The pharmacokinetics of vancomycin in cerebrospinal fluid (CSF) is an important basis for evaluating the bactericidal effect. The accuracy of using serum vancomycin concentrations only to estimate the CSF concentrations remains controversial, may lead to underdosing.The aims of this study were to evaluate the vancomycin exposure in CSF, investigate the factors affecting the vancomycin blood–brain barrier (BBB) penetration, and to establish the prediction model of vancomycin concentration in CSF.Eligible patients were included and given a standard dose of vancomycin. At the fifth dose, the blood and CSF samples were collected 0.5 h before the start of infusion of vancomycin, and 1, 2, 3, and 8 h from the start of infusion, and were measured by the enzyme-multiplied immunoassay technique using the Siemens Viva-E Drug Testing System.serumCSF/ of patients with intracranial infection was higher than that of patients without (p = 0.001). The CSF concentration was relatively stable between dosing periods (p = 0.095). The area under the concentration–time curve (AUC) ratio of CSF to serum (AUCCSF/serum) in patients with intracranial infection ranged from 15.1 to 80.1% . The CSF vancomycin AUC levels were affected by the serum trough concentration , and were mainly affected by the CSF white blood cells (WBCs)/total cells  .The AUCAfter intravenous administration of vancomycin, the CSF concentration curve was fluctuated gently. The CSF vancomycin concentration in patients with postoperative intracranial infection can be predicted by the WBCs to total cells ratio and the serum trough concentration, and help to adjust the administration of vancomycin. Postoperative intracranial bacterial infection is a well-known potential life-threatening complication in neurosurgical therapy, and it often seriously affects the clinical prognosis, and increases the mortality –3. Altho+) pathogens  clinical signs and symptoms: fever (>38°C) and meningeal irritation sign (+); (B) a positive result of CSF culture; (C) two or more changes in CSF specimens: (1) CSF glucose <2.2 mmol/L or <40% of serum glucose; (2) protein concentrations >600 mg/L; (3) white blood cells (WBCs) count more than 1,000 × 10− bacilli.Exclusion criteria included: (1) the patient or guardian refused to participate; (2) the patient with hypersensitivity to vancomycin; (3) the patient received vancomycin treatment by any means within 1 month; (4) the patient with abnormal hepatic and renal function; (5) CSF could not be collected; (6) age < 18 years old; (7) the result of CSF culture was GBasic demographic data of the patients were obtained from their guardians. The body temperature, weight, and height of the patients were collected before vancomycin treatment. Collected and recorded the laboratory parameters on the same day, such as blood WBCs, serum albumin, creatinine clearance rate (CLcr), serum procalcitonin (PCT), serum protein, and serum glucose.The estimated creatinine clearance was calculated based on the Cockcroft–Gault formula. Infusion reaction and anaphylaxis were monitored during each infusion.All patients received I.V. vancomycin  alone or in combination with meropenem. Vancomycin was administrated I.V. 1,000 mg every 12 h  as the recommended daily dose for adult patients with normal renal function. No patients received corticosteroids during anti-infection treatment. Samples of blood and CSF were collected at the fifth dose (day 3). At least 2 ml of venous blood and 2 ml of CSF were collected 0.5 h before the start of infusion of vancomycin, and 1, 2, 3, and 8 h from the start of infusion. CSF samples were obtained by a temporary lumbar catheterization. About 2 ml of CSF remaining in the drainage tube was discarded, and 2 ml of fresh CSF was collected. The blood and CSF samples were collected using normal biochemical test tubes.At the fifth CSF collection, appropriate amount of CSF was collected for CSF biochemical analysis, cytological analysis, PCT analysis, and microbial culture. Bacteriological antibiotic sensitivity test was determined using the Vitek-2 Compact automatic system . The temporary lumbar tube was removed or retained according to treatment needs after the collection of CSF samples.All blood and CSF samples were centrifuged and the supernatants were collected for assay. The vancomycin concentration in serum and CSF was measured by the enzyme-multiplied immunoassay technique using the Siemens Viva-E Drug Testing System with the Emit 2,000 Vancomycin Assay Test Kit . This method had a calibration range between 2.0 and 50.0 μg/ml, with a lowest limit of 2.0 μg/ml. The variation coefficients were <10% for both Intra- and inter-assay. All samples were tested by the same pharmaceutical technician and reviewed by another.z) and area under the 24-h concentration–time curve (AUC) were calculated by a non-compartmental analysis method using the DAS 3.0.2 pharmacokinetic program. As observable parameters, the time to reach the maximum concentration (Tmax) and the maximum concentration (Cmax) were obtained directly. Vancomycin concentration–time curve was measured and the relationship between vancomycin CSF and serum level was calculated.The PK parameters including drug clearance , ANOVA for repeated measurement data was used to assess differences between variables. Pearson's correlation analysis was used to assess the correlation between two variables. Multiple linear regression was used to analyze the relationship between multiple factors. The p-values of < 0.05 were considered as statistically significant.Statistical analysis was performed using SPSS 24.0 . All quantitative data were expressed as mean ± standard deviation. The student's The covariance analysis of vancomycin concentration in CSF and serum and AUC of patients in the two groups was conducted with body weight as covariable, and it was found that the significant factors of body weight were all >0.05, so covariance analysis was not applicable.The maximum limit of CSF protein was 3,000 mg/L, the lowest limit of CSF glucose was 1.1 mmol/L, and the lowest limit of CSF vancomycin concentration was 2.0 μg/ml in the laboratory department of our hospital. Therefore, for statistical purposes, CSF protein >3,000 mg/L was considered to be 3,000 mg/L, CSF glucose ≤1.1 mmol/L was regarded as 1.1 mmol/L, and CSF vancomycin concentration <2.0 μg/ml was considered to be 0 μg/ml for statistical purposes.p > 0.05). All patients received standard pharmacological treatment for the primary disease, and there was no differences between-group. During the treatment period, no adverse reactions such as vancomycin infusion reaction, decreased WBCs, or renal impairment were observed. The clinical and bacteriological cures were achieved for all patients. No correlation was found between body weight and drug concentration or AUC (p > 0.05).A total of 22 patients  with 110 blood samples and 110 CSF samples were included in this study . Twelve F = 109.61, p < 0.001). The concentration at 0.5 h before the fifth dose was the serum vancomycin trough concentration. The Tmax of the serum concentration was 2 h after the start of infusion of vancomycin. No significant differences were found in the serum vancomycin concentration between the patients with and without intracranial infection . The serum trough concentration levels were affected by the CLcr . The serum AUC was negatively correlated with CLcr .The mean serum vancomycin concentration of patients evaluated during the fifth 12 h of treatment is presented in + bacteria /MIC ratios ranged from 72.90 to 196.00  for these 6 patients.Of the 12 patients with intracranial infection, there were 6 positive cultures of Gbacteria . The MICF = 2.051, p = 0.095). The CSF Cmax of patients with intracranial infection was ranged from 3.6 to 20.5 μg/ml and of patients without intracranial infection ranged from 0 to 4.8 μg/ml . The CSF trough levels of patients with and without intracranial infection were, respectively, ranged from 2.1 to 11.1 μg/ml and from 0 to 3.0 μg/ml . Differences in CSF vancomycin concentration between patients with and without intracranial infection were significant .The CSF vancomycin concentration was <2.0 μg/ml in 4 patients without intracranial infection. The mean CSF vancomycin concentration of patients evaluated during the fifth 12 h of treatment is presented in vs. 288.59 ± 84.36 μg/ml·h, t = 0.94, p = 0.357). The CSF AUC of patients with intracranial infection (ranging from 50.28 to 270.67 μg/ml·h) was significant higher than that of patients without (ranging from 0 to 61.19 μg/ml·h)  (CSF/serum) in patients with intracranial infection ranged from 15.1 to 80.1% , and for patients without intracranial infection ranged from 0 to 18.3% . The AUCCSF/serum of patients with intracranial infection was higher than that of patients without .The serum AUC of patients with and without intracranial infection, respectively, ranged from 222.55 to 426.01 μg/ml·h and from 192.08 to 423.76 μg/ml·h (319.42 ± 69.28 μg/ml·h = 0.005) . The ratr = 0.503, p = 0.017), serum trough concentration , CSF proportion of polynuclear cells , and CSF WBCs/total cells . The CSF trough concentration was negatively correlated with CSF glucose . The CSF trough concentration levels were affected by the serum trough concentration , and was mainly affected by the CSF WBCs/total cells  .The CSF trough concentration was positively correlated with CSF PCT , CSF PCT , serum trough concentration , CSF proportion of polynuclear cells , and CSF WBCs/total cells . The CSF AUC was negatively correlated with CSF glucose . The CSF AUC was not significant correlated with the serum AUC . The CSF AUC levels were affected by the serum trough concentration , and were mainly affected by the CSF WBCs/total cells  .The CSF AUC was positively correlated with CSF WBCs  of patients with intracranial infection was 33.23% and the median was 26.25% in our study. And this two parameters for patients without intracranial infection were only 8.83 and 10.15%. Both the CSF vancomycin AUC and AUCCSF/serum were significantly higher in patients with intracranial infection than those in patients without intracranial infection. Similar to the previous reports, the vancomycin BBB penetration rate was significantly correlated with the degree of intracranial bacterial infection , this may be the main reason of individual difference of vancomycin concentration in CSF.A clinical trial to show the rate of vancomycin BBB penetration in 22 patients with postsurgical meningitis was carried out by Wang et al.  in 2015.r = 0.502, p = 0.017). Similarly with our result, several previous studies also reported this correlation. Taheri et al., . Cai et al. , and thought that this could direct the treatment of intracranial infection. Another study carried out by Li et al. . The patients/participants provided their written informed consent to participate in this study.M-CF and WF conceived, designed, and supervised the study. M-CF, J-LS, JS, and NW enrolled patients and collected data. MF and J-WM finalized the analysis. M-CF, WF, and J-LS drafted and revised the manuscript. All authors critically reviewed the manuscript. All authors read and approved the final manuscript.This work was supported, in part, by the National Natural Science Foundation of China (Grant No. 81903872). Author J-LS has received research support from the National Natural Science Foundation of China.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


