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PDF file - 199K, Nocodazole induces microtubular network disarray and morphologic elongation in multiple myeloma cells.

PDF file - 43K, Effects of nocodazole alone or in combination with dexamethasone on myeloma tumor growth.

We identified nocodazole as a potent antimyeloma drug from a drug screening library provided by the Multiple Myeloma Research Foundation. Nocodazole is a benzimidazole that was originally categorized as a broad-spectrum anthelmintic drug with antineoplastic properties. We found that nocodazole inhibited growth and induced apoptosis of primary and multiresistant multiple myeloma cells cultured alone and in the presence of bone marrow stromal cells. Nocodazole caused cell-cycle prophase and prometaphase arrest accompanied by microtubular network disarray. Signaling studies indicated that increased expression of Bim protein and reduced X-linked inhibitor of apoptosis protein and Mcl-1L levels were involved in nocodazole-induced apoptosis. Further investigation showed Bcl-2 phosphorylation as a critical mediator of cell death, triggered by the activation of c-jun-NH2 kinase (JNK) instead of p38 kinase or extracellular signal–regulated kinases. Treatment with JNK inhibitor decreased Bcl-2 phosphorylation and subsequently reduced nocodazole-induced cell death. Nocodazole combined with dexamethasone significantly inhibited myeloma tumor growth and prolonged survival in a human xenograft mouse model. Our studies show that nocodazole has potent antimyeloma activity and that targeting the microtubular network might be a promising new treatment approach for multiple myeloma. Mol Cancer Ther; 10(10); 1886–96. ©2011 AACR.

Selinexor (KPT-330) is a small molecule inhibitor of XPO1 (exportin 1, Chromosome Region Maintenance 1/CRM1), which mediates the transport of tumor suppressor proteins, oncogene mRNAs and other proteins involved in governing cell growth, from the cell nucleus to the cytoplasm. It is often overexpressed in many cancer types. Since eukaryotic translation initiator factor 4E (eIF4E) plays a critical role in cancer protein translation in multiple myeloma (MM), we evaluated the effectiveness of combined inhibition of protein translation and with a nuclear export inhibitor in MM. Selinexor, an nuclear protein inhibitor of nuclear protein export, dose-dependently decreased eIF4E, IKZF1 and c-MYC protein levelsinhibits eIF4E-directed translation of oncoproteins such as IKZF1 and c-MYC.. Using a doxycycline- inducible pLKO-Tet-On vector, knockdown of eIF4E significantly enhanced the antiproliferative effects of selinexor, sensitized resistant MM cells to selinexor, and increased apoptosis in MM cells. Immunofluorescent analysis of MM cells showed that the combined treatment increased localization of residual eIF4E to the nucleus compared with selinexor treatment alone. In vivo studies showed that eIF4E knockdown enhanced the anti-tumor activity of selinexor in mice. Overexpression of eIF4E at least partially rescued the effects of selinexor in MM cells by reducing G1 cell cycle arrest as well as increasing the selinexor-IC50 10-fold. Moreover, the combination of selinexor with pharmacologic inhibitors of protein translation showed synergistic anti-MM effects. These results suggest a synergistic anti-MM effects of selinexor combined with eIF4E inhibitors in vitro. Our work provides a better understanding of the potential mechanism of resistance to selinexor and a rationale for combining selinexor with eIF4E inhibitors for the treatment of MM.

Introduction Tigecycline is a potential alternative to trimethoprim–sulfamethoxazole in treating Stenotrophomonas maltophilia infections due to its potent in vitro antimicrobial activity. Clinical evidence regarding the use of tigecycline in the treatment of S. maltophilia infections is scarce. In this study, we assessed the efficacy of tigecycline treating ventilator-associated pneumonia (VAP) due to S. maltophilia in comparison with fluoroquinolones. Methods This is a multicenter retrospective cohort study of patients admitted between January 2017 and December 2020 with the diagnosis of VAP caused by S. maltophilia receiving either tigecycline or fluoroquinolones as the definitive therapy ≥ 48 h. Clinical outcomes including 28-day mortality, clinical cure and microbiological cure were analyzed. Results Of 82 patients with S. maltophilia VAP included, 46 received tigecycline, and 36 received fluoroquinolones; 70.7% of patients had polymicrobial pneumonia, and the appropriate empiric therapy was applied to only 14.6% of patients. The overall 28-day mortality was 39%. Compared with patients receiving fluoroquinolones, tigecycline therapy resulted in worse clinical cure (32.6% vs. 63.9%, p = 0.009) and microbiological cure (28.6% vs. 59.1%, p = 0.045), while there was no statistical difference between 28-day mortality (47.8% vs. 27.8%, p = 0.105) in the two groups. Similar results were also shown in the inverse probability of treatment weighted univariable regression model and multivariable regression model. Conclusions The standard dose of tigecycline therapy was associated with a lower clinical and microbiological cure rate but not associated with an increased 28-day mortality in patients with S. maltophilia VAP compared with fluoroquinolones. Considering the unfavorable clinical outcomes, we therefore recommend against using the standard dose of tigecycline in treating S. maltophilia VAP unless new clinical evidence emerges. Supplementary Information The online version contains supplementary material available at 10.1007/s40121-021-00516-5.

