Association between Iodized Salt Intake and Cognitive Function in Older Adults in China.

OBJECTIVES: To investigate the association between iodized salt intake and cognitive function in older adults. DESIGN: A cross-sectional study. SETTINGS: Individuals from the Zhejiang Major Public Health Surveillance Program (ZPHS). PARTICIPANTS: Data of 10,217 participants (including 4,680 coastal residents and 5,537 inland residents) aged ≥ 60 years were analyzed. MEASUREMENTS: Salt intake was evaluated using a questionnaire, and participants were stratified into the following three groups: iodized salt, non-iodized salt, and mixed salt. Cognitive function was assessed through the Mini-Mental State Examination and defined using education-specific cut-off points. Logistic regression models controlling for an extensive range of potential confounders were generated to examine the association between salt intake and cognitive function among all participants. RESULTS: Data from 10,217 participants with a 16.1% prevalence of cognitive impairment were analyzed. Compared with non-iodized salt intake, consumption of iodized salt was inversely associated with cognitive impairment (odds ratio [OR], 0.410; 95% confidence interval [CI], 0.351-0.480; P < 0.001) in all participants after multivariable adjustment. An association between iodized salt intake and cognitive impairment was observed in coastal (OR, 0.441; 95% CI, 0.340-0.572; P < 0.001) and inland residents (OR, 0.569; 95% CI, 0.439-0.738; P < 0.001). Despite the insufficient sample size, the results for individuals consuming mixed salt suggested an inverse association between mixed salt intake and cognitive impairment among coastal residents (OR, 0.598; 95% CI, 0.405-0.885; P = 0.010) after multivariable adjustment. CONCLUSION: Our results indicate that iodized salt intake may reduce the risk of cognitive impairment in older adults living in coastal or inland areas, and the protective effect of iodized salt intake is greater in coastal areas than in inland areas.

[Expression of tuftelin protein and its clinical value in HBV-related liver cancer tissues].

Objective: To analyze the expression of tuftelin protein (TUFT1) and its clinical value in hepatocellular carcinoma (HCC)-related liver cancer tissues. Methods: The biological information data of TUFT1 mRNA expression in liver cancer and non-cancer tissues were analyzed from the TCGA and Oncomine database. After the approval of the ethics committee, the self-pairing method was used to collect the postoperative cancer and para-carcinoma tissues of 132 HCC cases hospitalized between January 2009 and December 2014. Tissue microarray and immunohistochemistry (IHC) were used to analyze the expression of TUFT1 in liver tissues. According to IHC staining, liver cancer was divided into high TUFT1 and low/no expression group. Combined with clinical data, the clinicopathological characteristics were statistically analyzed between and within the groups. The 5-year overall survival (OS) and disease-free survival (DFS) was analyzed by correlation analysis. Results: IHC staining showed that TUFT1 in cancer tissue was localized in the cytoplasm and cell membrane, and its positive expression rate was significantly higher in the liver cancer group (87.1%) than the para-carcinoma group (64.4%) (χ (2) = 18.563, P < 0.001). TUFT1 expression intensity in patients with liver cancer was significantly correlated with HBeAg positive (χ (2) = 4.080, P = 0.043), tumor size (χ (2) = 9.388, P = 0.002), vascular invasion (χ (2) = 14.885, P < 0.001), TNM stage (χ (2) = 13.516, P < 0.001) and ascites (χ (2) = 5.940, P = 0.015). TUFT1 high expression was negatively correlated with OS and DFS (P < 0.001). Conclusion: The overexpression of TUFT1 is closely related to HBV replication, vascular invasion and poor prognosis, and it is expected to become a useful marker for liver cancer diagnosis and prognosis.

[Therapeutic efficacy analysis of immunotherapy in small cell lung cancer].

