CRISPR-Cas13 Inhibitors Block RNA Editing in Bacteria and Mammalian Cells.

Cas13 has demonstrated unique and broad utility in RNA editing, nucleic acid detection, and disease diagnosis; however, a constantly active Cas enzyme may induce unwanted effects. Bacteriophage- or prophage-region-encoded anti-CRISPR (acr) gene molecules provide the potential to control targeting specificity and potency to allow for optimal RNA editing and nucleic acid detection by spatiotemporally modulating endonuclease activities. Using integrated approaches to screen acrVI candidates and evaluate their effects on Cas13 function, we discovered a series of acrVIA1-7 genes that block the activities of Cas13a. These VI-A CRISPR inhibitors substantially attenuate RNA targeting and editing by Cas13a in human cells. Strikingly, type VI-A anti-CRISPRs (AcrVIAs) also significantly muffle the single-nucleic-acid editing ability of the dCas13a RNA-editing system. Mechanistically, AcrVIA1, -4, -5, and -6 bind LwaCas13a, while AcrVIA2 and -3 can only bind the LwaCas13-crRNA (CRISPR RNA) complex. These identified acr molecules may enable precise RNA editing in Cas13-based application and study of phage-bacterium interaction.

Cryo-EM structures of full-length Tetrahymena ribozyme at 3.1 Å resolution.

Single-particle cryogenic electron microscopy (cryo-EM) has become a standard technique for determining protein structures at atomic resolution1-3. However, cryo-EM studies of protein-free RNA are in their early days. The Tetrahymena thermophila group I self-splicing intron was the first ribozyme to be discovered and has been a prominent model system for the study of RNA catalysis and structure-function relationships4, but its full structure remains unknown. Here we report cryo-EM structures of the full-length Tetrahymena ribozyme in substrate-free and bound states at a resolution of 3.1 Å. Newly resolved peripheral regions form two coaxially stacked helices; these are interconnected by two kissing loop pseudoknots that wrap around the catalytic core and include two previously unforeseen (to our knowledge) tertiary interactions. The global architecture is nearly identical in both states; only the internal guide sequence and guanosine binding site undergo a large conformational change and a localized shift, respectively, upon binding of RNA substrates. These results provide a long-sought structural view of a paradigmatic RNA enzyme and signal a new era for the cryo-EM-based study of structure-function relationships in ribozymes.

Dissecting positive selection events and immunological drives during the evolution of adeno-associated virus lineages.

Adeno-associated virus (AAV) serotypes from primates are being developed and clinically used as vectors for human gene therapy. However, the evolutionary mechanism of AAV variants is far from being understood, except that genetic recombination plays an important role. Furthermore, little is known about the interaction between AAV and its natural hosts, human and nonhuman primates. In this study, natural AAV capsid genes were subjected to systemic evolutionary analysis with a focus on selection drives during the diversification of AAV lineages. A number of positively selected sites were identified from these AAV lineages with functional relevance implied by their localization on the AAV structures. The selection drives of the two AAV2 capsid sites were further investigated in a series of biological experiments. These observations did not support the evolution of the site 410 of the AAV2 capsid driven by selection pressure from the human CD4+ T-cell response. However, positive selection on site 548 of the AAV2 capsid was directly related to host humoral immunity because of the profound effects of mutations at this site on the immune evasion of AAV variants from human neutralizing antibodies at both the individual and population levels. Overall, this work provides a novel interpretation of the genetic diversity and evolution of AAV lineages in their natural hosts, which may contribute to their further engineering and application in human gene therapy.

Cryo-EM structures of human GPR34 enable the identification of selective antagonists.

GPR34 is a functional G-protein-coupled receptor of Lysophosphatidylserine (LysoPS), and has pathogenic roles in numerous diseases, yet remains poorly targeted. We herein report a cryo-electron microscopy (cryo-EM) structure of GPR34 bound with LysoPS (18:1) and Gi protein, revealing a unique ligand recognition mode with the negatively charged head group of LysoPS occupying a polar cavity formed by TM3, 6 and 7, and the hydrophobic tail of LysoPS residing in a lateral open hydrophobic groove formed by TM3-5. Virtual screening and subsequent structural optimization led to the identification of a highly potent and selective antagonist (YL-365). Design of fusion proteins allowed successful determination of the challenging cryo-EM structure of the inactive GPR34 complexed with YL-365, which revealed the competitive binding of YL-365 in a portion of the orthosteric binding pocket of GPR34 and the antagonist-binding-induced allostery in the receptor, implicating the inhibition mechanism of YL-365. Moreover, YL-365 displayed excellent activity in a neuropathic pain model without obvious toxicity. Collectively, this study offers mechanistic insights into the endogenous agonist recognition and antagonist inhibition of GPR34, and provides proof of concept that targeting GPR34 represents a promising strategy for disease treatment.