N = 39), HIV CSF (N = 79), or other suspected and confirmed neuroinflammatory CSF cases . Therefore, AnkG autoantibodies in CSF are rare but extend the catalog of AIS and NoR autoantibodies associated with neurological autoimmunity.Neuroinvasive infection is the most common cause of meningoencephalitis in people living with human immunodeficiency virus (HIV), but autoimmune etiologies have been reported. We present the case of a 51-year-old man living with HIV infection with steroid-responsive meningoencephalitis whose comprehensive pathogen testing was non-diagnostic. Subsequent tissue-based immunofluorescence with acute-phase cerebrospinal fluid revealed anti-neural antibodies localizing to the axon initial segment (AIS), the node of Ranvier (NoR), and the subpial space. Phage display immunoprecipitation sequencing identified ankyrinG (AnkG) as the leading candidate autoantigen. A synthetic blocking peptide encoding the PhIP-Seq-identified AnkG epitope neutralized CSF IgG binding to the AIS and NoR, thereby confirming a monoepitopic AnkG antibody response. However, subpial immunostaining persisted, indicating the presence of additional autoantibodies. Review of archival tissue-based staining identified candidate AnkG autoantibodies in a 60-year-old woman with metastatic ovarian cancer and seizures that were subsequently validated by cell-based assay. AnkG antibodies were not detected by tissue-based assay and/or PhIP-Seq in control CSF ( Immunocompromised individuals are vulnerable to neuroinvasive pathogens that can cause meningitis, encephalitis, and myelitis. Infections of the central nervous system (CNS) are a leading cause of death in persons with acquired immunodeficiency syndrome (AIDS) but are uncommon in those with well-controlled HIV. Yet, because CNS infection may still occur in well-controlled HIV , 2, compOther causes of neuroinflammation in persons with well-controlled HIV include macrophage-mediated HIV encephalitis and CD8 encephalitis —a hallmaHere, we present the case of a man with well-controlled HIV who developed steroid-responsive meningoencephalitis in the absence of HIV escape. Subsequent research studies identified a novel anti-ankyrinG (AnkG) antibody and a second antibody directed at a subpial antigen in his CSF. A search of archival neuroinflammatory cases identified an HIV-negative woman with a history of cancer and seizures who harbored the same anti-neuronal antibody but not the subpial antibody, indicating that AnkG may in rare cases be the target autoantigen in seronegative autoimmune neurological syndromes.+ count 791 cells/μL and undetectable viral load) on tenofovir alafenamide, emtricitabine, and dolutegravir presented to the emergency room with several weeks of headache, right-sided hearing loss, and memory impairment. He smoked one pack of cigarette per day and marijuana but did not use other drugs. He had lived in the San Francisco Bay Area for over 20 years and was an avid gardener. He did not have a history of incarceration, homelessness, or international travel  testing for infectious etiologies was non-diagnostic including serum toxoplasmosis IgG and CSF fungal cultures, microbial antigen and antibody testing, VDRL, universal bacterial, fungal, and mycobacterial PCR, and metagenomic next-generation sequencing (mNGS). Diagnostic biopsy of the leptomeninges overlying the right cerebrum revealed dense connective tissue with a mixed inflammatory infiltrate including granulocytes, and B and T cells , suggestOne week after discharge (week 7), the patient presented to the emergency department with new-onset expressive aphasia, hyponatremia, and ataxia. EEG revealed right greater than left diffuse background slowing and disorganization without epileptiform activity. Repeat LP again showed a neutrophilic pleocytosis (WBC 194/μL with 50% neutrophils), protein 75 mg/dL, elevated IgG index , and five restricted oligoclonal bands (OCBs). Brain MRI showed persistent meningoencephalitis with new ventriculomegaly. A large volume LP was performed. The opening pressure was 25 cm and the patient regained his speech after 1 h later, had improved gait, and improved mental status. He proceeded to get a ventriculoperitoneal shunt and continued on anti-TB therapy.A CSF autoimmune encephalopathy panel was reported as positive for NMDA receptor autoantibodies (1:8 tissue titer) as well as putatively non-specific additional staining on tissue. The whole body PET and scrotal ultrasound were negative. A repeat autoimmune encephalopathy panel was NMDA receptor antibody positive on cell-based assay but negative on reflex tissue staining. Because the clinical phenotype was markedly inconsistent with anti-NMDAR encephalitis, empiric anti-TB therapy was initially continued. Nonetheless, an autoimmune etiology was increasingly considered as a result of the repeatedly negative infectious workup and lack of improvement in antibiotic therapy. In this context, he received a 5-day course of methylprednisolone 1 gram daily on week 8 followed by a prednisone taper with dramatic improvement in his memory and language. Anti-TB therapy was ultimately discontinued after ~1 month of treatment.Over the following months, attempts to taper off prednisone resulted in clinical and radiological deterioration. Surveillance brain MRIs demonstrated worsening of meningoencephalitis with a new focus on enhancement in the left amygdala. A second brain biopsy of glucocorticoids at month 10 showed severe granulomatous and suppurative meningoencephalitis with a perivascular and intraparenchymal lymphocytic infiltrate, diffuse astrogliosis, and microglial activation. The pattern of inflammation was again suspicious for mycobacterial infection; however, AFB and AFB-FITE stains and AFB culture of the brain tissue were negative, as were mycobacterial universal PCR and metagenomic next-generation sequencing.Three weeks after the second biopsy, the patient was retreated with IV methylprednisolone followed by oral prednisone maintenance with subsequent clinical improvement over the next few months. Repeat imaging 3 months after the resumption of glucocorticoid treatment demonstrated near the total resolution of leptomeningeal enhancement and non-progression of his encephalitis. The patient was then transitioned empirically to mycophenolate mofetil and successfully weaned off prednisone without recurrence of his meningoencephalitis. He remained seizure-free on antiseizure therapy. Repeat CSF at 26 months post-presentation showed normal cellularity (3 WBC/μL with 87% lymphocytes and 17% monocytes), normal glucose (57 mg/dL), mildly elevated protein (67 mg/dL), persistent elevated IgG index (0.7 ref. ≤0.6), and two oligoclonal bands. MRI brain every 6 months showed stable multifocal T2/FLAIR cortical and white matter hyperintensities without associated enhancement. Following 3 years of immunosuppressive therapy, 4.5 years after his initial presentation, mycophenolate mofetil was stopped and the patient remained stable. Neurological assessment documented fixed sequelae consisting of dysarthria, executive dysfunction, and episodic memory impairment.A 60-year-old woman with a history significant for smoking, non-insulin-dependent type II diabetes, renal cell carcinoma status post-left radical nephrectomy and node dissection and essential hypertension, and no family history of autoimmune neurologic or oncologic disease was diagnosed with stage IIIC metastatic ovarian cancer. She underwent bilateral salpingoopherectomy and supracervical hysterectomy, and carboplatin and taxol therapy. Two years later, in the context of recurrent ovarian cancer again undergoing carboplatin, taxol, and avastin therapy, she developed seizures with interictal abnormalities noted in the left frontotemporal region on EEG. Her MRI at the time had bilateral parieto-occipital FLAIR abnormalities without evidence of meningeal involvement. Her spinal fluid was notable for elevated protein. Her seizures were attributed to posterior reversible encephalopathy syndrome (PRES) in the setting of carboplatin use. Six years after her initial diagnosis, she had a recurrence of her ovarian cancer. She was placed on tamoxifen, and over the next 18 months, she was treated with topotecan and bevacizumab (15 cycles), paclitaxel (five cycles), gemcitabine (two doses), and doxorubicin. Eight years after her initial diagnosis, she again developed seizures characterized by unresponsiveness, rightward head and eye deviation, and right upper extremity tonic–clonic movements. EEGs at the time captured left temporal seizures and interictal abnormalities and were treated with levetiracetam. An MRI brain with contrast at the time showed non-specific ischemic changes without metastatic disease. Her spinal fluid was non-inflammatory, and autoantibody testing at the time was negative. Her CSF protein was elevated (100 mg/dL), and oligoclonal bands were not sent. Four months later, she was admitted to the hospital with neutropenic fever (absolute neutrophil count 0.3), mouth sores, and dysphagia, and treated for sepsis. Due to ongoing failure to thrive, the patient elected to pursue hospice and she subsequently died.CSF from cases 1 and 2 was screened for autoantibodies by tissue-based immunofluorescence (TBIF). At a 1:100 dilution, acute-phase CSF from case 1 produced a pattern of immunoreactivity that localized to axon initial segment (AIS)-like structures throughout the cortex and node of Ranvier (NoR)-like structures in the optic tract. In contrast, case 1's convalescent CSF was not immunoreactive at a 1:100 dilution and