Introduction. The emergence of antibiotic-resistant Salmonella isolates is a global concern and has been attributed to the indiscriminate use of antibiotics in humans and animals. Integrons are mobile gene elements closely related to bacterial drug resistance. Among them, class 1 integrons containing various resistance gene cassettes could play an important role in disseminating and maintaining antibiotic resistance in Salmonella isolates. Hypothesis. Salmonella class 1 integrons have a relationship with Salmonella drug resistance. Aim. This study aims to investigate the distribution of class 1 integrons and their variable regional molecular characteristics, as well as the diversity of the promoters and drug sensitivity among Salmonella strains. Methodology. A total of 111 Salmonella strains, collected between 2018 and 2020, underwent fully automated bacterial identification using the VITEK 2 Compact system and an antibiotic sensitivity test. PCR was employed to screen class 1 integrase genes (IntI1) and integron variable regions, while promoter type and variable region gene cassette characteristics were determined using sequencing analysis. Results. A total of 24 IntI1-positive strains were detected in 111 Salmonella strains. Moreover, IntI1-positive strains exhibited statistically significant resistance to ceftazidime, ciprofloxacin, levofloxacin, ceftriaxone, trimethoprim/sulfamethoxazole and azithromycin compared to integron-negative strains (PIntI1-positive strains was significantly higher than that of negative strains. Variable regions were observed in 6 of the 24 IntI1-positive strains. Four gene cassettes were detected, namely dfrA17-aadA5, dfrA12-aadA2, aadA22 and aar-3-dfrA27. Finally, 3 types of class 1 integron variable region promoters were identified in 24 strains, including PcW, PcH1 and PcWTGN-10; they are all relatively weak promoters. Conclusion. The integron and the drug resistance genes carried by integron have a certain relationship with drug resistance.

Yi Zhang,1,* Shenglei Yu,1,* Chen Chen,1,* Feng Sun,1,* Lei Zhou,1 Haijun Yao,2 Jin Hu,2 Shirong Li,3 Jingwen Ai,1 Ning Jiang,1 Jing Wang,1 Qihui Liu,1 Jialin Jin,1 Wenhong Zhang1,4,5 1Department of Infectious Diseases, National Medical Center for Infectious Diseases, Shanghai Key Laboratory of Infectious Diseases and Biosafety Emergency Response, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China; 2Department of Critical Care Medicine, Huashan Hospital, Fudan University, Shanghai, China; 3Department of Clinical Laboratory, Huashan Hospital, Fudan University, Shanghai, China; 4National Clinical Research Center for Aging and Medicine, Huashan Hospital, Fudan University, Shanghai, China; 5Key Laboratory of Medical Molecular Virology (MOE/MOH), Shanghai Medical College, Fudan University, Shanghai, China*These authors contributed equally to this workCorrespondence: Wenhong Zhang; Jialin Jin, Email wenhongzhang_hs@126.com; jinjialin@fudan.edu.cnPurpose: Carbapenem-resistant organisms (CROs) have posed a great threat to antibiotic use and induce multi-drug resistance. Contamination of the hospital environment and infection of healthcare workers (HCWs) are reported as sources of nosocomial infections. Here, we performed a comprehensive environment sampling and timely epidemiological investigation during outbreaks to investigate the role of the environment and HCWs in CRO transmission.Patients and Methods: We enrolled carbapenem-resistant organism outbreaks in ICU-1 of Huashan Hospital from January 2019 to March 2019, and ICU-2 located at west branch of Huashan Hospital from October 2019 to November 2019. Carbapenem-resistant Klebsiella pneumoniae (CRKP) and carbapenem-resistant Acinetobacter baumannii (CRAB) isolates were collected from the patients. We performed a real-time comprehensive environmental and HCW sampling in the two ICUs. Isolated strains from patients and the positive colonies from the screening were sent for whole-genome sequencing. Finally, phylogenetic trees were constructed.Results: CRAB and CRKP outbreaks simultaneously occurred in ICU-1; the outbreak involved 13 patients. Meanwhile, the CRKP outbreak in ICU-2 included 11 patients. Twelve out of 146 environment and HCWs samples in ICU-1 were carbapenem-resistant bacteria, including six CRKP and six CRAB strains. For ICU-2, hospital surfaces and HCWs were negative for CRKP. Phylogenetic analyses showed that CRKP strains in ICU-1 were classified into two clades: Clade 1 and Clade 2, sharing a high similarity of isolates from the environment and HCWs. The same phenomenon was observed in CRAB.Conclusion: A timely comprehensive sampling combined with genome-based investigation may aid in tracking the transmission route of and controlling the infections. The environment and HCWs could be contaminated during CRO transmission, which calls for strengthened prevention and control measures.Keywords: carbapenem-resistant, environment, whole-genome sequencing, prevention, control

