Clinical Genetic Features and Neoadjuvant Chemotherapy Response in HER2-Low Breast Cancers: A Retrospective, Multicenter Cohort Study.

BACKGROUND: The objective of this work is to reveal differences in clinical and genetic features, as well as neoadjuvant chemotherapy (NAC) response, between HER2-low and HER2-zero or HER2-positive breast cancers. PATIENTS AND METHODS: A total of 245 female patients with breast cancer were retrospectively enrolled from seven hospitals. Core needle biopsy (CNB) samples were collected before NAC and used for next-generation sequencing by a commercial gene panel. Clinical and genetic features, as well as NAC response, were compared between HER2-low and HER2-zero or HER2-positive breast cancers. The nonnegative matrix factorization (NMF) method was applied to cluster the C-Score of enrolled cases to reveal the intrinsic features of each HER2 subgroup. RESULTS: A total of 68 (27.8%) cases are HER2-positive, 117 (47.8%) cases are HER2-low, and 60 (24.5%) cases are HER2-zero. HER2-low breast cancers have a significantly lower pathologic complete response (pCR) rate than HER2-positive and HER2-zero breast cancers (p < 0.050 for all comparisons). Compared with HER2-low breast cancers, HER2-positive cases have higher rates of TP53 mutation, TOP2A amplification, and ERBB2 amplification, as well as lower rates of MAP2K4 mutation, ESR1 amplification, FGFR1 amplification, and MAPK pathway alteration (p < 0.050 for all comparisons). After clustering HER2-low cases by the NMF method, 56/117 (47.9%) are in cluster 1, 51/117 (43.6%) are in cluster 2, and 10/117 (8.5%) are in cluster 3. HER2-low cases in cluster 2 have the lowest pCR rate among the three clusters (p < 0.050). CONCLUSIONS: HER2-low breast cancers have significant genetic differences from HER2-positive cases. Genetic heterogeneity exists in HER2-low breast cancers and impacts on NAC response in this subgroup.

Pseudo Polyampholytes with Sensitively Ion-Responsive Conformational Transition Based on Positively Charged Host-Guest Complexes.

Biological polyampholytes are ubiquitous in living organisms with primary functions including serving as transporters for moving chemical molecular species across the cell membranes. Synthetic amphoteric macromolecules that can change their phase states depending on the environment to simulate some properties of natural polyampholytes are of great interest. Here, the implementation of synthetic pseudo polymeric ampholytes is explored with ion-recognition-triggered conformational change. The phase transition behaviors of the ion-recognition-creative polyampholytes that contain deprotonated carboxylic acid groups as negative charges and 18-crown-6 units for forming positively charged host-guest complexes are systematically investigated. The ion-recognition-triggered phase transition behaviors of pseudo polyampholytes significantly depend on cation species and concentrations. Only those specific ions such as K+ , Ba2+ , Sr2+ and Pb2+ ions that can form 1:1 host-guest complexes with 18-crown-6 units in polymers enable control over conformational change like that of traditional pH-dependent polyampholytes. By regulating the content of carboxylic acid groups to match the content of ion-recognized positive charges provided by the host-guest complexes, the pseudo polyampholytes are more sensitive to the recognizable cations. Such ion-recognition-triggered amphoteric characteristics make the pseudo polyampholytes act like biological proteins, nucleic acids, and enzymes as molecular transporters, genetic code storage, and biocatalysts in artificial systems.

TRAF4, a new substrate of SIAH1, participates in chemotherapy resistance of breast cancer cell by counteracting SIAH1-mediated downregulation of ß-catenin.