stained only weekly at a 1:4 dilution, suggesting a decrease in the autoantibody titer following immunosuppressive therapy .To verify immunoreactivity to the AIS and NoR, we immunostained mouse brain tissue with acute-phase CSF and commercial antibodies to the AIS and nodal marker AnkG and the paranodal marker protein contactin-associated protein 1 (CASPR). Case 1 CSF IgG colocalized with AnkG at the AIS and NoR, thereby confirming AIS-NoR immunoreactivity . A revieCase 2 was identified as part of a screen for TRIM46 autoimmunity . CSF wasWe did not identify another case of anti-AIS/NoR immunoreactivity among the 39 control and 1,236 candidate CNS neuroinflammatory patients screened by TBIF in our laboratory.To identify candidate anti-AIS/NoR autoantibodies, we screened both cases in technical replicate by phage display immunoprecipitation sequence (PhIP-Seq) . We restWe hypothesized that spurious enrichment of non-target peptides could attenuate the contribution of individual target peptides to total protein enrichment values. Therefore, we filtered for AIS/NoR peptides that were enriched at least 10-fold in both technical replicates. For case 1, more than half of enriched peptides mapped to ankyrinG, six of which shared an epitope and had Z-scores ranging between 18 and 47 . MultiplN = 15 well-controlled and N = 18 untreated HIV) and found that neither AnkG nor epitope AnkG937−958 were enriched above negative controls  suggesting that case 2's autoantibodies recognize conformational or post-translationally modified epitopes that are not represented in our PhIP-Seq library.937−958 epitope . However, a comparison of human, rat, and murine ankyrinG revealed that rat AnkG differs from human and murine AnkG by a single amino acid (proline in rat vs. threonine in human and mouse) in the center of the putative AnkG epitope .Σ1 and Σ6 HEK 293T cell-based assays (Σ1 or Σ6 (data not shown).Based on the PhIP-Seq and TBIF findings, we hypothesized that case 2's target epitopes were conformational or post-translationally modified. We screened case 2's CSF against previously established AnkG and βIV spectrin variant d assays . We found assays  but not Advillin promoter is active in dorsal sensory neurons but inactive in ventral motor neurons (Ank3flfl) and dorsal root sensory neurons (DR-Ank3−/−) from Advillin-Cre:Ank3fl/fl mice with CSF from cases 1 and 2. In both cases, CSF IgG localized to VR-Ank3flfl NoR but not DR-Ank3−/− NoR, thereby confirming that the staining was due to the presence of AnkG autoantibodies in both cases' CSF samples  and a peptide encoding the adjacent C-terminal 25 AA as a non-epitope peptide control (control peptide: AnkG962−989)  .In technical quadruplicate, we immunostained rodent brain tissue with acute-phase CSF without peptide, or after pre-incubation with control or blocking peptide in five molar excess. Preincubation of case 1 CSF with the blocking peptide, but not the control peptide, completely abrogated CSF IgG binding to both the AIS and NoR without affecting the binding of a commercial AnkG antibody (NeuroMab N106/36) that binds outside of the control and blocking epitopes .We observed that the AnkG blocking peptide abrogated AIS and NoR immunoreactivity but not the limited nuclear and subpial CSF IgG immunoreactivity . HoweverThis case demonstrates the complexity of diagnosis and clinical management of people living with well-controlled HIV who present with neuroinflammatory syndromes. Although broad-based testing of blood, CSF, and brain tissue for infections was consistently negative, it is possible that a preceding CNS infection was the precipitant of this patient's autoimmune meningoencephalitis. Nonetheless, this case also highlights the need for continued autoantibody discovery in seronegative autoimmune CNS syndromes to decrease the time to treatment.Despite negative infectious testing, case 1's low CSF glucose sustained concern for an undetected microorganism. Hypoglycorrhachia is common in infectious meningitis and is unlikely to be due to pleocytosis alone in aseptic meningitis except for specific non-infectious syndromes such as systemic lupus erythematosus , which cAlthough IgG class NMDAR autoantibodies were detected in the CSF of our patient, they were low titer and inconsistently detected on clinical testing; this, along with the lack of typical clinical features of anti-NMDAR encephalitis, led us to conclude that these represented false-positive anti-NMDAR testing, which is known to occur . NMDAR aInstead, research testing identified a high-titer antibody to AnkG that decreased in titer during convalescence. AnkG is enriched in the AIS and NoR, subcellular structures with similar molecular profiles that are targeted by autoantibodies in diverse autoimmune neurological disorders including antibodies that bind to gangliosides , neurofa937−957 antibodies directly alter AnkG function.Although intracellular, AnkG is a membrane-associated protein owing to its interaction with many AIS-enriched membrane proteins, an N-terminal palmitoyl moiety , and an 937−957 blocking peptide also allowed us to identify a distinct population of subpial autoantibodies in the CSF of case 1. Although we were unable to identify the antigen, this subpial immune response may explain the different clinical phenotypes between case 1 and case 2. Alternatively, the difference may be because case 1 and case 2 target different AnkG target epitopes  numbers 13-12236 and 15-18425, and by Mayo Clinic Institutional Review Board (IRB) numbers 08-006647 and 08-007846. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. All procedures used in this study complied with federal guidelines and the institutional policies of the UCSF, Mayo Clinic, and Baylor College of Medicine Institutional Animal Care and Use Committees.CB: conceptual design, experimental design, tumor histology, IHC, CBAs, PhIP-Seq analysis, data interpretation, and writing. TN: CBAs, IHC, confocal microscopy, and writing. CC: neuropathological studies and editing. AH: clinical data and writing and editing. BA and IH: PhIP-Seq assays. RS: PhIP-Seq and writing and editing. KZ: clinical data collection and patient sample collection. TH and MR: AnkyrinG-deficient mice and writing and editing. AK and JD: PhIP-Seq database design. SS, JG, FC, and SeP: clinical data and editing. DD: tissue-based assay, clinical data, and writing and editing. MW and SaP: conceptual design, experimental design, data interpretation, and writing. All authors contributed to the article and approved the submitted version.


Neurofilament light (NfL) and neurogranin (Ng) are promising candidate AD biomarkers, reflecting axonal and synaptic damage, respectively. Since there is a need to understand the synaptic and axonal damage in preclinical Alzheimer’s disease (AD), we aimed to determine the cerebrospinal fluid (CSF) levels of NfL and Ng in cognitively unimpaired elderly from the Gothenburg H70 Birth Cohort Studies classified according to the amyloid/tau/neurodegeneration (A/T/N) system.T-test and ANCOVA.The sample consisted of 258 cognitively unimpaired older adults  from the Gothenburg Birth Cohort Studies. We compared CSF NfL and Ng concentrations in A/T/N groups using Student’s p=0.001) and the A−T+N+ group (p=0.006) compared with A−T−N−. CSF Ng concentration was higher in the A−T−N+, A−T+N+, A+T−N+, and A+T+N+ groups (p<0.0001) compared with A−T−N−. We found no difference in NfL or Ng concentration in A+ compared with A− (disregarding T− and N− status), whereas those with N+ had higher concentrations of NfL and Ng compared with N− (p<0.0001) (disregarding A− and T− status).CSF NfL concentration was higher in the A−T−N+ group (CSF NfL and Ng concentrations are increased in cognitively normal older adults with biomarker evidence of tau pathology and neurodegeneration. Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by intracerebral accumulation of amyloid (Aβ) and abnormally phosphorylated tau, followed by neurodegeneration leading to progressive cognitive decline. Cerebrospinal fluid (CSF) amyloid β42 (Aβ42), total tau (T-tau), and phosphorylated tau (P-tau) concentrations are established diagnostic and/or prognostic biomarkers for AD, reflecting the core pathological hallmarks of AD, amyloid plaques, neurodegeneration and the hyperphosphorylation of tau, respectively . In receThe sample was derived from the 2014–2016 examinations of the population-based H70 Gothenburg Birth Cohort Studies in Gothenburg, Sweden. Residential addresses were obtained from the Swedish Population Registry. The sample was obtained using systematic selection, where every 70-year-old living in Gothenburg , born on specific dates in 1944 was invited to partake in examination in 2014–2016 . 1203 suParticipants took part in a full-day examination at the Psychiatry, Cognition, and Old Age outpatient clinic at Sahlgrenska University Hospital in Gothenburg, Sweden, or in their homes as described previously .A comprehensive general examination was performed that included blood sampling and genotyping, use of medications, self-rating questionnaires, social factors, key informant interviews, and neuropsychiatric examination , 19. TheExaminations included the Mini-Mental State Examination (MMSE) and CDR. The final ratings were assigned by research nurses and a geriatric psychiatrist and neurologist (SK). All examinations were performed by trained and experienced research staff. Dementia was diagnosed according to DSM-III-R , keepingStroke and transient ischemic attack (TIA) information was acquired from self-reports and key informants. Education, defined in years of education, was assessed by self-report or key informant information , 16.APOE (gene map locus 19q13.2) were genotyped using KASPar® PCR SNP genotyping system . Genotype data for these two SNPs were used to define ε2, ε3, and ε4 alleles [APOE genotype data was missing for five individuals.The single nucleotide polymorphisms (SNPs) rs7412 and rs429358 in  alleles , and APOLumbar punctures (LP) to collect CSF samples were performed in the L3/L4 or L4/L5 inter-space in the morning. The procedure has been described elsewhere , 16.1–42), specifically constructed to measure the 1–42 isoform of Aβ [CSF T-tau, P-tau, and Aβ42 were analyzed as part of clinical routine diagnostics . CSF T-trm of Aβ  was usedrm of Aβ .The A/T/N classification scheme was used to classify participants . ParticiAPOE ε4 carriership status, including the Bonferroni adjustment for multiple comparisons. We also performed pairwise