Development of a short-term prognostic model for anti-N-methyl-D-aspartate receptor encephalitis in Chinese patients.

BACKGROUND: Recognizing the predictors of poor short-term prognosis after first-line immunotherapy in patients with anti-N-methyl-D-aspartate receptor (anti-NMDAR) encephalitis is essential for individualized treatment strategy. The objective of this study was to ascertain the factors that forecast short-term prognosis in patients with anti-NMDAR encephalitis, develop a prognostic prediction model, and authenticate its efficacy in an external validation cohort. Further, all patients were followed-up long-term to assess the factors of long-term outcome and relapses. METHODS: A prospective enrollment of patients diagnosed with anti-NMDAR encephalitis was conducted across five clinical centers in China from June 2014 to Mar 2022. The enrolled patients were divided into the derivation and validation sets based on enrollment time. The short-term prognostic model was visualized using a nomogram. Further, all patients were followed-up long-term to assess the factors of long-term outcome. RESULTS: This study found that poor short-term prognosis was a risk factor for poor long-term outcome (6-month prognosis, OR 29.792, 95%CI 6.507-136.398, p < 0.001; 12-month prognosis, OR 15.756, 95%CI 3.384-73.075, p < 0.001; 24-month prognosis, OR 5.500, 95%CI 1.045-28.955, p = 0.044). Abnormal behavior or cognitive dysfunction (OR 8.57, 95%CI 1.48-49.79, p = 0.017), consciousness impairment (OR19.32, 95%CI 3.03-123.09, p = 0.002), autonomic dysfunction or central hypoventilation (OR 5.66, 95%CI 1.25-25.75, p = 0.025), CSF pleocytosis (OR 4.33, 95%CI 1.48-12.65, p = 0.007), abnormal EEG (OR 5.48, 95% CI 1.09-27.54, p = 0.039) were independent predictors for a poor short-term prognosis after first-line immunotherapy. A nomogram that incorporated those factors showed good discrimination and calibration abilities. The area under the curve (AUC) for the prognostic model were 0.866 (95%CI: 0.798-0.934) with a sensitivity of 0.761 and specificity of 0.869. CONCLUSION: We established and validated a prognostic model that can provide individual prediction of short-term prognosis after first-line immunotherapy for patients with anti-NMDAR encephalitis. This practical prognostic model may help neurologists to predict the short-term prognosis early and potentially assist in adjusting appropriate treatment timely.

A comparison of clinical features between neurobrucellosis and tuberculous meningitis.

BACKGROUD: This study aims to compare the clinical manifestations, imaging findings, routine tests, biochemistry indicators and cerebrospinal fluid cytology between neurobrucellosis and tuberculous meningitis. The objective is to evaluate the similarities and differences of these two diseases and improve early diagnosis. METHODS: A comprehensive evaluation was conducted by comparing clinical data, imaging results, routine tests findings, biochemistry indicators and cerebrospinal fluid cytology of patients admitted to the Department of Neurology, the Second Hospital of Hebei Medical University from 2019 to 2021. Statistical analysis was applied to identify significant differences and similarities between the two diseases. RESULTS: Preliminary analysis demonstrated both diseases commonly present with symptoms such as fever, headache. However, there were no statistical differences between neurobrucellosis and tuberculous meningitis in early clinical data, imaging results, routine tests findings, biochemistry indicators. Further analysis indicates there is a statistically significantly difference in the lymphocyte ratio and neutrophil ratio in the cerebrospinal fluid between the two groups. CONCLUSIONS: Neurobrucellosis and tuberculous meningitis share similarities in early clinical manifestations, imaging findings and initial cerebrospinal fluid parametes, making early-stage differentiation challenging. The ratio of lymphocytes and neutrophil in the cerebrospinal fluid and a detailed medical history investigation can provide clues for early clinical diagnosis. So the examination of CSF cytology might be a potential to distinguish these two diseases and become a powerful tool in the future.

Interfacial Micro-Environment of Electrocatalysis and Its Applications for Organic Electro-Oxidation Reaction.