Objective: Recently, increasing number of lung cancer patients benefit from immune-checkpoint inhibitors (ICIs). However, the data of Chinese small cell lung cancer (SCLC) patients is limited. This study aims to analyze the response and survival data of ICIs treatment in SCLC and to explore the predictive biomarkers. Methods: Forty-seven SCLC patients who received ICIs treatment from Peking University Cancer Hospital from May 2017 to September 2019 was recruited. Clinical characteristics including sex, age, smoking status, ICIs strategy, PD-L1 expression and therapeutic efficacy were collected to explore the clinical predictive biomarkers for SCLC ICIs treatment. Results: Among the 47 patients, 18 (38.3%) cases were partial repose (PR), 11 (23.4%) were stable disease (SD), 18 (38.3%) were progressive disease (PD), and the objective response rate (ORR) was 38.3%, disease control rate (DCR) was 61.7%, the median progression-free survival (PFS) was 5.3 months. ICIs monotherapy accounts for 27.7%, the ORR was 15.4%, DCR was 53.8%, median PFS was 2.7 months. Combined therapy accounts for 72.3%, the ORR was 47.1%, DCR was 64.7%, median PFS was 5.4 months. Fourteen (29.8%) patients received ICIs as the first line treatment, their ORR was 85.7%, DCR was 100%, median PFS was 9.1 month. The ORR was not related to the age, sex, body mass index (BMI), smoking status and programmed death-ligand 1 (PD-L1) expression (P>0.05). The ORRs were higher in patients underwent PD-L1 monotherapy (P=0.001), combined therapy (P=0.002) and received ICIs as the first line treatment (P<0.001). Log-rank analysis indicated that the PFS of female patients were 12.0 months, significantly longer than 4.4 months of male patients in ICIs treatment (P=0.038). Patients who received PD-L1 monotherapy, combined treatment, or ICIs as the first line treatment had longer PFS than their counterparts, though no statistical significant was observed (P>0.05). Cox multivariate analysis showed that, the gender was not an independent predictor for PFS in ICIs treatment (HR=3.777, 95%CI=0.974~30.891, P=0.054). Conclusions: Immunotherapy is an effective treatment strategy for SCLC. Patients who receive combined ICIs treatment, first line ICIs treatment and PD-L1 treatment may get greater benefits. PD-L1 expression cannot predict the response and PFS in SCLC ICIs treatment.

[Hepatocellular carcinoma-derived exosomal miRNA expression and its clinical value].

The early diagnosis and effective treatment of hepatocellular carcinoma (HCC) still remains a difficult problem that plagues the medical community. Exosomes are microvesicles with a diameter of 40~100 nm, and contains proteins, lipids and nucleic acids (mRNAs, lncRNAs, circRNAs, and microRNAs). They serve as an information exchange carrier, and play an important role in regulating and controlling the biomolecular function to maintain the stability of the intracellular environment. The function of exosomes in HCC includes intercellular communication, neoangiogenesis, cancer cell metastasis and multidrug resistance, which mediates the transformation of microRNAs (miRNA) and regulate the microenvironment of tumor progression, and then affect the pathophysiological behavior of cancer cells. Exosome-derived miRNA can be used for HCC monitoring or potential specific markers of early diagnosis. In addition, with the development and application prospects it could be a therapeutic goal for HCC. This paper summarizes the recent progress in the study of HCC-derived exosomal miRNA.

Characterization of antibody and memory T-cell response in H7N9 survivors: a cross-sectional analysis.

OBJECTIVES: Despite the importance of immunological memory for protective immunity against viral infection, whether H7N9-specific antibodies and memory T-cell responses remain detectable years after the original infection is unknown. METHODS: A cross-sectional study was conducted to investigate the immune memory responses of H7N9 patients who contracted the disease and survived during the 2013-2016 epidemics in China. Sustainability of antibodies and T-cell memory to H7N9 virus were examined. Healthy individuals receiving routine medical examinations in a physical examination centre were recruited as control. RESULTS: A total of 75 survivors were enrolled and classified into four groups based on the time elapsed from illness onset to specimen collection: 3 months (n = 14), 14 months (n = 14), 26 months (n = 28) and 36 months (n = 19). Approximately 36 months after infection, the geometric mean titres of virus-specific antibodies were significantly lower than titres in patients 3 months after infection, but 16 of 19 (84.2%) survivors in the 36-month interval had microneutralization (MN) titres ≥40. Despite the overall declining trend, the percentages of virus-specific cytokine-secreting memory CD4+ and CD8+ T cells remained higher in survivors at nearly all time-points in comparison with control individuals. Linear regression analysis showed that severe disease (mean titre ratio 2.77, 95% CI 1.17-6.49) was associated with higher haemagglutination inhibition (HI) titre and female sex for both HI (1.92, 1.02-3.57) and MN (3.33, 1.26-9.09) antibody, whereas female sex (mean percentage ratio 1.69, 95% CI 1.08-2.63), underlying medical conditions (1.94, 95% CI 1.09-3.46) and lack of antiviral therapy (2.08, 95% CI 1.04-4.17) were predictors for higher T-cell responses. CONCLUSIONS: Survivors of H7N9 virus infection produced long-term antibodies and memory T-cell responses. Our findings warrant further serological investigation in general and high-risk populations and have important implications for vaccine design and development.