comparisons between the ATN groups as a post hoc test, and the same covariates were included.The NfL and Ng variables were both skewed to different degrees, and they were both log-transformed (log-10) prior to statistical analysis. Spearman correlation analysis was used to analyze correlations between biomarkers. We also performed ANOVA  and ANCOVA (ANOVA with covariates) to analyze differences in means of log(NfL) and log(Ng) in the ATN groups. In ANOVA analysis we used ATN groups as an independent variable and log(NfL) level as a dependent variable, without covariates. The same procedure was used for log(Ng). ANCOVA was performed with log(NfL) and log(Ng) as a dependent variable, ATN groups as an independent variable, and the covariates age, sex, stroke, and p<0.05) was selected. Statistical analyses were performed in IBM SPSS Statistics for Windows A two-tailed significance level (APOE ε4 carriers. Eight participants (3.1%) had a history of stroke. The median NfL concentration was 724 pg/mL (IQR=384), and the median Ng concentration was 196 pg/mL (IQR=80) [The 258 participants of this study had a mean age of 70.6 (SD=0.3) years and 129 (50%) were female. Mean education was 13.1 (SD=3.9) years and 86 (33.3%) were heterozygote or homozygote (IQR=80) .p= 0.001). NfL also correlated with T-tau  and P-tau . NfL did not correlate with Aβ42 (rho=−0.06 p=0.4). Ng correlated strongly with T-tau  and P-tau  and weakly with Aβ42 . The distribution of the ATN groups among the whole group of cognitively normal participants has been described previously [Correlations between NfL, Ng, T-tau, P-tau, and Aβ42 are displayed in Fig. eviously , 31.Fig.Our sample with data on CSF NfL and Ng concentrations comprised 258 cognitively unimpaired participants who were distributed among the ATN groups as follows: A−T−N−: 138 (53.5%), A−T−N+: 49 (19.0) A−T+N+: 12 (4.7%) A+T−N−: 33 (12.8 %) A+T−N+: 20 (7.8%) A+T+N+: 6 (2.3%), while there were no participants with A−T+N− and A+T+N− , 31..Concentrations of CSF-NfL and CSF-Ng in the ATN groups are given in Table There were no other statistically significant differences.p=0.001) and A-T+N+ (p=0.006) had higher concentrations than the A-T-N- group  concentrations in the different ATN groups using t-tests, we found that the groups A-T-N+  concentrations, we found that those with A−T−N+, A−T+N+, A+T−N+, and A+T+N+  had higher concentrations of CSF Ng than A−T−N−, all 001 Fig. .p=0.002, F=3.98, DF=5) and log(Ng) .We performed an ANOVA that showed differences between the ATN groups in log(NfL) , when using age, sex, APOE ε4 carriership, and stroke as covariates in an ANCOVA. CSF log(Ng) concentrations also differed significantly between A/T/N groups , when using age, sex, APOE ε4 carriership, and stroke as covariates in ANCOVA.The CSF log(NfL) concentrations between A/T/N groups differed significantly (p<0.001) lower concentrations than all other groups except A+/T−/N− (p=0.25), and A+/T+/N+ had significantly (p=0.03 or lower) higher concentrations than all other groups except A−/T+/N+ (p=1.0) (Table p=0.036).In groupwise comparisons, most A/T/N groups differed significantly from each other in log(Ng) concentrations Table . A−/T−/N0) Table . N+ had −/N− p=0.5, and A+tly p=0.0 or lowerp=0.503). Participants with tau pathology had higher levels of CSF log(NfL) than participants without pathology .To analyze the effect of Aβ pathology, we compared A+ and A− groups (irrespective of tau status) in relation to their CSF NfL and Ng levels. Similarly, we compared T+ and T− individuals irrespective of Aβ status. There was no difference in CSF log(NfL) concentration between A+ and A− participants . However, those with tau pathology had higher levels of CSF log(Ng) than those without tau pathology .There were no differences in CSF log(Ng) levels in those with amyloid pathology compared with those without amyloid pathology (2.28 vs 2.29, In this study, we assessed CSF NfL and Ng in cognitively unimpaired older adults from the Gothenburg H70 Birth Cohort studies classified according to the A/T/N scheme  and accoThe canonical and scientifically well-backed-up sequence of events in AD development is laid out in the amyloid cascade hypothesis, stating that an imbalance in the production or clearance of Aβ is the instigating event in AD leading to the subsequent formation of amyloid plaques, tau tangles, and ultimately resulting in neuronal death . As Aβ pSynapse loss may play a role in neurodegeneration and perhaps cognitive decline in dementia. Synapse loss and dysfunction occur in many neurodegenerative diseases  and can We have chosen a study design where we examined the relationship between the ATN system and the biomarkers NfL and Ng, using T-tau as the “N” biomarker rather than NfL, although it could be argued that NfL could have been used in the N category instead. In our analysis we wanted to study the traditional ATN classification, with T-tau concentrations representing N, to enhance knowledge on the relationship between the newer CSF biomarkers NfL /Ng and the hallmark AD biomarkers . Further., stable MCI, vascular dementia, dementia with Lewy bodies, frontotemporal dementia, Parkinson’s disease dementia, and other neurodegenerative disease processes behave differently regarding biomarker patterns. It should also be noted that Aβ, T-tau, P-tau, and Ng are all known to be associated with AD pathology and each other, making it difficult to establish causal relationships between them. NfL, on the other hand, also shows an AD-independent association with neurodegeneration, making it slightly different than the other biomarkers, a result that agrees with earlier findings [Some of the strengths of the study include the population-based sample with CSF data from a relatively large number of individuals. However, there were a small number of participants in some of the ATN groups, which may lead to lowering of statistical power. Some ATN patterns are hard to explain in terms of AD pathology, A−T+N− for example, as individuals with no amyloid pathology normally do not present with NFTs. A possible explanation for this is that these participants might be misclassified due to biomarker assessment errors, which highlights another weakness of this study, namely the vulnerability of dichotomizing biomarker readouts into healthy and pathological classifications. However, as all biomarker measurements were carried out at the same world-renowned laboratory, by accredited personnel, and with well-proven commercial test kits this weakness is attenuated as far as possible. Furthermore, the lack of follow-up information indicating which types of pathology were developed by each participant is also a weakness of this study, e.gfindings .In conclusion, this study showed that concentrations of especially CSF-Ng but also CSF-NfL were higher in cognitively healthy subjects classified as N+ and/or T+ according to A/T/N criteria. This finding corroborates the literature proposing CSF NfL and Ng as early biomarkers of neurodegeneration and synaptic dysfunction in AD. However, further studies with longer follow-ups and detailed characterizations of symptom and biomarker trajectories are needed.


P. trituberculatus (PtCFSH) was identified, cloned and expressed in the recombinant form. The physiological functions of PtCFSH on the AG and testis were investigated using the RNAi and recombinant protein injection in a series of in vivo and in vitro experiments. It was confirmed that PtCFSH can negatively regulate the spermatogenesis and testicular development via a CFSH-IAG-testis endocrine axis. PtCFSH may also act directly on the testis, dependent or independent of IAG. The signaling system of PtCFSH involves the participation of cAMP, cGMP, and NO, which resembles the MIH signaling pathway. In summary, our results demonstrated the mode of CFSH action in P. trituberculatus.The crustacean female sex hormone (CFSH) plays a crucial role in the development of secondary sexual characteristics in female Decapoda crustaceans, but its physiological function in males is less reported. In the present study, the CFSH of CFSH in the eyestalk-AG-testicular endocrine axis has been inadequate. We cloned and identified a homolog of CFSH, PtCFSH, in this study. RT-PCR showed that PtCFSH was mainly expressed in the eyestalk. A long-term injection of dsPtCFSH and recombinant PtCFSH (rPtCFSH) in vivo showed opposite effects on spermatogenesis-related gene expression and histological features in the testis of P. trituberculatus, and was accompanied by changes in AG morphological characteristics and PtIAG expression. In addition, the phosphorylated-MAPK levels and the expression of several IIS pathway genes in the testis was changed accordingly in two treatments, suggesting that PtCFSH may regulate the testicular development via IAG. The hypothesis was further validated by a mixed injection of both dsPtCFSH and dsPtIAG in vivo. The following in vitro studies confirmed the negatively effects of PtCFSH on AG, and revealed that the PtCFSH can also act directly on the testis. Treatment with rPtCFSH reduced the cAMP and cGMP levels as well as the nitric oxide synthetase activity. These findings provide vital clues to the mechanisms of CFSH action in both the eyestalk-AG-testis endocrinal axis and its direct effects on the testis.It has been shown in recent studies that the crustacean female sex hormone (CFSH) plays a crucial role in the development of secondary sexual characteristics in Decapoda crustaceans. However, research on the function of  MrIAG expression in young Macrobrachium rosenbergii [IAG in Cherax quadricarinatu and Eriocheir sinensis to affect the male phenotype was further validated [The traditional view is that sexual differentiation in crustaceans is a male-dominated process. For example, in crustaceans, a male-specific androgenic gland (AG) determines male sexual differentiation and the maintenance of secondary sexual characteristics; after the removal of the AG, crustaceans undergo feminization ,2, and venbergii . This alalidated ,6. Thus,alidated .Khalaila previously suggested that the eyestalk-AG-testicular endocrine axis occurs in decapod crustaceans . AdditioMacrobrachium nipponense, both the gonadal inhibitory hormone (GIH) and molt inhibitory hormone (MIH) have a repressive effect on the expression of IAG genes [Callinectes sapidus, and is preferentially expressed in the eyestalk. It contains structures not found in other known neuropeptides, is considered unique to crustaceans, and has a