PURPOSE: TRAF4 plays an important role in the development and progression of breast cancer, but its impact on chemotherapy resistance is as yet, however, poorly understood. METHODS: Western blotting, immunoprecipitation, and immunofluorescence staining were used to identify and verify that TRAF4 was a novel substrate of SIAH1 and prevented SIAH1-mediated ß-catenin degradation. Cell proliferation analysis and Flow cytometry analysis were utilized to detect TRAF4's function on the growth-inhibitory effect of etoposide. Immunohistochemistry was used to detect the expression of TRAF4, SIAH1, and ß-catenin. Statistical analysis was used to analyze the relationships between them with clinical parameters and curative effect of chemotherapy pathologically. RESULTS: Our results suggested that TRAF4 prevents SIAH1-mediated ß-catenin degradation. TRAF4 was a novel substrate of SIAH1 and the TRAF domain of TRAF4 was critical for binding to SIAH1. TRAF4 reduced the growth-inhibitory effect of etoposide via reducing the number of S-phase cells and suppressing cell apoptosis. Concordantly, we found that breast cancer patients with a low-TRAF4 expression benefited most from chemotherapy, who had higher tumor volume reduction rate and better pathological response, while, the high-TRAF4 expression group had lower tumor volume reduction rate and poor pathological response. CONCLUSIONS: TRAF4 was a novel substrate of SIAH1 and prevented SIAH1-mediated ß-catenin degradation, which explains the protective effect of TRAF4 on ß-catenin during cell stress and links TRAF4 to chemotherapy resistance in tumors. These findings implicated a novel pathway for the oncogenic function of TRAF4.

Chlorpyrifos-induced toxicity has no gender selectivity in the early fetal brain.

Organophosphorus pesticides induce gender-specific developmental neurotoxicity after birth, especially in adolescents and adults. However, whether and when the selectivity occurs in fetus remains unclear. In this study, we analyzed chlorpyrifos (CPF)-induced neurotoxicity in the early fetal brains of male and female mice. The gestational dams were administered 0, 1, 3, and 5 mg/(kg.d) CPF during gestational days (GD)7-11, and brains from the fetuses were isolated and analyzed on GD12. Fetal gender was identified by PCR technique based on male-specific Sry gene and Myog control gene. The body weight and head weight, the activity of acetylcholinesterase (AChE), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and the content of malondialdehyde (MDA), as well as the oxidative stress-related gene expression were examined. Our results showed that CPF pretreatment induced AChE inhibition in GD12 fetal brain. CPF treatment activated SOD and GPX but not CAT and MDA. For oxidative stress-related gene expression, CPF pretreatment increased mRNA expression of Sod1, Cat, Gpx1, and Gpx2 in the fetal brain on GD12. The statistical analysis did not show gender-selective CPF-induced toxicity. Moreover, our results showed that although the gestational exposure to CPF could elicit abnormalities in the early fetal brain, the toxicity observed was not gender-specific.

Assembly of Peroxisomal Membrane Proteins via the Direct Pex19p-Pex3p Pathway.

Correct targeting of peroxisomal membrane proteins (PMPs) is essential for the formation and maintenance of functional peroxisomes. Activities of Pex19p to interact with PMPs on one hand and Pex3p on the other, including formation of ternary complexes between Pex19p, PMP and Pex3p, strongly support posttranslational translocation of PMPs via the Pex19p- and Pex3p-dependent direct pathway, termed the class I pathway. However, it remains elusive whether Pex19p-PMP complexes are indeed capable of being imported into peroxisomal membranes through the interaction between Pex19p and Pex3p. We resolve this issue by investigating the targeting process of several topologically distinct PMPs, including multimembrane spanning PMPs. We show here that Pex19p forms cytosolic complexes with PMPs and directly translocates them to peroxisomes. Using a semi-intact mammalian cell-based import assay system, we prove that PMPs in the cytosolic complexes are imported into peroxisomes via the interaction between cargo-loaded Pex19p and Pex3p. Furthermore, we demonstrate for the first time that peroxisomal targeting of ATAD1, an N-terminally signal-anchored protein that resides on both mitochondria and peroxisomes, is also achieved through the Pex19p- and Pex3p-dependent class I pathway. Together, our results suggest that translocation of PMPs via the class I pathway is a common event in mammalian cells.

Overexpression of AIB1 correlates inversely with E-cadherin expression in pancreatic adenocarcinoma and may promote lymph node metastasis.