Association between Depressive Symptoms and Supplemental intake of Calcium and Vitamin D in Older Adults.

OBJECTIVES: We explored the associations between depressive symptoms and supplemental calcium and vitamin D intake in older adults. DESIGN: This was a prospective cohort study. PARTICIPANTS: 8,527 older adults aged ≥60 years from Zhejiang Major Public Health Surveillance Program (ZPHS) without depressive symptoms at baseline survey. MEASUREMENTS: Participants were divided into non-supplementation, calcium (Ca), vitamin D, and calcium plus vitamin D (CaD) groups based on their supplemental intake during the past year. Depressive symptoms were assessed using the Patient Health Questionnaire-9 (PHQ-9). Binary logistic regression analyses were performed to examine the association between depressive symptoms and supplemental intake. RESULTS: When compared to the non-supplementation group, the Ca group exhibited a significant odds ratio (OR) of 0.731 (95% CI: 0.552-0.967, P=0.028). After adjusting for age, sex, and Ca food sources, the OR was even smaller for the CaD group (OR: 0.326; 95% CI: 0.119-0.889, P=0.029). Additionally, our results indicated that taking Ca supplements ≥4 days/week yielded a significant OR of 0.690 (95% CI: 0.492-0.968) after full adjustment. Taking CaD supplements ≥4 days/week yielded a significant OR of 0.282 (95% CI: 0.089-0.898) after adjusting for age, sex, and Ca food sources. CONCLUSIONS: Supplemental intake of Ca or CaD ≥4 days/week can decrease the risk of depressive symptoms in older adults, although CaD supplements may be more effective.

Seven genes for the prognostic prediction in patients with glioma.

PURPOSE: Glioma is a common malignant tumor of the central nervous system, which is characterized by a low cure rate, high morbidity, and high recurrence rate. Consequently, it is imperative to explore some indicators for prognostic prediction in glioma. METHODS: We obtained glioma data from The Cancer Genome Atlas (TCGA). Differentially expressed genes (DEGs) were obtained by R software from TCGA data sets. Through Cox regression analysis, risk scores were obtained to assess the weighted gene-expression levels, which could predict the prognosis of patients with glioma. The validity and the prognostic value of this model in glioma were confirmed by the manifestation of receiver-operating characteristic (ROC) curves, area under the curve (AUC), and 5-year overall survival (OS). RESULTS: In total, 920 DEGs of transcriptome genes in glioma were extracted from the TCGA database. We identified a novel seven-gene signature associated with glioma. Among them, AL118505.1 and SMOC1 were positively related to the 5-year OS of patients with glioma, showing a better prognosis for glioma; however, RAB42, SHOX2, IGFBP2, HIST1H3G, and IGF2BP3 were negatively related to 5-year OS, displaying a worse prognosis. In addition, according to risk scores, AL118505.1 was also a protective factor, while others were risk factors. Furthermore, the expression levels of SHOX2, IGFBP2, and IGF2BP3 were significantly positively correlated with glioma grades. Receiver-operating characteristic (ROC) curve assessed the accuracy and sensitivity of the gene signature. Each of the seven genes for patients with the distribution of the risk score was presented in the heat map. CONCLUSION: We identified a novel seven-gene signature in patients with glioma, which could be used as a predictor for the prognosis of patients with glioma in the future.

[Abnormal expression of Wnt3a and inhibiting role of its molecular-targeted intervening in hepatocellular carcinoma].