regulatory role in secondary sexual characteristics [CFSH has also been identified in several species, including Sagmariasus verreauxi [C. quadricarinatus [Marsupenaeus japonicus [Scylla paramamosain [M. rosenbergii [Lysmata vittate [SpIAG versus SpCFSH in S. paramamosain in the eyestalk during the AG developmental cycle showed an opposite pattern [SpIAG. At the same time, a reciprocal inhibitory effect of LvCFSH1 with LvIAG in L. vittate was revealed [EsCFSH1 and EsIAG by in vivo injection into E. sinensis [Recently, several neuropeptides, such as the crustacean hyperglycemic hormone (CHH), the molt-inhibiting hormone (MIH), and the red pigment concentrating hormone (RPCH), have been found to be involved in crustacean development ,14. AmonAG genes .Moreovereristics . CFSH haerreauxi , C. quadarinatus , Marsupeaponicus , Scylla mamosain , M. roseenbergii  and Lysm vittate . Recentl pattern . Additiorevealed . The samsinensis .CFSH on IAG has been mostly described, the majority of reports on CFSH function are studied in females [L. vittate [E. sinensis [CFSH in males is also related to IAG. However, in L. vittate, the long-term RNA silencing of the CFSH fails to affect testicular maturation [CFSH is involved in the “eyestalk-AG-testis” endocrine axis. Therefore, the function of the CFSH in male crustaceans needs to be studied in more species, and its mode of action and molecular mechanism need to be more clearly elucidated.Although the negative regulatory effect of  females ,22,23. IPortunus trituberculatus is an economically essential crab species on the southeast coast of China, but the endocrine regulatory mechanism of its testicular development remains largely unclear. In this study, the homolog of the CFSH in P. trituberculatus (PtCFSH) was cloned and characterized. The function of PtCFSH in male P. trituberculatus was studied using a series of in vivo and in vitro experiments, based on the double-strand RNA-mediated RNAi technology and recombinant-CFSH treatments. We confirmed that the PtCFSH is involved in the “eyestalk-AG-testis” endocrine axis, and can also act directly on the testis.The swimming crab Portunus trituberculatus, which belongs to invertebrates and is a kind of crab. In China, ethical approval is not required for experiments on crabs. All of our crabs were purchased from the local aquatic market. Prior to dissection, we anesthetized the crabs on ice. All the experiments comply with the requirements of the governing regulation for the use of experimental animals in Zhejiang Province  and the Animal Care and Use Committee of Ningbo University. Healthy wild adult swimming crabs with a body weight of 100 to 140 g were purchased from the local aquatic market in Xianxiang, Ningbo. Some individuals in the intermolt period were temporarily reared in a capacious 120 L tank bottom containing 30 cm of fine sand and shelter in our laboratory; the crabs were fed and their water was changed periodically until the 20-day (long-term) and 3-day (short-term) treatment of RNAi and the in vivo recombinant proteins injection was completed. Other crabs were collected for gene cloning and in vitro treatment, respectively. All samples were stored in RNA preservation fluid  at −80 °C until RNA extraction.The experimental animal in this study is g for 10 min at 4 °C. The supernatant was sucked and stored at −80 °C for western blotting.In addition, the testis and AG-related tissues were sampled from the crab and cut into 3–5 mm pieces and fixed in 4% Paraformaldehyde Fix Solution for 24 h at 4 °C. Then, the tissues were dehydrated with serial ethanol, cleared with serial xylene, and finally embedded in paraffin. Tissues were cut into 5–7 μm sections for hematoxylin and eosin (H&E) staining. Next, 30 to 50 mg of testis tissue was placed in a 1.5 mL EP tube containing 300 microliters of PBS and  , after which the homogenate was centrifuged at 8000 ® reagent  according to the manufacturer’s instructions. RNA concentrations were determined using a NanoDrop 2000 UV Spectrophotometer . The genomic DNA removed with (10× gDNA Remover Mix) and the first strand of cDNA was synthesized using a HiFiScript gDNA Removal cDNA Synthesis Kit  according to the manufacturer’s protocol, followed by storage at −80 °C until use.Total RNA was isolated from different samples using the RNA-SolvCFSH; this was validated by reverse transcription polymerase chain reaction (RT-PCR) and elongated by the 3′ and 5′ rapid amplification of cDNA ends (RACE)  using cDNA generated from muscle-extracted RNA. Escherichia coli cells. After transformation, three positive clones were picked for sequencing . The amplified sequences from 3′ and 5′ RACE were assembled with the partial cDNA sequence using Vector NTI 10.0 software, and the open reading frame (ORF) was identified using ORF finder (http://www.ncbi.nlm.nih.gov/gorf/) (accessed on 22 January 2022). Predictions of the conserved domains were performed using the SMART program (http://smart.emblheidelberg.de/) (accessed on 22 January 2022). The 3D structure of the PtCFSH protein was predicted by the Alphafold2 web server  (accessed on 15 February 2022) and edited with the PyMOL Software (https://pymol.org/2/) (accessed on 27 February 2022). A multiple sequence alignment and phylogenic analysis was performed using ClustalW1.8 and MEGA7.0 (https://www.megasoftware.net/) (accessed on 28 February 2022).A keyword-based screening of our RNAseq library (unpublished) yielded a short transcript homologue of the PtCFSH in different tissues, primers PtCFSH-RTF/RTR (To detect the expression levels of -RTF/RTR  were desE. coli cells. The confirmed recombinant plasmids were extracted using the E.Z.N.A. Plasmid Mini Kit I (Omega Bio-Tek) and then transformed into E. coli Rosetta-gami cells to express the rGST-CFSH protein. The vector pGEX-4t-2 as a control was also expressed as the GST tag protein (rGST). The recombinant proteins were purified using the GST-tag Purification Resin  and verified three times by SDS-PAGE.The double-strand RNA for PtCFSH, PtIAG, and green fluorescent protein (GFP) were synthesized as previously described . The plaFor long-term treatments in vivo, the double-strand RNA of CFSH (dsCFSH) and the recombinant CFSH protein (rCFSH) were injected into crabs at 3 ug/g every three days for 20 days, with dsGFP and rGST serving as controls. The testes and AGs were collected to observe tissue changes. In the short-term RNAi treatment in vivo, dsCFSH, dsIAG, and dsGFP were all injected into different individuals at a dose of 3 ug/g dose, and samples were collected after 48 h. For the in vitro experiments, the desired eyestalks, AGs and testis tissues were all precultured in M199 medium for 1 h. Afterwards, dsCFSH and dsGFP were added at a dose of 3 μg/g dose [® Premix Ex Taq™ II Kit  according to the manufacturer’s instructions. PCR conditions were as follows: 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s and 56 °C for 20 s. Relative mRNA expression levels were normalized to β-actin mRNA expression [−ΔΔCt) method [We determined the relative gene expression levels using quantitative real-time PCR (qPCR). The template cDNA was prepared as described above and then amplified with qPCR primers . PCR waspression  and calc) method .For the Western blot assay, the protein concentration of the testis was measured using the BCA Protein Assay Kit . For SDS-PAGE, 30 mg of protein from each sample was used, followed by electrophoretic transfer to a 0.45 βm PVDF membrane using a Mini Trans-Blot . After blocking with Y-Tec Rapid Blocking Buffer  at room temperature for 1 h, the membranes were incubated with Phospho-p44/42 mos/mitogen-activated protein kinase (MAPK) (Erk1/2) or p44/42 MAPK (Erk1/2)  monoclonal antibodies diluted to 1:1000 in (Y-Tec Stabilizing Antibody Diluent)  overnight at 4 °C. The membranes were washed three times with TBST  and then incubated with β-Tubulin Rabbit IgG polyclonal antibody (CST) diluted to 1:2000 in Y-Tec Rapid Blocking Buffer  at room temperature for 1 h. After washing three times with TBST for 10 min each, the membrane was incubated in a Western Bright ECL substrate  before exposure to ChemiScope 6000 . The protein bands were quantified using the ChemiScope figure software package, and the results were derived from the statistical analysis of three independent experiments.−6 M) and forskolin (10−5 M) treatment (three replicates per treatment). The cAMP levels and the cGMP levels of the sampled testis at 5 and 15 min were measured using the cAMP ELISA kit  and cGMP ELISA kit , respectively. Samples collected after 6 h of treatment were used to detect changes in nitric oxide synthase (NOS) levels using a NOS-typed assay kit .The testis was collected at 5, 15 min and 6 h after rCFSH .At the end of the long-term RNAi and recombinant protein injection experimental period, crabs from four groups with a body width of 12.2–14.6 cm were collected. Testis were dissected from the individuals, cut into 3 mmThis hypothetical pathway was constructed with reference to the classical insulin pathway and the general neuropeptide signaling pathway . In thist-test or Tukey’s multiple-group comparison test. The data were analyzed using SPSS 24.0 software. Significant differences were accepted at p < 0.05. Asterisk denotes statistically significant differences: * p < 0.05; ** p < 0.01; *** p < 0.001.All data are expressed as mean ± SEM and were subjected to a one-way analysis of variance (ANOVA), followed by Student’s PtCFSH cDNA (Genbank accession number: ON929327) comprising a 19 bp 5′-untranslated region (UTR), 678 bp ORF, and 542 bp 3′-UTR with a poly (A) tail (We obtained a 1241 bp (A) tail . A seque(A) tail A,B. The (A) tail C.PtCFSH were measured in different tissues obtained from the adult male. It was demonstrated by these results that the PtCFSH transcripts were mainly expressed in the eyestalk, with a slight expression in the brain and testis , ATP-dependent RNA helicase (Vasa), CyclinB and cyclin-dependent kinase-2 (Cdc-2) [PtCFSH silencing  , was redilencing B. The coIAG itself, insulin-like growth factor banding protein-related peptide (IGFBP-rp), insulin-like hormone receptor 1 (IR1), IR2, protein kinase B (Akt), mammalian target of rapamycin (mTOR), and forkhead transcription factors (Foxo) [PtCFSH silencing  ,31, weres (Foxo) , the chailencing B,C. TherIAG and insulin pathway genes in the testis were down-regulated  as their membrane receptor, and the signaling system needs the participation of second messengers . To explCFSH gene sequence