BACKGROUND: It was reported that the nuclear receptor coactivator amplified in breast cancer1 (AIB1) could regulate cancer cell invasion and migration in a nuclear receptor signaling-independent manner. Meanwhile, the process of epithelial mesenchymal transition (EMT) is critical for tumor invasion and metastasis. The present study aimed to determine the role of AIB1 and EMT markers in human pancreatic adenocarcinoma. METHODS: AIB1, ZO-1, E-cadherin, vimentin, and N-cadherin protein expression in 76 pancreatic adenocarcinomas were assessed using immunohistochemistry and analyzed for clinicopathological significance. RESULTS: The frequency of AIB1 overexpression in pancreatic adenocarcinomas with lymph node metastasis is 68 % (19/28), which is significantly higher than in pancreatic adenocarcinomas without lymph node metastasis (42 %; 20/48). In addition, the frequency of low expression of E-cadherin in pancreatic carcinomas with lymph node metastasis (68 %; 19/28) was significantly higher than in tumors without lymph node metastasis (44 %; 21/48). Correlation analysis demonstrated that the overexpression of AIB1 was inversely correlated with low expression of E-cadherin in pancreatic adenocarcinomas. CONCLUSION: Overexpression of AIB1 might promote invasion and metastasis of cancer cells and is associated with down-regulation of E-cadherin in pancreatic adenocarcinomas.

Peroxisome biogenesis factor PEX14 is crucial for survival and fecundity of female brown planthopper, Nilaparvata lugens (Stål).

Peroxisomes are ubiquitous cellular organelles participating in a variety of critical metabolic reactions. PEX14 is an essential peroxin responsible for peroxisome biogenesis. In this study, we identified the human PEX14 homolog in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae). N. lugens PEX14 (NlPEX14) showed significant topological similarity to its human counterpart. It is expressed throughout all developmental stages, with the highest expression observed in adult insects. Down-regulation of NlPEX14 through injection of NlPEX14-specific double-strand RNA impaired nymphal development. Moreover, females subjected to dsNlPEX14 treatment exhibited a significantly reduced lifespan. Additionally, we found abnormal ovarian development and a significant decrease in the number of eggs laid in NlPEX14-downregulated females. Further experiments support that the shortening of lifespan and the decrease in female fecundity can be attributed, at least partially, to the accumulation of fatty acids and reduced expression of vitellogenin. Together, our study reveals an indispensable function of NlPEX14 for insect reproduction and establishes a causal connection between the phenotypes and peroxisome biogenesis, shedding light on the importance of peroxisomes in female fecundity.

Overexpression of phosphatase regenerating liver 3 in oesophageal squamous cell carcinoma associated with metastasis and its comparison with phosphatase regenerating liver 1.

Expression of PRL3 (phosphatase of regenerating liver 3) protein was examined with immunohistochemistry in 60 cases of ESCC (oesophageal squamous cell carcinoma) with matched lymph node metastasis (n  =  40) and 6 cases of oesophageal adenocarcinoma. Its associations with PRL1 and clinicopathological parameters were analysed. The results showed the frequency of PRL3 protein expression was significantly higher in ESCC (39/60, 65%) than in normal oesophageal mucosa (0/20, P < 0.001); higher in ESCC with lymph node metastasis (30/40, 75%) than in ESCC without lymph node metastasis (9/20, P = 0.022), as well as higher in metastatic ESCC in lymph node (38/40, 95%) than in the primary ESCC (39/60, 65%, P < 0.001). PRL3 was expressed in 1 out of 6 oesophageal adenocarcinomas, but showed no nuclear staining of PRL1. Expression of PRL3 protein was positively associated with the grade and partially with the stage of ESCC. These results suggest that expression of PRL3 protein may be involved in the metastasis of ESCC and serve as a biomarker for prediction of ESCC metastasis.

Assimilation of blended in situ-satellite snow water equivalent into the National Water Model for improving hydrologic simulation in two US river basins.

This study investigates the potential of assimilating a 1/8° blended in situ-satellite snow water equivalent (SWE) product for improving snow and streamflow predictions of the National Water Model (NWM). The blended product is assimilated into the NWM via a three-dimensional variational (3DVAR) scheme and a direct insertion (DI) scheme, with a daily (1d) and a every 5 days (5d) assimilation frequencies. The experiments are for the Upper Colorado River Basin (UCRB) and Susquehanna River Basin (SRB), which feature seasonal and ephemeral snow covers, respectively. Results indicate that 3DVAR with a 5d assimilation frequency generally outperforms the other scenarios. The assimilation of the blended SWE product mitigates the underestimation of SWE evident in the open-loop simulations for both basins and its impacts are more pronounced for UCRB than for SRB since snowfall is the main source of precipitation in the former. Assimilation leads to improved streamflow over a majority of SRB subbasins, but over a minority of UCRB subbasins. The degradations in streamflow for UCRB subbasins are mainly caused by the overestimated SWE. In addition, the open-loop simulation often produces an earlier streamflow peak in UCRB, and this error is mitigated to a limited extent by assimilation. These findings in aggregate suggest that the efficacy of snow assimilation is strongly dependent upon the types of snowpack and differential assimilation methods and frequencies.