Objective: To investigate the Wnt3a expression in tissues of HCC and its gene knockout on effects of HepG2 cell proliferation or xenograft tumor growth. Methods: Hepatic Wnt3a expressions in 87 HCC and their matched surrounding tissues were observed by tissue microarray and immunohistochemistry for analyzing its clinicopathological characteristics; Wnt3a-knockout HepG2 cell lines were established by Crispr/cas9-sgRNA system and genomic cleavage efficiency was verified at gene level by surveyor assay. The relative proteins were confirmed by Western blotting; Cell Counting Kit-8 assay was used to examine cell proliferation after knocking-out Wnt3a successfully, and the nude mice HepG2 cell xenograft tumors delete that the relationship between Wnt3a and HCC growth. Results: The positive Wnt3a with brown staining particles was mainly distributed in cytosol and membrane of hepatocytes. The incidence of hepatic Wnt3a expression in cancerous tissues (95.4%) was significantly higher (χ (2) = 47.754, P < 0.001) than that in their surrounding tissues (49.4%). The high Wnt3a expression was 70.1% in the HCC and only 14.9% in the surrounding tissues. High Wnt3a expression was associated with poorly-differentiated grade, liver cirrhosis, HBV infection, portal vein invasion, TNM stage and 5-year survival rate. After knocked-out by Crispr/cas9-sgRNA system successfully, Wnt3a expression was down-regulated significantly at gene or protein level. Key molecule ß-catenin in cytoplasma was obviously inhibited. HepG2 cell lines proliferation was suppressed in time-dependent manner. The nude mice HepG2 cell xenograft tumors confirmed that the knock-out of Wnt3a could significantly supressed HCC growth with slower speed (t = 6.418, P < 0.001), smaller volume(869.4 ± 222.5 mm(3) vs 355.0 ± 99.9 mm(3), t = 5.168, P < 0.001), and lighter weight (0.88 ± 0.20 g vs 0.35 ± 0.11 g, t = 5.628, P < 0.001)compared with the control group. Conclusion: Abnormal expression of Wnt3a could be expected as a promising target for HCC gene therapy.

[Quantitative detection and clinical value of CD44 in patients with nonalcoholic fatty liver disease].

Objective: To quantitatively detect CD44 expression in patients with nonalcoholic fatty liver disease (NAFLD) for comparative analysis. Methods: Patients with chronic liver diseases accompanied with or without NAFLD, including chronic hepatitis B, cirrhosis and hepatocellular carcinoma after chronic hepatitis B, and healthy blood donors as normal controls who admitted to the Affiliated Hospital of Nantong University from May to October 2018 were selected. The proportion of CD44 positive cells was analyzed by flow cytometry. CD44 level was quantified by an enzyme-linked immunosorbent assay, and the biochemical indicators such as serum aspartate aminotransferase, alanine aminotransferase activity, total cholesterol and triglyceride were routinely analyzed. The cancerous and adjacent cancerous tissues of patients accompanied with or without NAFLD were collected by self-matching method and analyzed by immunoblotting and histochemistry and compared by CD44 integrated optical density. Image-Pro Plus version 6.0, Image J, GraphPad Prism 5.0, Photoshop, Microsoft Excel and IBM SPSS statistics 23 were used to analyze and draw pictures. An independent sample t-test was used to compare the differences between groups. Results: Patients accompanied with NAFLD had hepatocyte injury and dyslipidemia. NAFLD and chronic liver disease patients had significantly elevated serum CD44 levels than normal control group (P < 0.01). CD44 positive lymphocyte ratio was 78.19 % ± 16.33 % in NAFLD patients and 68.47% ± 20.91% in chronic hepatitis B group, which was higher than the control group (46.51% ± 20.52%). Chronic hepatitis B group with steatosis had significantly higher CD44 concentration (181.42 ± 49.36) ng/ml than chronic hepatitis B group (142.52 ± 53.87) ng/ml and normal control group (99.47 ± 15.23) ng/ml. CD44/GAPDH ratio in the liver cancer group (1.306 ± 0.614) was significantly higher than paracancerous group (0.477 ± 0.291) and the difference between the two groups was statistically significant (t = 3.451, P = 0.004). The integrated optical density of CD44 in the NAFLD-related liver cancer and paracancerous group were 25.721 ± 5.881 and 14.155 ± 4.001 and the difference between the groups was statistically significant (t = 14.544, P < 0.001). The pathological features of high expression of CD44 in patients with hepatocellular carcinoma were significantly correlated with HBV infection, tumor size, single/multi-center, and lymph node metastasis, degree of differentiation, TNM grade, Child-Pugh score, portal vein tumor thrombus and extrahepatic metastasis. HCC patients with NAFLD had significantly higher serum CD44 (234.62 ± 69.40) ng/ml than patients without NAFLD (186.49 ± 58.89) ng/ml (t = -3.191, P = 0.002), but there was no statistically significant difference in the clinicopathological characteristics between the high/low CD44 groups of HCC patients with NAFLD. Conclusion: The results suggest that CD44 is abnormally activated and its mechanism may play an important role in the progression of NAFLD.