of P. trituberculatus, encoding 225 amino acids, including the signal peptide, a cleavage KR site, and an IL-7 structural domain containing eight cysteines, was investigated in this study. It was shown that it has the same structural organization as type I CFSHs. Multiple sequence comparisons and a phylogenetic tree analysis supported that PtCFSH belongs to type I CFSHs . Thus, the CFSH may play a more extensive role in decapod crustaceans [The CFSH is a primary hormone involved in developing relevant phenotypes in female crustaceans ,22,23,33 I CFSHs . Most tyntiation ,22,33. Istaceans ,21.PtCFSH in male P. trituberculatus was investigated in a series of in vivo and in vitro experiments, based on the dsPtCFSH RNA silencing and rPtCFSH treatment. Consistent with the reported negative regulation of IAG by CFSH [PtCFSH silencing and rPtCFSH treatment both suggested that PtCFSH can significantly inhibit the expression level of PtIAG in vitro and in vitro. Furthermore, the histological study on the AG showed that PtCFSH had a negative effect on the number of IAG-producing cells. This would be reminiscent of the bilateral eyestalk ablation of the P. pelagicus that caused an increase in the cells producing IAG [Macrobrachium nipponense, RNAi in several eyestalk neuropeptides revealed that the gonad-inhibiting hormone (GIH) and molt-inhibiting hormone (MIH) had an inhibitory effect on IAG expression [In the present study, the physiological role of  by CFSH ,23, PtCFcing IAG . In Macrpression . Our reskifc-1, vasa, cyclinB, and cdc-2 [P. trituberculatus. The effects of PtCFSH on testis might be achieved through IAG, since several classical insulin pathway genes, including IAG itself, insulin-like growth factor binding protein-related peptide (IGFBP-rp), insulin-like hormone receptor 1 (IR1), IR2, protein kinase B (Akt), mammalian target of rapamycin (mTOR), and forkhead transcription factors (Foxo) [PtCFSH silencing and simultaneously reduced by rPtCFSH. As one of the two major intracellular pathways in the conserved insulin signaling system, the MAPK pathway mainly controls development and reproduction [It was revealed in our in vivo studies that rPtCFSH injection could reduce the conversion of spermatocytes to spermatids F,G, whilnd cdc-2 ,39, chans (Foxo) ,31,40,41oduction . TherefoIAG expression, dsPtCFSH was also injected in the short-term experiments, with the purpose of causing a transient increase in PtIAG expression; this obtained opposite effects to the PtIAG silencing. In fact, the short-term silencing in PtCFSH confirms the regulatory relationship between PtCFSH with PtIAG and related genes, which had been revealed in the long-term experiments. The co-injection of both dsPtCFSH and dsPtIAG showed similar effects on the testis to the single injection of dsPtIAG; it could be understood that the regulatory effect of dsPtCFSH on the testis was blocked after the injection of dsPtIAG, demonstrating the central role of IAG in connecting the CFSH and testis.To confirm the existence of the CFSH–IAG–testis endocrine axis, a short-term injection of dsPtIAG was further performed. It was found that this treatment reduced the levels of gene expression and MAPK phosphorylation. Given the inhibitory effect of PtCFSH on PtIAG and insulin pathway gene expression in the testis, as well as the MAPK phosphorylation. Although the presence of IAG in testis has been less reported, it was revealed by our previous study that PtIAG is expressed in the testis of P. trituberculatus [In addition to the indirect effects of PtCFSH on the testis, the in vitro treatment of rPtCFSH on the testis explants suggested that the PtCFSH was able to act directly on the testis. Interestingly, however, this direct effect might also be related to the IAG. Our results showed that rPtCFSH can also inhibit the levels of rculatus . Since trculatus ,45,46, trculatus .2+ pathway, a “summation phase” involving the NO/cGMP pathway, and the calmodulin (CaM), which links the two phases by activating NOS [To exert its physiological functions, whether on the AG or the testis, the CFSH must have its own set of signaling mechanisms. As a neuropeptide, GPCR is a primary candidate for the CFSH receptor . Howeverting NOS . It is lP. trituberculatus CFSH was identified, cloned and expressed in the recombinant form. The physiological functions of PtCFSH on the AG and testis were investigated using the RNAi and recombinant protein injection in a series of in vivo and in vitro experiments. It was confirmed that PtCFSH can negatively regulate the spermatogenesis and testicular development via a CFSH–IAG–testis endocrine axis. PtCFSH may also act directly on the testis, dependent or independent of IAG. The signaling system of PtCFSH involved the participation of cAMP, cGMP, and NO, which resembles the MIH signaling pathway. In order to summarize, a hypothetical pathway for the mode of the CFSH action in P. trituberculatus was constructed by figdraw (In conclusion, the  figdraw .


External ventricular drains (EVDs) have been used for acute relief of hydrocephalus and also for reliable monitoring of intracranial pressure and are thus one of the most common neurosurgical interventions. An EVD can be inserted almost regardless of the cause of the hydrocephalus, for example, in subarachnoid hemorrhage (SAH), in intraventricular hemorrhage, in brain tumors, in shunt failure, or in the case of a stroke. However, any EVD insertion is associated with the risk of infection, with typical ventriculostomy-related infection (VRI) rates of approximately 10% described . HoweverCSF samples are typically drawn at the proximal port closest to the head see Fig. . The CSFThis prospective, monocentric study in two intensive care units  at the tertiary care hospital of the University of Munich, Ludwig Maximilian University, was approved by the responsible local Ethics Committee . Written informed consent was given by all patients or their legal representatives. Withdrawal of diagnostic samples from the proximal port of the VentrEX EVD  was synchronized with CSF supernatant collection from a distal port . The overflow reservoir was first completely emptied, and the CSF supernatant was withdrawn after a fill state of 2 ml. All CSF samples were taken by foot to the clinical laboratory for immediate analysis. The measurement results were compared by applying graphical  and statistical evaluation methods (Lins concordance of correlation coefficient [CCC]) with the R programming language (version 4.0.2) using the R package DescTools. A CCC of > 0.8 demonstrated that diagnostic CSF sampling from the proximal and distal reservoir ports are analytically equivalent [A total of 20 patients were included in the study , with up to two samples per patient being subjected to the comparative measurements . The median age of the patients was 56 years, and 15 patients suffered from SAH, 2 patients from postoperative hydrocephalus, and 1 patient each from intracerebral hemorrhage, subdural empyema, and traumatic brain injury. The agreement of the comparative measurements is illustrated in goodness-of-fit plots in Fig. None of the biochemical laboratory parameters showed a structural deviation from the line of identity over the investigated concentration range. In accordance with the graphical results, the numerical results revealed a CCC ≥ 0.88 for all biochemical analytes indicating a good agreement between the direct sampling from the proximal port and the analysis of the supernatant obtained from the distal port below the collection chamber (see Table In contrast, comparative measurements for cellularity had a very low correlation between the proximal and distal port sampling. The red and white blood cell counts were significantly lower in supernatant samples when compared with the proximal counterpart. In addition, the quality of supernatant cytology smears was insufficient for morphological workup. Given the good agreement of NSE measurements, profound in vitro hemolysis and white cell lysis are unlikely. We therefore assume that CSF-repellant properties of the CSF filter system that are supposed to prevent ascending infections do cause an inconsistent downstream drainage of cells into the overflow system. In addition, intraventricular gravity sedimentation of cells could also affect passive drainage of cells into the overflow chamber. Further statistical or graphical comparisons were therefore avoided. Due to the installed filter, our results are not directly comparable to the results by Wong , who fouThe decisive aspect to be answered remains whether, and if so, in which indication, analysis of the supernatant from the distal overflow system may replace direct sampling from the proximal port in clinical practice. For reliable assessment of neurodestruction, analysis for both NSE and protein S100 was feasible in our study. Ferritin, a sensitive diagnostic parameter for detection of suspected SAH, was also reliably quantified from CSF supernatant. According to the Centers for Disease Control and Prevention criteria of ventriculitis, total cell count, glucose, and total protein concentration define the laboratory findings of ventriculitis. In addition, high lactate concentrations are commonly used as a surrogate parameter for developing ventriculitis. Based on our study results, it can be stated that glucose, lactate, and total protein can be reliably determined. In addition to these typical “ventriculitis markers,” IL-6 in CSF could be determined from the supernatant and was previously included in a prediction model for drain-associated ventriculitis and might play an increasing role in the future . HoweverThis study demonstrates that CSF supernatant from the overflow system can be used for diagnostic workup with various biochemical parameters that are requested in several clinical conditions. Our findings indicate that other noncellularity parameters may also be assayed from CSF supernatant, for example, small molecules for therapeutic drug monitoring purposes. Harnessing CSF from the collection chamber may also come in handy for clinical studies, without the need of sampling from the proximal port. It should be noted, however, that this study was conducted with a rather small patient cohort. Larger studies are therefore required to elucidate and confirm our preliminary findings. As with any diagnostic procedure, CSF supernatant analysis from the overflow system must be validated separately for different EVD systems and preanalytical variables at each medical center.