A novel chemosensor for sensitive and facile detection of strontium ions based on ion-imprinted hydrogels modified with guanosine derivatives.

A novel chemosensor is developed for the sensitive and facile detection of trace strontium ions (Sr2+) based on the ion-imprinted hydrogels. With Sr2+ as the templates, the ion-imprinted hydrogels are synthesized by copolymerizing the ion-responsive units 5'-O-acryloyl-2',3'-O-isopropylidene guanosine (APG) and the thermo-responsive units N-isopropylacrylamide (NIPAM). In the presence of Sr2+, APG units can self-assemble to form planar G-quartets via the complexation with Sr2+, which are introduced into the gel network during polymerization. Then Sr2+ templates can be removed by multiple repeated washing. When re-exposed to Sr2+, the relaxed G-quartets can recognize Sr2+, leading to the weakening of electrostatic repulsion between the four oxygen atoms in the G-quartets and inducing the shrinkage of the hydrogels. In this work, the Sr2+-imprinted chemosensors are designed as the grating systems for detecting trace Sr2+. Based on the array of hydrogel strings synthesized on a nano-scale, the smart grating systems thus constructed can convert and amplify the Sr2+ concentration signals to the easily-measurable optical signals. With the Sr2+-imprinted hydrogel gratings, trace Sr2+ (10-11 M) in an aqueous solution can be detected sensitively. Moreover, the proposed Sr2+-imprinted chemosensors can be integrated with other smart systems for developing various detectors with high performance.

Novel Multifunctional Stimuli-Responsive Nanoparticles for Synergetic Chemo-Photothermal Therapy of Tumors.

In this study, a novel class of multifunctional responsive nanoparticles is designed and fabricated as drug nanocarriers for synergetic chemo-photothermal therapy of tumors. The proposed nanoparticles are composed of a thermo-/pH-responsive poly(N-isopropylacrylamide-co-acrylic acid) (PNA) nanogel core, a polydopamine (PDA) layer for photothermal conversion, and an outer folic acid (FA) layer as a targeting agent for the folate receptors on tumor cells. The fabricated nanoparticles show good biocompatibility and outstanding photothermal conversion efficiency. The proposed nanoparticles loaded with doxorubicin (DOX) drug molecules are stable under physiological conditions with low leakage of drugs, while rapidly release drugs in environments with low pH conditions and at high temperature. The experimental results show that the drug release process is mainly governed by Fickian diffusion. In vitro cell experimental results demonstrate that the PNA-DOX@PDA-FA nanoparticles can be phagocytized by 4T1 tumor cells and release drugs in tumor cell acidic environments, and confirm that the combined chemo and photothermal therapeutic efficacy of PNA-DOX@PDA-FA nanoparticles is higher than the photothermal therapeutic efficacy or the chemotherapeutic efficacy alone. The proposed multifunctional responsive nanoparticles in this study provide a novel class of drug nanocarriers as a promising tool for synergetic chemo-photothermal therapy of tumors.

Adalimumab ameliorates memory impairments and neuroinflammation in chronic cerebral hypoperfusion rats.

Vascular dementia (VaD) is the second most common type of dementia worldwide. Although there are five FDA-approved drugs for the treatment of Alzheimer's disease (AD), none of them have been applied to treat VaD. Adalimumab is a TNF-α inhibitor that is used for the treatment of autoimmune diseases such as rheumatoid arthritis. In a recent retrospective case-control study, the application of adalimumab for rheumatoid or psoriasis was shown to decrease the risk of AD. However, whether adalimumab can be used for the treatment of VaD is not clear. In this study, we used 2VO surgery to generate a VaD rat model and treated the rats with adalimumab or vehicle. We demonstrated that VaD rats treated with adalimumab exhibited significant improvements in memory. In addition, adalimumab treatment significantly alleviated neuronal loss in the hippocampi of VaD rats. Moreover, adalimumab significantly reduced microglial activation and reversed M1/M2 polarization in VaD rats. Furthermore, adalimumab treatment suppressed the activity of NF-κB, an important neuroinflammatory transcription factor. Finally, adalimumab displayed a protective role against oxidative stress in VaD rats. Our results indicate that adalimumab may be applied for the treatment of human patients with VaD.