Lung cancer (LC) is the leading cause of cancer‑related death, with high incidence and mortality rate. Early diagnosis and treatment of LC are imperative to improve the 5‑year survival rate for patients with LC. In recent years, miRNAs as promising biomarkers with high sensitivity and specificity for LC have been studied increasingly. In LC regulatory networks, miRNAs play crucial roles in the occurrence, development and metastasis of non‑small cell lung cancer (NSCLC), such as oncogenic factors, tumor suppressors and regulators. Dysfunctional miRNAs perform tumor‑suppressive or oncogenic functions in the regulation of cell proliferation, invasion, apoptosis, cell cycle disorder and angiogenesis by negatively regulating target genes. In the present review, the biological process of miRNAs was firstly summarized and recent advances in the mechanism of miRNAs involved in tumor formation and development were described. In addition, the present review concentrated on the latest findings on the miRNAs related with circulating free and extracellular vesicles in the early diagnosis of NSCLC. Additionally, the diagnostic performances of circulating free and extracellular vesicles‑associated miRNAs in NSCLC were contrasted. Owing to the increased stability and wide‑ranging practical applicability, miRNA may be one promising biomarker for NSCLC diagnosis.

Membrane-based technology has been confirmed as an effective way to treat emulsified oily wastewater, however, membrane fouling is still one of practical challenges in long-term operation. Herein, a novel passive-active combined strategy was proposed to control membrane fouling in continuous oily wastewater purification, where the δ-MnO

Jingfang Granule (JFG) is a Traditional Chinese Medicine prescription to empirically treat skin disease such as urticaria in clinical practice. However, the potential mechanisms of JFG on urticaria are not fully defined.The aim of this study is to investigate the mechanisms of JFG in treating urticaria through an OVA/aluminum hydroxide induced urticaria mice model.KM mice were injected intraperitoneally (i.p.) with OVA/aluminium hydroxide to establish the model with urticaria. After the mice were administered JFG, itching degree and hematoxylin and eosin (HE) staining were used to assess the protective effect of JFG on mice with urticaria. The regulatory networks were investigated by proteomics and central carbon metabolomics. Spleen T lymphocyte subsets were detected by flow cytometry. Peripheral blood cytokines were detected using ELISA kits or Cytometric Bead Array (CBA) kits. The protein expression of skin tissue was detected by western blot or immunohistochemical staining.JFG significantly relived skin tissue lesions and skin pruritus in mice with urticaria. Meanwhile, JFG significantly decreased IgE, IL-1β, IL-6, IL-4, TNF-α and IL-17A levels and increased IFN-γ levels in the serum of urticaria mice by inhibiting the expression of inflammation associated proteins including TLR4 and p-NF-κB p65, p-ERK1/2, p-JNK and p-p38, NLRP3, ASC and cleaved caspase-1. The results of proteomics, central carbon metabolomics, western blot and immunohistochemical staining confirmed that JFG inhibited Glycolysis/Gluconeogenesis and Pentose phosphate pathway in the skin tissue of urticaria mice by activating the LKB1/AMPK/SIRT1 axis and then downregulating the protein expressions of Glut1, TORC2, p-CREB, PEPCK, HNF4α and G6Pase.The current study demonstrates that JFG is effective in treating OVA/aluminum hydroxide-induced skin lesions and inflammation in mice, and JFG exhibits the clinical benefits via modulating LKB1/AMPK/SIRT1 axis, which in turn inhibits Glycolysis/Gluconeogenesis and Pentose phosphate pathway.