Conventional designing principal of electrocatalyst is focused on the electronic structure tuning, on which effectively promotes the electrocatalysis. However, as a typical kind of electrode-electrolyte interface reaction, the electrocatalysis performance is also closely dependent on the electrocatalyst interfacial micro-environment (IME), including pH, reactant concentration, electric field, surface geometry structure, hydrophilicity/hydrophobicity, etc. Recently, organic electro-oxidation reaction (OEOR), which simultaneously reduces the anodic polarization potential and produces value-added chemicals, has emerged as a competitive alternative to oxygen evolution reaction, and the role IME played in OEOR is receiving great interest. Thus, this article provides a timely review on IME and its applications toward OEOR. In this review, the IME for conventional gas-involving reactions, as a contrast, is first presented, and then the recent progresses of IME toward diverse typical OEOR are summarized; especially, some representative works are thoroughly discussed. Additionally, cutting-edge analytical methods and characterization techniques are introduced to comprehensively understand the role IME played in OEOR. In the last section, perspectives and challenges of IME regulation for OEOR are shared.

Cerebrospinal fluid exosomal protein alterations via proteomic analysis of NSCLC with leptomeningeal carcinomatosis.

PURPOSE: Leptomeningeal carcinomatosis (LC) is a rare complication of non-small cell lung cancer (NSCLC) with highly mortality. Cerebrospinal fluid (CSF) as a special kind of tumor microenvironment (TME) better represents alterations than plasma. However, the clinical value of protein profiles of exosome in CSF as liquid biopsy remains unclear. METHODS: In this study, CSF samples of NSCLC patients with (LC group) or without (NSCLC group) LC were collected and compared to patients without tumors (normal group). CSF exosomes were isolated by ultracentrifugation and protein profiles were performed by label-free proteomics. Differentially expressed proteins (DEPs) were detected by bioinformatics tools and verified by parallel reaction monitoring (PRM). RESULTS: A total of 814 proteins were detected. Bioinformatics analysis revealed their shared function in the complement activation, extracellular region, and complement and coagulation cascades. Between LC and NSCLC group, 72 DEPs were found among which FN1 demonstrated the highest betweenness centrality (BC) after protein-protein interaction network analysis. CONCLUSION: We investigated the application of label free and PRM based proteomics to detect key proteins related to LC. FN1 may serve as potential indicator to classify LC and NSCLC. Extracellular matrix (ECM) and epithelial-mesenchymal transition (EMT) are important in the process of LC. These data is promising for early prediction and diagnosis of LC.

Adult spinal cord diffuse midline glioma, H3 K27-altered mimics symptoms of central nervous system infection: a case report.

Diffuse midline gliomas, H3 K27-altered are infiltrative growth gliomas with histone H3K27M mutations. This glioma is more common in the pediatric population, and the prognosis is usually poor. We report a case of diffuse midline gliomas, H3 K27-altered in an adult patient that mimicked symptoms of central nervous system infection. The patient was admitted due to double vision for 2 months and paroxysmal unconsciousness for 6 days. Initially, lumbar puncture showed persistent high intracranial pressure, high protein, and low chlorine. Magnetic resonance imaging showed diffuse thickening and enhancement of meninges and spinal meninges, and later, fever occurred. The initial diagnosis was meningitis. We suspected central nervous system infection, so we started anti-infection treatment, but the treatment was ineffective. The patient's condition gradually worsened, with lower limb weakness and even the consciousness became unclear. A repeat magnetic resonance imaging and positron emission tomography-computed tomography scan showed space-occupying lesions in the spinal cord, which was considered a tumor. Following neurosurgery, pathological tests identified the tumor as diffuse midline gliomas, H3 K27-altered. The patient was recommended for radiotherapy and temozolomide chemotherapy. The patient's condition improved after chemotherapy treatment, and he survived for an additional 6 months. Our case shows that diagnosing diffuse midline gliomas, H3 K27-altered in the central nervous system is complex and can be confused with the clinical characteristics of central nervous system infection. Therefore, clinicians should pay attention to such diseases to avoid misdiagnosis.

Diagnosis, treatment and genetic analysis of a case of familial aldosteronism type II with WFS1 gene mutation.