Visual detection of trace lead(II) using a forward osmosis-driven device loaded with ion-responsive nanogels.

A simple and portable thermometer-type device based on forward osmosis-driven liquid column rising is developed for visual detection of trace Pb2+. The device consists of a top indicator tube, a chamber loaded with Pb2+-responsive poly(N-isopropylacrylamide-co-benzo-18-crown-6-acrylamide) (PNB) smart nanogels and a bottom semipermeable membrane. Upon the recognition of Pb2+, PNB smart nanogels undergo a Pb2+-induced hydrophobic to hydrophilic transition, which simultaneously causes the increase of osmotic pressure inside the device. Driven by this osmotic pressure difference, more Pb2+ solution flows into the device, causing the rise of the liquid column in the indicator tube, which can be directly observed by naked eyes. The relationship between the change of liquid column height and the Pb2+ concentration is investigated for the quantitative detection of Pb2+. With the proposed forward osmosis-driven device, trace Pb2+ as low as 10-10 M in aqueous solutions can be detected. This method provides a novel and simple strategy for the visual detection of trace Pb2+.

Exosomes Derived From MicroRNA-148b-3p-Overexpressing Human Umbilical Cord Mesenchymal Stem Cells Restrain Breast Cancer Progression.

Exosomes derived from human umbilical cord mesenchymal stem cells (HUCMSCs) expressing microRNAs (miRs) have been highlighted as important carriers for gene or drug therapy. Hence, this study aimed to explore the role of exosomal miR-148b-3p from HUCMSCs in breast cancer. Clinical samples subjected to RT-qPCR detection revealed that miR-148b-3p was poorly expressed, while tripartite motif 59 (TRIM59) was highly expressed in breast cancer tissues. Online analyses available at miRanda, TargetScan, and miRbase databases revealed that miR-148b-3p could bind to TRIM59, while dual-luciferase reporter gene assay further verified that TRIM59 was a target gene of miR-148b-3p. Next, miR-148b-3p mimic or inhibitor and siRNA against TRIM59 were delivered into the breast cancer cells (MDA-MB-231) to alter the expression of miR-148b-3p and TRIM59 so as to evaluate their respective effects on breast cancer cellular processes. Evidence was obtained demonstrating that miR-148b-3p inhibited cell proliferation, invasion, and migration, but promoted cell apoptosis in breast cancer by down-regulating TRIM59. Next, MDA-MB-231 cells were co-cultured with the exosomes derived from HUCMSCs expressing miR-148b-3p. The results of co-culture experiments demonstrated that HUCMSCs-derived exosomes carrying miR-148b-3p exerted inhibitory effects on MDA-MB-231 progression in vitro. In vivo experimentation further confirmed the anti-tumor effects of HUCMSCs-derived exosomes carrying miR-148b-3p. Taken together, HUCMSC-derived exosomes carrying miR-148b-3p might suppress breast cancer progression, which highlights the potential of exosomes containing miR-148b-3p as a promising therapeutic approach for breast cancer treatment.

[Effect of nanosize delivery system for ASODN against hTERT on the expression of telomerase in the esophageal cancer EC9706 cells].

OBJECTIVE: To investigate the inhibitory effect of nanoparticle-mediated antisense oligodeoxynucleotide (ASODN) of human telomerase reverse transcriptase (hTERT) on telomerase in the esophageal cancer EC9706 cells. METHODS: Line-polyethylenimine (L-PEI) was used to condense ASODN into nanoparticle and to couple NGR peptides into targeting nanoparticle, and the prepared L-PEI/ASODN complexes were transfected into the EC9706 cells. Cellular uptake of L-PEI/ASODN complexes was detected by laser confocal scanning microscopy. MTT assay was used to detect the inhibitory rate of EC9706 cell growth. The level of hTERT mRNA and its protein expression were measured by RT-PCR and immunohistochemistry, respectively. Annexin V FITC/PI double labeling was used to detect cell apoptosis. The distribution of drug in nude mice was observed by laser confocal scanning microscopy, and the growth and morphology of the tumor was examined. RESULTS: The L-PEI-mediated ASODN uptake was enhanced. After transfection, the inhibitory rate of EC9706 cells was time-dependant and there was a significant difference between control cell group and L-PEI/ASODN group (P < 0.05). At 48 h after transfection, the level of hTERT mRNA was decreased significantly compared with that of control cell group (P < 0.05), and the expression of hTERT protein was negative. There was apparent apoptosis in EC9706 cells after transfection with L-PEI/ASODN complexes. For the two NGR/L-PEI/ASODN groups, fluorescence was observed in the liver, kidney, lung and tumor tissues of nude mice, and their uptake intensity was time-dependent. The mean volume of tumors in the two NGR/L-PEI/ASODN groups was significantly smaller than those in blank control group and SODN group (P < 0.05). Apoptotic bodies were detected in the tumors of L-PEI/ASODN group. CONCLUSION: The NGR/L-PEI/ASODN nanoparticles can effectively reach into the human esophageal cancer xenograft and inhibit the tumor growth in nude mice, and this may provide a theoretical and experimental basis for gene therapy for human esophageal squamous cell carcinoma.