CXCL13 as a Novel Immune Checkpoint for Regulatory B Cells and Its Role in Tumor Metastasis.

Tumor metastasis is the primary cause of mortality in patients with cancer. Several chemokines are identified as important mediators of tumor growth and/or metastasis. The level of CXCL13 has been reported to be elevated in serum or tumor tissues in patients, which mainly functions to attract B cells and follicular B helper T cells. However, the role of CXCL13 in cancer growth and metastasis is not fully explored. In the current study, we found that CXCL13 is not a strong mediator to directly promote tumor growth; however, the mice deficient in CXCL13 had far fewer pulmonary metastatic foci than did the wild-type mice in experimental pulmonary metastatic models. In addition, Cxcl13 -/- mice also had fewer IL-10-producing B cells (CD45+CD19+IL-10+) in the metastatic tumor immune microenvironment than those of wild-type C57BL/6 mice, resulting in an enhanced antitumor immunity. Notably, CXCL13 deficiency further improved the efficacy of a traditional chemotherapeutic drug (cyclophosphamide), as well as that of anti-programmed death receptor-1 immunotherapy. These results suggested that CXCL13 has an important role in regulating IL-10-producing B cells in tumor metastasis and might be a promising target for improving therapeutic efficiency and stimulating tumor immunity in future cancer therapy.

Synergistic effects of chemical additives and mature compost on reducing H2S emission during kitchen waste composting.

Additives could improve composting performance and reduce gaseous emission, but few studies have explored the synergistic of additives on H2S emission and compost maturity. This research aims to make an investigation about the effects of chemical additives and mature compost on H2S emission and compost maturity of kitchen waste composting. The results showed that additives increased the germination index value and H2S emission reduction over 15 days and the treatment with both chemical additives and mature compost achieved highest germination index value and H2S emission reduction (85%). Except for the treatment with only chemical additives, the total sulfur content increased during the kitchen waste composting. The proportion of effective sulfur was higher with the addition of chemical additives, compared with other groups. The relative abundance of H2S-formation bacterial (Desulfovibrio) was reduced and the relative abundance of bacterial (Pseudomonas and Paracoccus), which could convert sulfur-containing substances and H2S to sulfate was improved with additives. In the composting process with both chemical additives and mature compost, the relative abundance of Desulfovibrio was lowest, while the relative abundance of Pseudomonas and Paracoccus was highest. Taken together, the chemical additives and mature compost achieved H2S emission reduction by regulating the dynamics of microbial community.

Enhanced organic degradation and microbial community cooperation by inoculating Bacillus licheniformis in low temperature composting.

Microbial activity and interaction are the important driving factors in the start-up phase of food waste composting at low temperature. The aim of this study was to explore the effect of inoculating Bacillus licheniformis on the degradation of organic components and the potential microbe-driven mechanism from the aspects of organic matter degradation, enzyme activity, microbial community interaction, and microbial metabolic function. The results showed that after inoculating B. licheniformis, temperature increased to 47.8°C on day 2, and the degradation of readily degraded carbohydrates (RDC) increased by 31.2%, and the bioheat production increased by 16.5%. There was an obvious enhancement of extracellular enzymes activities after inoculation, especially amylase activity, which increased by 7.68 times on day 4. The inoculated B. licheniformis colonized in composting as key genus in the start-up phase. Modular network analysis and Mantel test indicated that inoculation drove the cooperation between microbial network modules who were responsible for various organic components (RDC, lipid, protein, and lignocellulose) degradation in the start-up phase. Metabolic function prediction suggested that carbohydrate metabolisms including starch and sucrose metabolism, glycolysis / gluconeogenesis, pyruvate metabolism, etc., were improved by increasing the abundance of related functional genes after inoculation. In conclusion, inoculating B. licheniformis accelerated organic degradation by driving the cooperation between microbial network modules and enhancing microbial metabolism in the start-up phase of composting.

Targeting Metabolic-Redox Circuits for Cancer Therapy.

Metabolic alterations and elevated levels of reactive oxygen species (ROS) are two characteristics of cancer. The metabolic patterns of cancer cells are elaborately reprogrammed to fulfill the high biomass demands of rapid propagation. ROS, the byproducts of metabolic processes, are accumulated in cancer cells partially due to metabolic abnormalities or oncogenic mutations. To prevent oxidative damage, cancer cells can orchestrate metabolic adaptation to maintain reduction-oxidation (redox) balance by producing reducing equivalents. ROS, acting as second messengers, can in turn manipulate metabolic pathways by directly or indirectly affecting the function of metabolic enzymes. In this review we discuss how cancer cell metabolism and redox signaling are intertwined, with an emphasis on the perspective of targeting metabolic-redox circuits for cancer therapy.