Primary aldosteronism (PA) is a disease characterized by hypertension and hypokalemia due to the excessive aldosterone secretion from the adrenal cortex, which leads to the retention of both water and sodium, and the inhibition of the renin-angiotensin system as well. Familial hyperaldosteronism type II (FH-II) is known as an autosomal dominant hereditary disease, which is a scarce cause of PA. In this report, we cllected the clinical data of a patient with repeated hypertension and hypokalemia of uncertain diagnosis since 2014. Nevertheless, we discovered by genetic sequencing in 2021 that the CLCN2 and WFS1 gene mutation of the patient, whose mother belongs to heterozygote genotype and father belongs to wild-type genotype. Combined with a series of endocrine function tests and imaging studies, the patient was finally certified her suffering from FH-II and WFS1 gene mutation. By summarizing and analyzing the characteristics and genetic test results of this case, we recommended gene sequencing for patients with PA whose etiology is difficult to be determined clinically. This case also provides new clinical data for subsequent genetic studies of the disease.

Neurosyphilis with a rare magnetic resonance imaging pattern confirmed by metagenomic next-generation sequencing: a case report.

BACKGROUND: Neurosyphilis refers to infection of the central nervous system by Treponema pallidum. The clinical presentation is variable and nonspecific. Neuroimaging findings are complex and that the diagnosis is based on clinical presentation, cerebrospinal fluid (CSF) parameters, and serologic and CSF evidence of syphilis. To date, there is no case report describing Treponema pallidum detected by metagenomic next-generation sequencing (mNGS) in CSF. CASE PRESENTATION: In this report, we describe a case of neurosyphilis in a HIV-negative, 29-year-old man, who was admitted to our hospital with an epileptic seizure and progressive cognitive impairment. Brain magnetic resonance imaging (MRI) revealed fluid-attenuated inversion recovery (FLAIR) high signal intensities in bilateral medial and anterior temporal lobes, insula, right pulvinar of the thalami, precuneus, frontal and temporo-occipital lobes. Laboratory examination showed positive results by means of nontreponemal or specific treponemal test in serum and CSF. mNGS of the CSF was also performed to identify Treponema pallidum for the first time. CONCLUSIONS: This case underscores the importance of considering neurosyphilis as a potential cause of mesiotemporal abnormality. In addition, the rapid improvement and wide usability of mNGS technology will bring new breakthroughs in the clinical diagnosis of neurosyphilis.

Clinical efficacy and safety of different doses of intrathecal methotrexate in the treatment of leptomeningeal carcinomatosis: a prospective and single-arm study.

OBJECTIVE: To investigate the clinical efficacy and safety of different doses of intrathecal methotrexate in the treatment of leptomeningeal carcinomatosis. METHODS: 53 patients admitted to the Second Hospital of Hebei Medical University with leptomeningeal carcinomatosis were recruited. They were divided into two groups: 15-mg-group received 15 mg methotrexate intrathecally, while the other received 10 mg methotrexate. All patients were followed up to 31 December 2020 or until death. Primary endpoint was the response rate. Secondary endpoints were survival and safety. Treatment-related adverse events were recorded. RESULTS: The intrathecal chemotherapy was regularly maintained in 42 cases. Most primary cancers were lung (60.4%), stomach (18.9%) or breast (5.7%). The clinical response rate was higher in the 15 mg group than the 10 mg group (62.5 vs. 34.5%, P = 0.042). In the 15 mg group, two cases showed myelosuppression and one case showed seizures. In the 10 mg group, one patient appeared fever, three patients appeared myelosuppression and one showed leukoencephalopathy. However, there were no serious irreversible adverse reactions in neither of the two groups. In terms of survival, the median survival was 15.7 weeks in the 15 mg group and 27.1 weeks in the 10 mg group (P = 0.116). Multivariate analysis showed that only targeted therapy improved the survival (P < 0.0001, HR = 5.386). CONCLUSION: Increased dose of methotrexate did not prolong the overall survival, but it was more effective in relieving clinical symptoms with no increased adverse reactions. Targeted therapy might improve the survival.

A Diagnostic Scoring System for Distinguishing between Tuberculous and Bacterial Meningitis Based on Clinical and Laboratory Findings.