Evaluation of Cardiovascular Risk Factors and Restless Legs Syndrome in Women and Men: A Preliminary Population-Based Study in China.

STUDY OBJECTIVES: Many studies have investigated the association between restless legs syndrome (RLS) and cardiovascular risk factors, leading to conflicting results. Therefore, the aim of the current study was to determine whether RLS is associated with cardiovascular risk factors and disease. METHODS: This cross-sectional study included 5,324 consecutive subjects who visited the Physical Examination Center of The First Affiliated Hospital of Zhengzhou University for their yearly routine physical examination. Participants underwent a face-to-face interview with a neurologist for the assessment of RLS, based on the International Restless Legs Study Group criteria. They also completed a questionnaire related to cardiovascular risk factors and other health-related and demographic information. Logistic regression was used to assess which of the demographic and cardiovascular risk factors increased the odds of RLS. Then, unadjusted and adjusted models were designed to determine whether RLS was associated with increased odds of cardiovascular disease, coronary artery disease, or hypertension. RESULTS: RLS was observed in 9.2% of the participants. Multivariable logistic regression models, which included the covariates age, sex, body mass index, smoking status, hypercholesterolemia, and Pittsburgh Sleep Quality Index score (dichotomized at 5), demonstrated that female sex (odds ratio [OR]: 2.42, 95% confidence interval [CI]: 1.99-2.95), smoking (OR: 1.96, 95% CI: 1.31-2.92), high cholesterol (OR: 1.30, 95% CI: 1.03-1.64), and PSQI score > 5 (OR: 5.61, 95% CI: 2.14-14.69) are significantly associated with RLS. Additionally, RLS was associated with hypertension, after adjusting for age, sex, body mass index, smoking, hypercholesterolemia, Pittsburgh Sleep Quality Index score > 5, diabetes, anemia, and decreased renal function. CONCLUSIONS: RLS is associated with the prevalence of hypertension but not with that of cardiovascular disease or coronary artery disease.

In Vitro PMP Import Analysis Using Cell-Free Synthesized PMP and Isolated Peroxisomes.

The Pex19p- and Pex3p-dependent direct import of peroxisomal membrane proteins (PMPs), termed the class I pathway, can be reconstituted in vitro by incubating cell-free synthesized PMPs with highly purified peroxisomes at 26 °C for 1 h. This method ensures that the proteins targeted to peroxisomes are imported directly without involvement of other organelles.

Peroxisomal Membrane and Matrix Protein Import Using a Semi-Intact Mammalian Cell System.

Peroxisomes are essential intracellular organelles that catalyze a number of essential metabolic pathways including ß-oxidation of very long chain fatty acids, synthesis of plasmalogen, bile acids, and generation and degradation of hydrogen peroxide. These peroxisomal functions are accomplished by strictly and spatiotemporally regulated compartmentalization of the enzymes catalyzing these reactions. Defects in peroxisomal protein import result in inherited peroxisome biogenesis disorders in humans. Peroxisomal matrix and membrane proteins are synthesized on free ribosomes and transported to peroxisomes in a manner dependent on their specific targeting signals and their receptors. Peroxisomal protein import can be analyzed using a semi-intact assay system, in which targeting efficiency is readily monitored by immunofluorescence microscopy. Furthermore, cytosolic factors required for peroxisomal protein import can be manipulated, suggesting that the semi-intact system is a useful and convenient system to uncover the molecular mechanisms of peroxisomal protein import.