Regulation and signaling pathways in cancer stem cells: implications for targeted therapy for cancer.

Cancer stem cells (CSCs), initially identified in leukemia in 1994, constitute a distinct subset of tumor cells characterized by surface markers such as CD133, CD44, and ALDH. Their behavior is regulated through a complex interplay of networks, including transcriptional, post-transcriptional, epigenetic, tumor microenvironment (TME), and epithelial-mesenchymal transition (EMT) factors. Numerous signaling pathways were found to be involved in the regulatory network of CSCs. The maintenance of CSC characteristics plays a pivotal role in driving CSC-associated tumor metastasis and conferring resistance to therapy. Consequently, CSCs have emerged as promising targets in cancer treatment. To date, researchers have developed several anticancer agents tailored to specifically target CSCs, with some of these treatment strategies currently undergoing preclinical or clinical trials. In this review, we outline the origin and biological characteristics of CSCs, explore the regulatory networks governing CSCs, discuss the signaling pathways implicated in these networks, and investigate the influential factors contributing to therapy resistance in CSCs. Finally, we offer insights into preclinical and clinical agents designed to eliminate CSCs.

Pancreatic ß cell microRNA-26a alleviates type 2 diabetes by improving peripheral insulin sensitivity and preserving ß cell function.

Type 2 diabetes (T2D) is characterized by insulin resistance along with pancreatic ß cell failure. ß cell factors are traditionally thought to control glucose homeostasis by modulating insulin levels, not insulin sensitivity. Exosomes are emerging as new regulators of intercellular communication. However, the role of ß-cell-derived exosomes in metabolic homeostasis is poorly understood. Here, we report that microRNA-26a (miR-26a) in ß cells not only modulates insulin secretion and ß cell replication in an autocrine manner but also regulates peripheral insulin sensitivity in a paracrine manner through circulating exosomes. MiR-26a is reduced in serum exosomes of overweight humans and is inversely correlated with clinical features of T2D. Moreover, miR-26a is down-regulated in serum exosomes and islets of obese mice. Using miR-26a knockin and knockout mouse models, we showed that miR-26a in ß cells alleviates obesity-induced insulin resistance and hyperinsulinemia. Mechanistically, miR-26a in ß cells enhances peripheral insulin sensitivity via exosomes. Meanwhile, miR-26a prevents hyperinsulinemia through targeting several critical regulators of insulin secretion and ß cell proliferation. These findings provide a new paradigm for the far-reaching systemic functions of ß cells and offer opportunities for the treatment of T2D.

Bacillus licheniformis inoculation promoted humification process for kitchen waste composting: Organic components transformation and bacterial metabolic mechanism.

Kitchen waste (KW) composting always has trouble with slow humification process and low humification degree. The objective of this study was to develop potentially efficient solutions to improve the humification of KW composting, accelerate the humus synthesis and produce HS with a high polymerization degree. The impact of Bacillus licheniformis inoculation on the transformation of organic components, humus synthesis, and bacterial metabolic pathways in kitchen waste composting, was investigated. Results revealed that microbial inoculation promoted the degradation of organic constituents, especially readily degradable carbohydrates during the heating phase and lignocellulose fractions during the cooling phase. Inoculation facilitated the production and conversion of polyphenol, reducing sugar, and amino acids, leading to an increase of 20% in the content of humic acid compared to the control. High-throughput sequencing and network analysis indicated inoculation enriched the presence of Bacillus, Lactobacillus, and Streptomyces during the heating phase, while suppressing the abundance of Pseudomonas and Oceanobacillus, enhancing positive microbial interactions. PICRUSt2 analysis suggested inoculation enhanced the metabolism of carbohydrates and amino acids, promoting the polyphenol humification pathway and facilitating the formation of humus. These findings provide insights for optimizing the humification process of kitchen waste composting by microbial inoculation.

Untargeted metabolomics elucidated biosynthesis of polyhydroxyalkanoate by mixed microbial cultures from waste activated sludge under different pH values.