Background Autoimmune hepatitis (AIH) is an autoimmune mediated inflammatory disease that causes serious economic burden to mankind. Previous studies reported that Jingfang Granules (JFG) had an anti-inflammatory effect. However, there are few studies of JFG on AIH. The aim of the study was to investigate the therapeutic effect of JFG on AIH and its mechanisms of action. Methods The animal model of AIH was constructed by intravenous injection of Concanavalin A (Con A). JFG treatment was given by intragastric gavage (ig) after an hour later of injected Con A. Serum and liver tissues were collected after 24 h treatment. Mortality, histology, levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) in serum were to evaluate therapeutic effect of JFG. The apoptosis of hepatocyte was detected by terminal deoxynucleotidyl transferase mediated dUTP biotin nick end labeling assay (TUNEL). The expression of B-cell lymphoma-2 (Bcl-2), Bcl-2 Associated X (Bax), Cleaved-Caspase 3, Toll-like receptor 4 (TLR4)/nuclear factor kappa B p65 (NF-κB), Mitogen activated protein kinase (MAPK) pathways associated proteins, CS, IDH1, ACLY, SUCLG1, and SUCLA2 were measured by western blot. The inflammatory cytokines interleukin-1β (IL-1β), interleukin-4 (IL-4), interleukin-6 (IL-6), tumor necrosis factor (TNF)-α, interferon (IFN)-γ and anti-inflammatory cytokine interleukin-10 (IL-10) were checked by ELISA. The possible target mechanism of JFG in the treatment of AIH was explored by proteomics and metabolomics. Results The results showed that JFG substantially alleviated Con A-mediated hepatitis in mice, which was shown by increased survival rates, decreased of liver injury, aminotransferase, hepatocyte necrosis, and apoptosis. JFG could also regulate the production of inflammatory factors (IL-1β, IL-4, IL-6, TNF-α, IFN-γ and IL-10) by reduced the activation of TLR4/NF-κB and MAPK pathways. In addition, it was found that JFG could inhibit the inflammatory pathway by regulating tricarboxylic acid (TCA) cycle activity. Conclusions These results suggested that JFG played a therapeutic role in Con A-induced AIH by inhibiting liver injury, apoptosis, and inhibiting inflammatory pathway by regulating TCA cycle.

In multiple myeloma (MM), frequent mutations of NRAS, KRAS, or BRAF are found in up to 50% of newly diagnosed patients. The majority of the NRAS, KRAS, and BRAF mutations occur in hotspots causing constitutive activation of the corresponding proteins. Thus, targeting RAS mutation in MM will increase therapeutic efficiency and potentially overcome drug resistance. We identified germinal center kinase (GCK) as a novel therapeutic target in MM with RAS mutation. GCK knockdown (KD) in MM cells demonstrated in vitro and in vivo that silencing of GCK induces MM cell growth inhibition, associated with blocked MKK4/7-JNK phosphorylation and impaired degradation of IKZF1/3, BCL-6, and c-MYC. These effects were rescued by overexpression of a short hairpin RNA (shRNA)-resistant GCK, thereby excluding the potential off-target effects of GCK KD. In contrast, overexpression of shRNA-resistant GCK kinase-dead mutant (K45A) inhibited MM cell proliferation and failed to rescue the effects of GCK KD on MM growth inhibition, indicating that GCK kinase activity is critical for regulating MM cell proliferation and survival. Importantly, the higher sensitivity to GCK KD in RASMut cells suggests that targeting GCK is effective in MM, which harbors RAS mutations. In accordance with the effects of GCK KD, the GCK inhibitor TL4-12 dose-dependently downregulated IKZF1 and BCL-6 and led to MM cell proliferation inhibition accompanied by induction of apoptosis. Here, our data identify GCK as a novel target in RASMut MM cells, providing a rationale to treat RAS mutations in MM. Furthermore, GCK inhibitors might represent an alternative therapy to overcome immunomodulatory drug resistance in MM.