It is very difficult to diagnose and distinguish tuberculous meningitis, and the current laboratory methods are unsubstantial in developing countries. The study is aimed at creating a scoring system on the basis of basic laboratory and clinical achievements that could be used as diagnostic aid for tuberculous meningitis for Chinese patients. A retrospective study of cases was conducted for comparison between clinical characteristics and laboratory features of 241 patients on admission who conformed to inclusion criteria of tuberculous meningitis (n = 141) or bacterial meningitis (n = 100). Logistic regression was employed to establish a diagnostic formula to distinguish between tuberculous meningitis and bacterial meningitis. The receiver operating characteristic curve analysis was applied to determine the best diagnostic critical point of the diagnostic formula. It was found that five variables (disease course, white blood cell count, serum sodium, total white cell count of cerebrospinal fluid, and neutrophil proportion in cerebrospinal fluid) were independently associated with tuberculous meningitis. The 87% sensitivity and 94% specificity were included in the diagnostic scoring system derived from these variables. Especially in the case of limited microbial resources, doctors can use this diagnostic scoring system to distinguish tuberculous meningitis from bacterial meningitis.

Changes of microbial and metabolome of the equine hindgut during oligofructose-induced laminitis.

BACKGROUND: Laminitis is a common and serve disease which caused by inflammation and pathological changes of the laminar junction. However, the pathologic mechanism remains unclear. In this study we aimed to investigate changes of the gut microbiota and metabolomics in oligofructose-induced laminitis of horses. RESULTS: Animals submitted to treatment with oligofructose had lower fecal pH but higher lactic acid, histamine, and Lipopolysaccharide (LPS) in serum. Meanwhile, oligofructose altered composition of the hindgut bacterial community, demonstrated by increasing relative abundance of Lactobacillus and Megasphaera. In addition, the metabolome analysis revealed that treatment with oligofructose decreased 84 metabolites while 53 metabolites increased, such as dihydrothymine, N3,N4-Dimethyl-L-arginine, 10E,12Z-Octadecadienoic acid, and asparagine. Pathway analysis revealed that aldosterone synthesis and secretion, regulation of lipolysis in adipocytes, steroid hormone biosynthesis, pyrimidine metabolism, biosynthesis of unsaturated fatty acids, and galactose metabolism were significantly different between healthy and laminitis horses. Furthermore, correlation analysis between gut microbiota and metabolites indicated that Lactobacillus and/or Megasphaera were positively associated with the dihydrothymine, N3,N4-Dimethyl-L-arginine, 10E,12Z-Octadecadienoic acid, and asparagine. CONCLUSIONS: These results revealed that disturbance of gut microbiota and changes of metabolites were occurred during the development of equine laminitis, and these results may provide novel insights to detect biomarkers for a better understanding of the potential mechanism and prevention strategies for laminitis in horses.

Development and validation of a predictive model for the diagnosis of neural antibody-mediated epilepsy/ seizure in patients with new-onset seizure or established epilepsy.

PURPOSE: Currently, the diagnosis of neural antibody-mediated epilepsy/seizure (NAME/S)relies heavily on neural antibody testing, which is time-consuming, costly and introduces diagnostic delays. A statistical tool to predict the probability of a patient with NAME/S is lacking. We aimed to construct a predictive model to help clinicians expedite the diagnostic process. METHODS: We retrospectively recruited subjects (206 in the development group and 62 in the validation group) with new-onset seizures or established epilepsy suspected to have presented with antibody-mediated seizures between January 2014 and December 2019. We collected data about demographics, medical history, clinical manifestations and follow up. Binary logistic regression was used to select potential predictors for the construction of a predictive model. Five-fold cross and bootstrap validation were applied to avoid overfitting. Concordance index, calibration plots and decision curve analysis were used to assess its performance. RESULTS: The model, incorporating presence/absence of tumour, psychiatric/cognitive/emotional changes, language disturbances, sensory auras, tonic-clonic seizures, multiple seizure events, hyponatremia and MRI inflammation, was visualized as a nomogram. The crude and adjusted concordance indices were both 0.88 with a cut-off value of 0.62, sensitivity of 83.2 % and specificity of 77.4 %. The slope and intercept of the calibration curve were 0 and 1, respectively. The model also showed good performance in the validation group with a concordance index of 0.82, cut-off value of 0.33, sensitivity of 75.5 % and specificity of 73.1 %. The slope was 0.86 and the intercept was 0.039. Decision curve analysis showed that the model was useful with an optimal threshold probability of ＞4 % in both groups. CONCLUSIONS: Despite limitations such as sample volume and selection bias in subject enrolment, this model may be used to estimate the individualized probability of having NAME/S, deserving further exploration and validation.

