing tAltered lipid levels, including sphingolipids and phosphatidylcholines (PCs), are associated with renal function impairment in diabetic patients . SphingoAltogether, the increased uric acid and lower paraxanthine levels in the fasting CSF samples of DKD patients, might suggest an enhanced oxidative stress and decreased neuroprotective effects of caffeine. The altered uric acid, acetylcarnitine, bilirubin, and PCs in the plasma samples of patients with DKD may imply a mitochondrial dysfunction and phospholipid remodeling.Theoretically, better glycemic and blood pressure control might be able to ameliorate the progression of DKD, but it is still unable to substantially decrease the annual incidence of DKD-related ESRD . Thus, aTo the best of our knowledge, this pilot study is the first to predict the development of DKD by integrating CSF and plasma metabolomic LC-MS signatures of Taiwanese patients with T2DM. Although the invasive nature of the CSF sampling may limit the utilization of CSF biomarkers, these profiled CSF and plasma metabolites could deepen our insight into the DKD pathophysiology. The method of CSF sampling undertaken in this study enabled us to detect the actual metabolite alterations in the cerebral circulation without causing unnecessary discomfort. The diagnosis of DKD in this cohort was solid because it was confirmed by specialists during inpatient or out-patient follow-ups for T2DM.However, this small cohort study had several limitations. First, the number of cases in this study were limited due to insufficient budget and loss to follow-up during the COVID-19 pandemic. Therefore, the interpretation of the current results should be made with caution because of individual variations. However, our novel findings regarding the DKD-correlated CSF and plasma metabolites deserve further validation in larger cohorts. Second, the enrolled participants had significant variations among different groups in sex, BMI, serum creatinine, and chronic diseases (hypertension and hyperlipidemia), which might confound our results, even though these factors were already adjusted. Moreover, the complex and dynamic nature of the metabolome could be affected by many factors, such as socioeconomic circumstances, diet, lifestyle, and medications. Further larger cohorts and longitudinal repeated samplings might be required to verify our findings and determine the causal relationship.In this longitudinal study, we profiled the CSF and plasma metabolomic signature of patients with T2DM complicated with current DKD or developing new-onset DKD, using the LC-MS analysis. The identified potential DKD biomarkers, such as uric acid, acetylcarnitine, PC 36:4, and PE 38:4, had a significant correlation with eGFR and UACR, suggesting their values in the predictions and risk stratification for DKD in patients with T2DM. These metabolic alterations imply a defective mitochondrial fatty acid oxidation, purine metabolism, and phospholipid remodeling during the DKD progression. Further verification of our results in a larger multi-omic cohort is required to confirm the causal relationship.