The prevalence of carbapenem-resistant

Basalt fibre-reinforced plastic sheet (BFRP) enhancement is one of the strengthening methods suitable for concrete structures due to the low price and high tensile strength of BFRPs. It is of great importance to study the mechanical performance of BFRP-reinforced low-strength concrete structures in freeze–thaw environments. Therefore, 15 concrete beam specimens were designed, and a fast freeze–thaw experiment was conducted in this research to consider different parameters, such as the number of freeze–thaw cycles, sequence of freezing–thawing and BFRP pasting and other factors. The mechanical performance of low-strength concrete beams reinforced with BFRPs under freeze–thaw cycles was investigated. The failure mode and load–deflection curves of specimens were also analysed. The test results revealed that the ultimate strength of concrete beams reinforced with BFRPs was greatly improved, with a maximum increase of 32.7%. With increasing number of freeze–thaw cycles, the ultimate strength of beams gradually decreased over the bearing capacity of non-frozen-thawed concrete beams reinforced with BFRPs (F0b), and the bearing capacity of low-strength concrete beams reinforced with BFRPs decreased 17.1% after 50 continuous freeze–thaw cycles, while the beam failure mode changed from bending failure to shear failure accompanied by de-bonding failure. A mechanical model of BFRP-reinforced low-strength concrete beams was constructed under the de-bonding failure mode, and a calculation equation to determine the magnitude of de-bonding failure was proposed. Finally, finite element analysis of specimens was performed with cohesive elements to simulate the nonlinear softening mechanical behaviour of the BFRP–concrete interface.

A modified apparatus for measuring surface tension with the bubble-pressure method is proposed. Compared with a traditional instrument, this apparatus improved the accuracy of maximum pressure difference measurement and achieved the hydrostatic pressure correction. The apparatus was employed to determine the surface tension values of distilled water and ethanol, respectively, at different temperatures. Additionally, the cross-sectional area of the ethanol molecule was obtained from the surface tension of ethanol solutions with different concentrations and Gibbs adsorption. It was found that all measured values were in accordance with literature data. Thus, the improved device exhibited excellent accuracy and reliability. Furthermore, all of the instrumental components were assembled into one body, making it easy for undergraduate students to operate.

Due to the damage of scalp because of chemical dyes, increasing attention had been paid to the ecological dyeing of human hair. To address the need for ecological restoration and dyeing of human hair, herein, a novel approach has been proposed via laccase-mediated in situ oxidation of dihydroxy phenylalanine (dopamine). As laccase can catalyze dopamine oxidation and then polymerization to produce strongly colored polymers, different biological coloration processes of human hair based on this process were investigated: (i) Human hair was treated with laccase alone; (ii) The absorption of human hair with dopamine, followed by further catalysis of air; (iii) The absorption of human hair with dopamine, followed by further catalysis of laccase; (iv) Simultaneous laccase-mediated polymerization and dyeing of human hair; and (v) Polymerization of Dopamine with laccase and then dyeing human hair. The surface properties of dyed hair were evaluated in terms of ultraviolet (UV)-protection, color fastness, Fourier transforming infrared spectrum (FT-IR), optical microscope, and scanning electron microscopy (SEM). The results show that best hair dyeing effect was achieved via simultaneous laccase-mediated polymerization and dyeing. The dyed hair obtained K/S values of 28, light fastness of level 5, and UPF of 100+. The optical microscope and SEM observations show that the surface of colored hair fibers was covered with thin burrs, which can be attributed to polydopamine attached with physical adhesive force and covalent reaction. FT-IR analysis shows that functional groups were present on the surface of the dyed hair which can be considered as introduction of hydroxyl radical groups. This biological stain technique may be widely used in dyeing of hair with black color.