Validation of the NEOS score in Chinese patients with anti-NMDAR encephalitis.

OBJECTIVE: The performance of anti-NMDAR Encephalitis One-Year Functional Status (NEOS) in predicting the 1-year functional status in Chinese patients with anti-NMDAR encephalitis is unknown. METHODS: We recruited patients with anti-NMDAR encephalitis from the Multicenter and Prospective Clinical Registry Study of Anti-NMDAR Encephalitis in Beijing Area. Patients were followed up for 1 year. We defined the poor functional status as a modified Rankin Scale score of more than 2 and good functional status as a modified Rankin Scale score of no more than 2. We performed a receiver-operator characteristic analysis to assess the discriminatory power of the NEOS score in predicting the 1-year functional status by using the area under the curve (AUC). Calibration was assessed by Pearson correlation coefficient and Hosmer-Lemeshow tests. RESULTS: Among the 111 patients with anti-NMDAR encephalitis recruited from 364 potentially eligible participants, 87 (78.4%) had good functional status at 1 year, whereas the remaining 24 (21.6%) had poor functional status. The AUC of the NEOS score for 1-year poor functional status was 0.86 (95% CI 0.78-0.93, p < 0.001). The increased NEOS was associated with higher risk of 1-year poor functional status in patients with anti-NMDAR encephalitis. CONCLUSIONS: The NEOS score is considered a reliable predictor of the risk of 1-year poor functional status in Chinese patients with anti-NMDAR encephalitis. This score could help to estimate the velocity of clinical improvement in advance. CLINICALTRIALGOV IDENTIFIER: NCT02443350. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that in patients with anti-NMDAR encephalitis, the NEOS score predicts 1-year functional status.

Knockdown of IKKß Inhibits Tumor Development in a Leptomeningeal Metastasis Mouse Model and Proliferation of Lung Cancer Cells.

OBJECTIVE: This study will explore the role of IKKß in the leptomeningeal metastasis (LM) of lung cancer cells. METHODS: In vitro studies were conducted in mouse Lewis lung carcinoma (LLC) cells with IKKß knockdown. Cell proliferation was explored using CCK-8 and tumor colony-forming assays. The expression of the extracellular signal-regulated kinase (ERK) signaling pathway was detected by Western blot. Tumor cell apoptosis was identified through Western blot detection of Bax and Bcl-2. The migration of tumor cells was identified by a wound healing assay. In vivo studies used an LM mouse model developed by injecting LLC cells with or without IKKß knockdown into the cisterna magna. Mouse brain and spinal samples were sectioned for hematoxylin and eosin staining. RESULTS: In vitro: IKKß knockdown inhibits tumor cell proliferation, initiates apoptosis, and attenuates cell migration. In vivo: IKKß knockdown inhibits tumor growth in the LM mouse model. In addition, the in vitro results showed that IKKß knockdown attenuated the expression of ERK phosphorylation in LLC cells. CONCLUSION: Blocking the NF-κB signaling pathway by IKKß knockdown in LLC cells inhibited tumor growth in the LM mouse model. IKKß supports leptomeningeal tumor progression by promoting cancer cell proliferation and migration and inhibiting cancer cell apoptosis, and these actions may be correlated to ERK signaling.

Angiostrongyliasis detected by next-generation sequencing in a ELISA-negative eosinophilic meningitis: A case report.

Next-generation sequencing (NGS) is an emerging method with the potential of pan-pathogen screening. This study described a case of eosinophilic meningitis (EoM) with enzyme-linked immunosorbent assay (ELISA)-negative results for Angiostrongylus cantonensis (A. cantonensis), Trichinella spiralis and Paragonimus westermani and a positive identification of A. cantonensis by NGS in the cerebrospinal fluid.

miR-210 in Exosomes Derived from Macrophages under High Glucose Promotes Mouse Diabetic Obesity Pathogenesis by Suppressing NDUFA4 Expression.