While there has been an increase in research on Chinese as a foreign language (CFL) teachers’ identity worldwide, limited attention has been drawn to CFL teachers’ positioning and affordances to interpret their identity construction in an overseas context. To fill the gap, this study investigated seven novice CFL teachers’ identity construction as Chinese language teachers in primary schools in New Zealand from positioning and affordance perspectives. Retrospectivesemi-structured interviews were adopted to understand how the novice CFL teachers were positioned, how they positioned themselves, and what affordances they perceived to be influential to their Chinese language teacher identity construction. The findings showed that the novice CFL teachers’ identity construction was subject to the social, institutional, and individual levels of being positioned, self-positioning, and affordances. Specifically, (1) consistent self-positioning at the social, institutional, and individual levels could largely determine the novice CFL teachers’ identity construction; (2) inconsistency of identities between being positioned and self-positioning at the social, institutional, and individual levels might weaken the novice CFL teachers’ identity construction; (3) affordances as opportunities at the social, institutional, and individual levels could strengthen the novice CFL teachers’ identity construction, whereas affordances as challenges could not. The study concluded with implications and limitations to inform future research. In an era characterized by internationalization and multilingualism, and in a global economy in which China has the second largest share of the world GDP, Chinese as a foreign language (CFL) learning has gained significant popularity around the world. Taking New Zealand as an example, Chinese has become the fastest-growing foreign language (L2) taught in both schools and universities . To suppAmong the recruited volunteer CFL teachers, most of them are novice in overseas Chinese language teaching. In other words, they may have little experience in terms of how to fit themselves in a different educational system, how to handle relationships with students, teachers, and school leaders in a cross-cultural context, and how to exploit resources and affordances to adapt their teaching to the local students. All the above may influence novice CFL teachers’ identity construction as Chinese language teachers and thus their future commitment to this profession. Since teachers’ identity construction and formation is a continuous process of interpretation and reinterpretation of their personal and professional experiences entrenched in complex social contexts, it is necessary to take teachers’ living and teaching experiences in their situated contexts into account to examine their teacher identity development.Teacher identity has emerged as a critical construct for examining teachers’ professional lives, teaching quality, teaching motivation, and career decision-making . A signiThe present study, therefore, examined novice CFL teachers’ identity construction as Chinese language teachers in primary schools in New Zealand from positioning and affordance perspectives. It was hoped that implications and insights could be drawn to inform schools and teacher training programs to better support novice CFL teachers’ teacher identity construction in an overseas context. The present study was guided by the following overarching research question (RQ).RQ: How do novice CFL teachers’ lived and teaching experiences as mandarin language assistants contribute to their Chinese language teacher identity construction in primary schools in New Zealand?Teacher identity refers to teachers’ understanding of themselves and their relationships with others in their situated communities. However, “teacher identity is not something that is fixed nor is it imposed; rather it is negotiated through experience and the sense that is made of that experience” , p. 15. To examine the complex and multifaceted nature of teacher identity, different theoretical lenses have been adopted over the decades, including the social identity theory , the theThis study, therefore, employed positioning and affordance as an integrated analytical lens to understand novice CFL teachers’ identity construction as Chinese language teachers in primary schools in New Zealand. Specifically, interactive positioning  and reflexive positioning , along with affordances  that accompanied by positions, were collected for the examination.Recent research in the field of language teacher education has witnessed a growing interest in the construction and formation of language teacher identity . For exaIn line with the EFL teacher identity literature, research on CFL teacher identity suggests that these teachers’ identity construction derives from their social engagements and linguistic practices in complex activity systems . For exaAlthough previous research has evidenced the important role of social engagements and linguistic practices in shaping language teacher identity, findings of CFL teachers’ identity in cross-cultural contexts have been revealed to be divergent. On the one hand, some studies found that CFL teachers’ identity tended to be authoritarian and teacher-centered under the influence of Confucianism e.g., . On the While there is a growing interest in CFL teachers’ identity, our understanding of this group of teachers’ identity construction from the positioning perspective is limited. Positioning can be simply understood as “assigning an identity position to one’s self or to an other” , p. 36. Informed by interactive and reflexive positioning, this study attempted to understand how novice CFL teachers were positioned as Chinese language teachers in primary schools in New Zealand and whether such being positioned teacher identities were in line with their self-positioning as Chinese language teachers. If not, how the disparities between being positioned and self-positioning may influence novice CFL teachers’ identity construction as Chinese language teachers in primary schools in New Zealand.The concept of affordance was first proposed by Regardless of the different types of affordances, the affordances embedded in the community are the same for all teachers. However, teachers may vary in utilizing affordances “due to disparities in their prior education experiences, knowledge, and understanding, a perceived sense of agency in taking control of teaching, identity recognition, and personal goals in work” . For exaAlthough the concept of affordance has been recognized as an influential ecological approach to teacher education, previous research mainly focuses on technology-assisted language instruction  and multThis qualitative case study was carried out to examine novice CFL teachers’ identity construction as Chinese language teachers in primary schools in New Zealand from positioning and affordance perspectives. Seven pre-service CFL teachers from China were randomly recruited for the present study. The seven participants were all novice female CFL teachers. According to the literature, teachers with less than 5 years of teaching experience fall in the novice category . These nTo become mandarin language assistants in New Zealand, candidates should hand in their application to Hanban. Hanban then selects strong candidates for final interviews. All the successful candidates are required to attend one to two months of training to equip them with essential knowledge and skills for teaching and living abroad. When candidates arrive and settle down in New Zealand, they will be informed by the Confucius Institute in terms of which school or schools they are going to teach. In New Zealand, primary school teachers teach a wide range of subjects such as arts, English, maths, and science in their own fixed classrooms. Given that mandarin language assistants are not fully registered teachers in New Zealand, they do not have their own classrooms for Chinese language teaching. Instead, they have to move between different classrooms with classroom teachers sitting in class to help classroom management if necessary.Data were collected through retrospective semi-structured interviews after obtaining ethical approval and participant consent. The seven participants were interviewed individually according to their schedules. The interviews ranged from 60 to 80 minutes. During the interviews, the first author asked casual questions to establish a rapport with the participants first. Afterwards, the first author asked a wide variety of open-ended questions designed to elicit information on how they positioned themselves and how they were positioned by others, as well as what external resources, support, and challenges influenced their identity construction in primary schools in New Zealand see .The interviews were organized chronologically. The first section collected the early experiences of the participants in order to determine what led them to the CFL teaching career. The second section focused on their positioning and affordances during their Chinese language teaching in primary schools in New Zealand. The third section centered on how their positioning and affordances influenced their identity construction as Chinese language teachers. The last section of the interview elicited the participants’ future commitments. All the retrospective semi-structured interviews were carried out at the end of their first-year teaching in New Zealand. The interviews were conducted in the participants’ native language (Chinese) in order to elicit in-depth information about their “experiences, memories, and their developing professional selves” .After the data collection, the first author transcribed all the audio-recorded interviews and returned the transcripts to the participants for their verification and clarification. The transcripts were then analyzed line by line by the second author through a systematic qualitative inductive approach . InitialTo enhance the trustworthiness of the data analysis, the rest authors double-checked the extracted codes, categories, and themes by the second author. When disagreement arose, the authors engaged in a discussion to reach a consensus. Since the transcripts were in Chinese, the excerpts presented in the findings were then translated into English. To preserve the original idea and avoid potential loss of meaning, the authors carefully examined back and forth the selected excerpts and their English translations.social level of being positioned, Ruby). Such an identity was further enhanced by the training sessions offered by Hanban for the novice CFL teachers before they were dispatched to New Zealand to teach Chinese . For example,Before joining as mandarin language assistants or Chinese language teachers in New Zealand, the participants were all majoring in Teaching Chinese to Speakers of Other Languages (TCSOL) in China. Their identities were “positioned by the society in China as Chinese language teachers serving as a cultural bridge between the East and the West” I have learned much cultural stuff from Hanban’s training, such as tea culture, martial arts, and paper cutting. The training is quite informative. I do not think I would have had the opportunity to learn these skills and knowledge if Hanban did not provide the training. Because of the training, I consciously try to embed cultural elements in my Chinese language teaching. .Although the cultural bridge or cross-cultural facilitator was an identity attributed to them by the Chinese society, all the novice CFL teachers tended to take up this identity position. As evidenced in the interviews, the participants pointed out that they were “honored to be on the mission not only to teach Chinese language but also to help learners understand Chinese people, Chinese culture, and Chinese society” .While Hanban’s training serves as a supportive social affordance for novice CFL teachers in terms of how to teach Chinese abroad, the training has some drawbacks. Firstly, the training is too general to include country-specific sessions. As a result, teachers who majored in TCSOL may find the training superficial. Secondly, the training mainly focuses on how to teach college students Chinese. Consequently, teachers who are assigned to primary or secondary schools may find the teaching theories, approaches, and contents offered by Hanban inappropriate for young learners. As Alice recalled, “the training is very general. In fact, it is not so suitable for New Zealand’s education, but it is better than nothing.” . Although homestay, to some extent, enhanced the cross-cultural communication between the novice CFL teachers and the local family, it made some of them feel self-conscious. For example,After the novice CFL teachers arrived in New Zealand, the Mandarin Language Assistants program helped the novice CFL teachers find a homestay to support their social and cultural adaptation to the new environment The family hosts not only me but also an American. Because they [the host family and the American] share similar cultures and use the same