Determinants of cerebral white matter changes in patients with stroke.

BACKGROUND/AIM: Cerebral white matter changes (WMC) are commonly observed in magnetic resonance imaging (MRI) scans of elderly people. Information about the prevalence of WMC is limited, and little is known about site-specific risk factors for the subcortical and periventricular regions in patients with ischaemic stroke. The study aims to analyse the prevalence and severity of WMC and investigate the risk factors of periventricular WMC (PVWMC) and deep WMC (DWMC) separately in patients with ischaemic stroke. METHODS: The data were collected between January and December 2013 from a medical centre in southern Taiwan. Every patient underwent a cerebral MRI scan, and WMC was separately rated as PVWMC and DWMC by using the modified Fazekas scale. RESULTS: In total, 527 patients who had experienced ischaemic stroke were included. The mean age of the patients was 67.0 ± 12.5 years (range: 31-94) and 62% of them were men. The mean age was significantly different among the four grades of severity in both the PVWMC (P < 0.001) and DWMC (P < 0.001) groups after adjustments for sex and vascular risk factors. Hypertension was independently correlated with severity of DWMC (P = 0.032) but not with PVWMC (P = 0.222). In multiple logistic regressions model, hypertension was a significant independent indicator of DWMC (odds ratio = 4.30; 95% confidence interval = 1.70-10.89). CONCLUSION: Our results suggest a region-specific pathogenesis of cerebral white matter in Asian patients with ischaemic stroke that may differ from those in the general population.

Alpha4beta2 nicotinic acetylcholine receptors are required for the amyloid beta protein-induced suppression of long-term potentiation in rat hippocampal CA1 region in vivo.

Amyloid beta protein (Abeta) is thought to be responsible for the deficit of learning and memory in Alzheimer's disease (AD), possibly through interfering with synaptic plasticity such as hippocampal long-term potentiation (LTP). Nicotinic acetylcholine receptors (nAChRs) participate in various cognitive brain functions. However, it is unclear whether nAChRs, especially alpha4beta2 subtype nAChRs, are involved in Abeta-induced impairment of hippocampal LTP. The present study investigates a possible role of nAChRs during the impairment of LTP by Abeta. Our results showed that: (1) intracerebroventricular injection of Abeta(1-40), Abeta(25-35) or Abeta(31-35) significantly suppressed high-frequency stimulation-induced LTP, while Abeta(35-31), a reversed sequence of Abeta(31-35), have no effect on the LTP; (2) epibatidine, a specific agonist of alpha4beta2 subtype of nAChRs, dose-dependently suppressed the induction of LTP; (3) co-injection of epibatidine together with Abeta(31-35) did not further enhance the suppression of LTP induced by Abeta(31-35) or epibatidine alone; (4) dihydro-beta-erythroidine, a selective antagonist against alpha4beta2 subtype of nAChRs, showed no effect on the induction of LTP, but significantly reversed Abeta(31-35)-induced LTP impairment. These results indicate that: (1) sequence 31-35 in Abeta molecule might be a shorter active center responsible for the neurotoxicity of full length of Abeta; (2) alpha4beta2 subtype of nAChRs is required for the suppressive action of Abeta on the hippocampal LTP in vivo. Thus, the present study provides further insight into the mechanisms by which Abeta impairs synaptic plasticity and cognitive function in the AD brain.

Targeted mutations in the syntaxin H3 domain specifically disrupt SNARE complex function in synaptic transmission.