Cancer stem cells (CSCs) are characterized by their ability to self-renew, to differentiate into multiple cell types and also drive tumor formation, altogether making them important cellular targets for therapeutic intervention. However, existing CSC-targeting drugs do not significantly improve clinical outcomes. More recently, preclinical studies of natural product-derived compounds have demonstrated their potential usefulness as a therapeutic cancer treatment through their cytotoxic actions on CSCs.Here, we identify CSC-specific compounds derived from natural products and characterize their putative mechanisms of action in CSCs.Glioblastoma stem cells (GSCs) were labeled with EGFP via homologous recombination and utilized for a high-throughput screen of 8,344 fractions from 386 herbal medicines. The fractions that extinguished EGFP fluorescence signal were then further characterized by LC-MS/MS. Next, several putative cytotoxic compounds were evaluated for their cytotoxic effects on GSCs, cancer cell lines and immortalized cells using a variety of methods to study cell proliferation (EdU incorporation assay), cell death (cleaved-Caspase-3 immunostaining), DNA damage (comet assay), mitochondrial membrane changes (JC-1 immunostaining), and tumor formation in vitro (soft agar colony forming assay). We also performed surface plasmon resonance analysis, western blotting, and immunohistochemistry to characterize the putative mechanisms underlying the cytotoxic effects of putative compounds on GSCs. Finally, we carried out xenograft tumor growth assays to study the cytotoxic potential of several candidates in vivo.Our high throughput screen led to the identification of the furostanol saponin taccaoside A and its two homologs from the rhizomatous geophyte Tacca. subflabellata that were cytotoxic to GSCs. Interestingly, the cytotoxic effect of taccaoside A on cell lines was significantly less compared to its homologs, owing to stereochemical differences of a carbon-carbon double bond between C-20 and C-22. Molecular studies revealed that taccaoside A binds to RAS to inhibit downstream effector signaling. Correspondingly, blockade of the interaction between taccaoside A and RAS abolished the inhibitory effect of this compound on CSCs. Furthermore, taccaoside A treatment was effective in limiting tumor cell growth in vivo.Our study yielded an effective approach to screen for CSC-specific agents. Through this approach, we identified taccaoside A from the rhizomatous geophyte Tacca. subflabellata are cytotoxic to CSCs through a molecular mechanism that involves RAS binding and suppression of its downstream signaling. Our findings indicate taccaoside A is a potential lead compound for anti-CSC drug discovery.

The durative endoplasmic reticulum stress (ERS) and subsequent apoptosis contributes to the development and progression of Alzheimer’s disease (AD). MiR-326 can reduce pyruvate kinase M2 (PKM2) exp...

Thermoelastic damping (TED) is one of the main internal energy dissipation mechanisms in micro-/nano-resonators. Accurate evaluation of TED is important in the design of micro-electromechanical systems and nano-electromechanical systems. In this paper, a theoretical analysis on the TED in functionally graded material (FGM) micro-beam resonators is presented. Equations of motion and the heat conduction equation governing the thermodynamic coupling free vibration of non-homogenous micro-beams are established based on the Euler–Bernoulli beam theory associated with the modified couple stress theory. Material properties of the FGM micro-beam are assumed to change in the depth direction as power-law functions. The layer-wise homogenization method is used for solving the heat conduction equation. By using the mathematical similarity of eigenvalue problem between the FGM beam and the reference homogeneous one, the complex natural frequency including TED is expressed in terms of the natural frequency of the isothermal homogenous beam. In the presented numerical results, influences of various characteristic parameters, such as beam thickness, material gradient index, structure size, vibration mode and boundary conditions, on TED are examined in detail. It shows that TED decreases with the increases in the values of length scale parameters because the latter lead to the increase in structural stiffness.

Prophenoloxidase (PPO), an important immunity protein in insects, is mainly produced by hemocytes and released into the hemolymph upon cell lysis. In addition, PPO can also be produced by epidermal cells in the foregut to detoxify the toxic plant secondary metabolites and in the hindgut to kill pathogens through PPO-induced melanization. Previously, we noticed a pair of tubes extended from the larval hindgut became melanized upon staining in dopamine dissolved in 30% ethanol. However, the structure and function of these tubes are largely unknown. In this study, we performed staining of the tubes and the neighboring Malpighian tubule for further confirmation. Eventually, we detected PPO inside epidermal cells of the tubes, and called them as PPO-positive tubes. We observed that the PPO-positive tubes are physically derived from the hindgut but strongly adhere to the Malpighian tubule. Inside the PPO-positive tubes, there is an acellular peritrophic membrane to protect the epidermal cells. Furthermore, the PPO-positive tubes act like a doorkeeper to firstly detoxify the metabolite wastes collected by the Malpighian tubule from the hemolymph.