language, they can talk to each other for a long time. Normally, I just keep quiet and listen to their conversation most of the time. I do not know what to talk about and what to share with them. This is a problem for me and the biggest challenge in my life. I want to improve it but feel hopeless. . In the pre-service training, the program introduced the education system and teaching mode of New Zealand to the novice CFL teachers. In the mid-term training, the program gathered the novice CFL teachers together for discussion and reflection on their Chinese language teaching in New Zealand. According to the novice CFL teachers, they found the former training “informative” and the latter training “beneficial but insufficient” .Apart from homestay support, the Mandarin Language Assistants program also organized two training sessions for novice CFL teachers, namely pre-service training and mid-term training I find the two training sessions helpful and necessary. The pre-service training provides us with an overview of New Zealand education and shows us the local teaching approaches and styles. It helps me quickly adapt to the local education system and become a local teacher here. The mid-term training offers us an opportunity to discuss teaching difficulties and share effective teaching techniques with each other. However, the mid-term training is only once a year. More sessions of such kind can help us become more competent Chinese language teachers. . According to the regulations of the New Zealand Ministry of Education, only those who have obtained local teaching certificates are qualified to teach in primary or secondary schools in New Zealand. In other words, these novice CFL teachers cannot teach alone until licensed to teach in New Zealand. Therefore, when they teach in schools, a local teacher should stay in class for supervision. Institutionally being positioned as “teaching assistants” or “illegitimate teachers,” to some extent, shaken the novice CFL teachers’ Chinese language teacher identity construction in primary schools in New Zealand. For example,During their teaching in primary schools in New Zealand, the novice CFL teachers realized that they were positioned by schools as language teaching assistants rather than regular teachers, given that they were not registered teachers in New Zealand I am confused about my position in my school as a teaching assistant or a teacher. The school may be unconcerned with my status [position], but the ambiguity of my position in the school affects my mood. It makes me question whether I am a member of this school and whether I am entitled to the same rights of expression and so forth. . Some novice CFL teachers also “believed that [their] efforts in Chinese language teaching in schools have changed the schools’ positioning of [them] from being assistants to teachers” .Although the novice CFL teachers felt perplexed due to being positioned by the schools as assistants, outsiders, and illegitimate teachers, the teachers challenged this idea and positioned themselves as professionally trained Chinese language teachers. As Helen recalled, “I am no less valued than the local teachers. I am a professional Chinese language teacher. What I teach will benefit children’s future growth” . The pre-service observation is usually one-week long. Through this affordance, novice teachers can learn from experienced teachers the methods of classroom management and familiarize themselves with the learning habits, styles, and characteristics of students with different ethnic backgrounds. As Olivia recalled, “observing local teachers’ classes helped me a lot because I gained an understanding of what strategies I can use to educate the local students, to attract their attention, and to maintain discipline in class” .In terms of the institutional level of affordances, “schools normally arrange classroom observations for [the novice CFL teachers] to help [them] get the first-hand understanding of the local teaching environment and classroom features” before they take their role as Chinese language teachers . Nevertheless, most novice CFL teachers felt awkward during the breaks. As some recalled, “when I am in the staff room for a morning tea break, I do not know what I can talk to my colleagues. I do not know how to chat with them” . The local teachers, on the other hand, “just think there is a new colleague. They do not pay attention to you” . “They just do not care to talk to you” .Schools also offered morning tea breaks, allowing teachers to mingle and build rapport  left these teachers feeling vulnerable. With no reference for Chinese language teaching, these novice CFL teachers had to design their own lesson plans and teaching materials. The struggling situation, to some point, waned these teachers’ self-efficacy and made them anxious about deciding what and how to teach Chinese to their students. For example,Although classroom observations and morning tea breaks were offered to help the novice CFL teachers adapt to the new environment, the lack of standardized curriculum and syllabus for Chinese language in New Zealand There is no textbook or curriculum I can refer to, and there is no one I can talk to in terms of Chinese language teaching. I am the only Chinese teacher in my school. This is the case for most schools. Without a curriculum or a syllabus as a guide, I am a bit lost. I have to design my lesson arbitrarily depending on my own preferences. It would be better if there were any achieved documents for reference . As Olivia pointed out, “I do not think my colleagues see me as a regular teacher but rather a trainee teacher. They are unwilling to engage in deep conversations. To them, I am a random person who will go back to China after just one or two years” . Similarly, other novice CFL teachers found that they were positioned by the school as “tools” and “teaching assistants” .Teacher identity is established through negotiating different positions . In the individual level of self-positioning, Tina) and a “cross-cultural promotion ambassador”  between China and New Zealand. As Jane and Emma pointed out, “through our teaching, students may understand Chinese language, culture, and people better. It is meaningful to teach Chinese abroad” . “The profession [as a Chinese language teacher] is a great cause to introduce and promote Chinese language and culture worldwide. I am eager to attract more people to learn Chinese” .Although being positioned by their colleagues as temporary helpers rather than teachers was discouraging, the novice CFL teachers did not lose their identity as professionally trained Chinese language teachers. In effect, they were proud to be “Chinese teachers” and identified themselves as a “cultural bridge” . These novice CFL teachers were also positioned as friends by their students over time due to their strengthened bond. As Alice recalled, “I am more than a teacher to my students now. I am a friend in my students’ eyes who can play with them and teach them some Chinese” . However, the friend identity caused classroom management issues for the novice CFL teachers, as “students may not follow instructions accordingly” .Apart from colleagues, students also played a role in the novice CFL teachers’ self-positioning. Specifically, the novice CFL teachers expressed that they were respected and liked by their students. This, in turn, “motivated [them] to commit more to [their] Chinese language teaching” (Tina). The above positive relationship enabled the novice CFL teachers to “gain a great sense of accomplishment and satisfaction from being a Chinese language teacher”  and being positioned  can help make sense of how different positions experienced by teachers may influence their teacher identity construction . SpecifiThe above positioning findings also support the literature that positioning is relational; can shape teachers’ agency and identity; and can be beneficial to learning and teaching . For insIn terms of affordances in teacher identity construction, the present study showed that affordances, such as the social level of teacher training and the institutional level of classroom observation, helped the novice CFL teachers adapt to Chinese language teaching in primary schools in New Zealand. This, in turn, enhanced the novice CFL teachers’ identity construction as professionally trained Chinese language teachers rather than teaching assistants or illegitimate teachers in New Zealand. Nevertheless, there were also circumstances that challenged the novice CFL teachers’ identity as professionally trained Chinese language teachers. For example, the lack of the institutional level of affordances, such as referable syllabus and appropriate textbooks, made the novice CFL teachers feel anxious and vulnerable. Additionally, the lack of the individual level of affordances, such as students’ classroom cooperation, led the novice CFL teachers to question their classroom management competence and thus shaken their identity construction as professionally trained Chinese language teachers.The above affordance findings support To support novice CFL teachers’ identity construction as Chinese language teachers in overseas contexts, schools and teacher training programs may take the following suggestions into consideration. Firstly, it is important to equip novice CFL teachers with solid professional knowledge and diverse teaching skills to help them establish concrete self-positioning such as professionally trained teachers. For example, schools and teacher training programs may consider opening a course tailored for prospective CFL teachers in terms of how to teach Chinese overseas. Schools and teacher training programs may also consider inviting veteran CFL teachers to share their overseas teaching experiences and tips to strengthen their overseas teaching ability.Secondly, it is important to create an inclusive and welcoming environment  for noviLastly, it is advised to enhance novice CFL teachers’ awareness of affordances so as “to enable them to profit maximally from the resources at their command” , p. 83. Summing up, this study reports on the novice CFL teachers’ identity construction in primary schools in New Zealand from an integrated lens of positioning and affordance. The findings show that the novice CFL teachers’ identity construction can be subject to the social, institutional, and individual levels of being positioned, self-positioning, and affordances. Specifically, (1) consistent self-positioning at the social, institutional, and individual levels could, to a large extent, determine the novice CFL teachers’ identity construction; (2) inconsistency of identities between being positioned and self-positioning at the social, institutional, and individual levels might weaken the novice CFL teachers’ identity construction; and (3) affordances as opportunities at the social, institutional, and individual levels could strengthen the novice CFL teachers’ identity construction, whereas affordances as challenges could not.There are some limitations of the present study. First, the participants of the study are all females with a similar educational background. The findings of the study, therefore, may not be generalizable to other samples. Future research may consider examining other types of CFL teachers, such as novice in-service teachers, experienced in-service teachers, and non-TCSOL major teachers, to obtain a more comprehensive understanding of how positioning and affordances may influence CFL teachers’ identity construction. Second, the findings of this single sourced qualitative study may not be convincing. Other qualitative data collection methods, such as focus groups and naturalistic observation, could have been adopted to cross-validate the findings of the present study. Lastly, this study focuses on novice CFL teachers’ identity construction from positioning and affordance perspectives. Future research is advised to carry out comparative studies to investigate whether there are any disparities between different types of CFL teachers’ identity construction as Chinese language teachers in an overseas context.luapnus@zju.edu.cn.The datasets presented in this article are not readily available because the raw data supporting the conclusions of this article may be requested from the first author. Requests to access the datasets should be directed to PS, The studies involving human participants were reviewed and approved by the University of Auckland Human Ethics Committee (014726). The patients/participants provided their written informed consent to participate in this study.PS contributed to the data collection, data analysis, and manuscript writing. YW contributed to data analysis and manuscript writing. YL contributed to the manuscript review. ZL contributed to the data collection. All authors contributed to the article and approved the submitted version.The study was supported by the Research Project on International Chinese Education (Grant no. 20YH05D) and the Fundamental Research Funds for the Central Universities in China.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.