OBJECTIVE: Type 2 diabetes mellitus (T2DM) is featured by insulin resistance and lipid metabolism dysregulation. A large number of miRNAs were identified in exosomes derived from adipose tissue macrophages associated with T2DM pathogenesis, but its pathogenic roles remain unknown. This study is aimed at investigating the function of miR-210 in diabetic obesity. METHODS: Exosomes from mouse macrophage RAW264.7 cells were characterized by electron microscopy, combined with biomarker expression by western blot. Expression of miR-210 was determined by quantitative RT-PCR. Glucose uptake was measured by a fluorometric method, and the mitochondrial respiratory chain activity was evaluated by ELISA. The target gene of miR-210 was validated by dual-luciferase reporter and pull-down assays. A mouse obese diabetic model was established by a high-fat diet and streptozocin treatment. RESULTS: miR-210 was highly expressed in exosomes derived from high glucose-induced macrophage RAW264.7 cells. Macrophage-derived exosomes impaired glucose uptake and mitochondrial CIV complex activity and suppressed NADH dehydrogenase ubiquinone 1 alpha subcomplex 4 (NDUFA4) expression in 3T3-L1 adipocytes. miR-210 directly bind with mRNA sequences of NDUFA4 gene. Inhibition of miR-210 mitigated the effects of macrophage-derived exosomes on the glucose uptake and complex IV (CIV) activity in 3T3-L1 adipocytes, and NDUFA4 overexpression offset the inhibition of glucose uptake and CIV activity by macrophage-derived exosomes. Furthermore, mice with miR-210 knockout showed greatly repressed diabetic obesity development. CONCLUSION: miR-210 derived from adipose tissue macrophages promotes mouse obese diabetes pathogenesis by regulating glucose uptake and mitochondrial CIV activity through targeting NDUFA4 gene expression.

High electrochemical performance carbon nanofibers with hierarchical structure derived from metal-organic framework with natural eggshell membranes.

Carbon nanofibers with hierarchical structure were synthesized by combining Co-containing zeolitic imidazolate frameworks (ZIF-67) with natural eggshell membranes (ESMs). Benefiting from the hierarchical structure and element modification of Co/N, the obtained nanofibers exhibited excellent oxygen reduction reaction (ORR) performance. The reusing of ESMs trash made this strategy meaningful for environment.

Evaluating the cerebrospinal fluid ctDNA detection by next-generation sequencing in the diagnosis of meningeal Carcinomatosis.

BACKGROUND: Meningeal carcinomatosis (MC) is the most severe form of brain metastasis and causes significant morbidity and mortality. Currently, the diagnosis of MC is routinely confirmed on the basis of clinical manifestation, positive cerebrospinal fluid (CSF) cytology, and/or neuroimaging features. However, negative rate of CSF cytology and neuroimaging findings often result in a failure to diagnose MC from the patients who actually have the disease. Here we evaluate the CSF circulating tumor DNA (ctDNA) in the diagnosis of MC. METHODS: A total of 35 CSF samples were collected from 35 patients with MC for CSF cytology examination, CSF ctDNA extraction and cancer-associated gene mutations detection by next-generation sequencing (NGS) at the same time. RESULTS: The most frequent primary tumor in this study was lung cancer (26/35, 74%), followed by gastric cancer (2/35, 6%), breast cancer (2/35, 6%), prostatic cancer (1/35, 3%), parotid gland carcinoma (1/35, 3%) and lymphoma (1/35, 3%) while no primary tumor could be found in the remaining 2 patients in spite of using various inspection methods. Twenty-five CSF samples (25/35; 71%) were found neoplastic cells in CSF cytology examination while all of the 35 CSF samples (35/35; 100%) were revealed having detectable ctDNA in which cancer-associated gene mutations were detected. All of 35 patients with MC in the study underwent contrast-enhanced brain MRI and/or CT and 22 neuroimaging features (22/35; 63%) were consistent with MC. The sensitivity of the neuroimaging was 88% (95% confidence intervals [95% CI], 75 to 100) (p = 22/25) and 63% (95% CI, 47 to 79) (p = 22/35) compared to those of CSF cytology and CSF ctDNA, respectively. The sensitivity of the CSF cytology was 71% (95% CI, 56 to 86) (n = 25/35) compared to that of CSF ctDNA. CONCLUSIONS: This study suggests a higher sensitivity of CSF ctDNA than those of CSF cytology and neuroimaging findings. We find cancer-associated gene mutations in ctDNA from CSF of patients with MC at 100% of our cohort, and utilizing CSF ctDNA as liquid biopsy technology based on the detection of cancer-associated gene mutations may give additional information to diagnose MC with negative CSF cytology and/or negative neuroimaging findings.