The cytoplasmic H3 helical domain of syntaxin is implicated in numerous protein-protein interactions required for the assembly and stability of the SNARE complex mediating vesicular fusion at the synapse. Two specific hydrophobic residues (Ala-240, Val-244) in H3 layers 4 and 5 of mammalian syntaxin1A have been suggested to be involved in SNARE complex stability and required for the inhibitory effects of syntaxin on N-type calcium channels. We have generated the equivalent double point mutations in Drosophila syntaxin1A (A243V, V247A; syx(4) mutant) to examine their significance in synaptic transmission in vivo. The syx(4) mutant animals are embryonic lethal and display severely impaired neuronal secretion, although non-neuronal secretion appears normal. Synaptic transmission is nearly abolished, with residual transmission delayed, highly variable, and nonsynchronous, strongly reminiscent of transmission in null synaptotagmin I mutants. However, the syx(4) mutants show no alterations in synaptic protein levels in vivo or syntaxin partner binding interactions in vitro. Rather, syx(4) mutant animals have severely impaired hypertonic saline response in vivo, an assay indicating loss of fusion-competent synaptic vesicles, and in vitro SNARE complexes containing Syx(4) protein have significantly compromised stability. These data suggest that the same residues required for syntaxin-mediated calcium channel inhibition are required for the generation of fusion-competent vesicles in a neuronal-specific mechanism acting at synapses.

Crystal structure of the VHS and FYVE tandem domains of Hrs, a protein involved in membrane trafficking and signal transduction.

We have determined the 2 A X-ray structure of the 219-residue N-terminal VHS and FYVE tandem domain unit of Drosophila Hrs. The unit assumes a pyramidal structure in which the much larger VHS domain (residues 1-153) forms a rectangular base and the FYVE domain occupies the apical end. The VHS domain is comprised of an unusual "superhelix" of eight alpha helices, and the FYVE domain is mainly built of loops, two double-stranded antiparallel sheets, and a helix stabilized by two tetrahedrally coordinated zinc atoms. The two-domain structure forms an exact 2-fold-related homodimer through antiparallel association of mainly FYVE domains. Dimerization creates two identical pockets designed for binding ligands with multiple negative charges such as citrate or phosphatidylinositol 3-phosphate.

Syntaxin 1A interacts with multiple exocytic proteins to regulate neurotransmitter release in vivo.

Biochemical studies suggest that syntaxin 1A participates in multiple protein-protein interactions in the synaptic terminal, but the in vivo significance of these interactions is poorly understood. We used a targeted mutagenesis approach to eliminate specific syntaxin binding interactions and demonstrate that Drosophila syntaxin 1A plays multiple regulatory roles in neurotransmission in vivo. Syntaxin mutations that eliminate ROP/Munc-18 binding display increased neurotransmitter release, suggesting that ROP inhibits neurosecretion through its interaction with syntaxin. Syntaxin mutations that block Ca2+ channel binding also cause an increase in neurotransmitter release, suggesting that syntaxin normally functions in inhibiting Ca2+ channel opening. Additionally, we identify and characterize a syntaxin Ca2+ effector domain, which may spatially organize the Ca2+ channel, cysteine string protein, and synaptotagmin for effective excitation-secretion coupling in the presynaptic terminal.

ROP, the Drosophila Sec1 homolog, interacts with syntaxin and regulates neurotransmitter release in a dosage-dependent manner.

The Sec1 family of proteins is thought to function in both non-neuronal and neuronal secretion, although the precise role of this protein family has not been defined. Here, we study the function of ROP, the Drosophila Sec1 homolog, in neurotransmitter release. Electrophysiological analyses of transgenic lines overexpressing ROP and syntaxin, a presynaptic membrane protein, indicate that ROP interacts with syntaxin in vivo. Characterization of four point mutations in ROP shows that they fall into two phenotypic classes. Two mutations cause a dramatic reduction in both evoked and spontaneous neurotransmitter release. In contrast, the other two mutations reveal an increase in evoked neurotransmission. Our data further show that neurotransmission is highly sensitive to the levels of ROP function. Studies on heterozygote animals indicate that half the amount of wild-type ROP results in a dramatic decrease in evoked and spontaneous exocytosis. Taken together, these results suggest that ROP interacts with syntaxin in vivo and is a rate-limiting regulator of exocytosis that performs both positive and inhibitory functions in neurotransmission.

Genetic dissection of synaptic transmission in Drosophila.

Recent studies of mutations of Drosophila proteins implicated in synaptic transmission have yielded new insights into the roles of these proteins and the pathways in which they function. Analysis of mutant embryos lacking syntaxin or synaptobrevin suggests that these proteins perform distinct functions after vesicle docking with the presynaptic membrane. In addition, characterization of Drosophila endocytotic mutants provides in vivo evidence for the presence of different endocytotic pathways at a single synapse.