Waste activated sludge has been frequently used as mixed substrate to produce polyhydroxyalkanoate (PHA). However, insufficient research on microbial metabolism has led to difficulties in regulating PHA accumulation in mixed microbial cultures (MMCs). To explore the variation of functional genes during domestication and the effect of different pH conditions on metabolic pathways during PHA accumulation, MMCs were domesticated by adding acetate and propionate with aerobic dynamic feeding strategy for 60 days. As the domestication progressed, the microbial community diversity declined and PHA-producing bacteria, Brevundimonas, Dechloromonas and Hyphomonas, were enriched. Through bacterial function prediction by PICRUSt the gene rpoE involved in starvation resistance of bacteria was enriched after the domestication. The pH value of 8.5 was the best condition for PHA accumulation in MMCs, under which a maximum PHA content reached 23.50% and hydroxybutyric (HB)/hydroxyvaleric (HV) reached 2.22. Untargeted metabolomics analysis exhibited that pH conditions of 7 and 8.5 could promote the up-regulation of significant differential metabolites, while higher alkaline conditions caused the inhibition of metabolic activity. Functional annotation showed that pH condition of 8.5 significantly affected Pyrimidine metabolism, resulting in an increase in PHA production. Regarding the pathways of PHA biosynthesis, acetoacetate was found to be significant in the metabolism of hydroxybutyric, and the alkaline condition could restrain the conversion from hydroxybutyric (HB) to the acetoacetate to protect PHB accumulation in MMCs compared with neutral condition. Taken together, the present results can advance the fundamental understanding of metabolic function in PHA accumulation under different pH conditions.

Humic acid and phosphorus fractions transformation regulated by carbon-based materials in composting steered its potential for phosphorus mobilization in soil.

This study investigated the effects of different carbon-based additives including biochar, woody peat, and glucose on humic acid, fulvic acid, and phosphorus fractions in chicken manure composting and its potential for phosphorus mobilization in soil. The results showed that the addition of glucose effectively increased the total humic substance content (90.2 mg/g) of composts, and the fulvic acid content was significantly higher than other groups (P < 0.05). The addition of biochar could effectively improve the content of available phosphorus by 59.9% in composting. The addition of carbon-based materials to the composting was beneficial for the production of more stable inorganic phosphorus in the phosphorus fraction. The highest proportion of soluble inorganic phosphorus components of sodium hydroxide was found in group with woody peat addition (8.7%) and the highest proportion of soluble inorganic phosphorus components of hydrochloric acid was found in group with glucose addition (35.2%). The compost products with the addition of biochar (humic acid decreased by 17.9%) and woody peat (fulvic acid decreased by 72.6%) significantly increased soil humic acid mineralization. The compost products with the addition of biochar was suitable as active phosphate fertilizer, while the compost products with the addition of glucose was suitable as slow-release phosphate fertilizer.

Genomic evolution and diverse models of systemic metastases in colorectal cancer.

OBJECTIVE: The systemic spread of colorectal cancer (CRC) is dominated by the portal system and exhibits diverse patterns of metastasis without systematical genomic investigation. Here, we evaluated the genomic evolution of CRC with multiorgan metastases using multiregion sequencing. DESIGN: Whole-exome sequencing was performed on multiple regions (n=74) of matched primary tumour, adjacent non-cancerous mucosa, liver metastasis and lung metastasis from six patients with CRC. Phylogenetic reconstruction and evolutionary analyses were used to investigate the metastatic seeding pattern and clonal origin. Recurrent driver gene mutations were analysed across patients and validated in two independent cohorts. Metastatic assays were performed to examine the effect of the novel driver gene on the malignant behaviour of CRC cells. RESULTS: Based on the migration patterns and clonal origins, three models were revealed (sequential, branch-off and diaspora), which not only supported the anatomic assumption that CRC cells spread to lung after clonally expanding in the liver, but also illustrated the direct seeding of extrahepatic metastases from primary tumours independently. Unlike other cancer types, polyphyletic seeding occurs in CRC, which may result in late metastases with intermetastatic driver gene heterogeneity. In cases with rapid dissemination, we found recurrent trunk loss-of-function mutations in ZFP36L2, which is enriched in metastatic CRC and associated with poor overall survival. CRISPR/Cas9-mediated knockout of ZFP36L2 enhances the metastatic potential of CRC cells. CONCLUSION: Our results provide genomic evidence for metastatic evolution and indicate that biopsy/sequencing of metastases may be considered for patients with CRC with multiorgan or late postoperative metastasis.

Targeting the tumor stroma for cancer therapy.

Tumors are comprised of both cancer cells and surrounding stromal components. As an essential part of the tumor microenvironment, the tumor stroma is highly dynamic, heterogeneous and commonly tumor-type specific, and it mainly includes noncellular compositions such as the extracellular matrix and the unique cancer-associated vascular system as well as a wide variety of cellular components including activated cancer-associated fibroblasts, mesenchymal stromal cells, pericytes. All these elements operate with each other in a coordinated fashion and collectively promote cancer initiation, progression, metastasis and therapeutic resistance. Over the past few decades, numerous studies have been conducted to study the interaction and crosstalk between stromal components and neoplastic cells. Meanwhile, we have also witnessed an exponential increase in the investigation and recognition of the critical roles of tumor stroma in solid tumors. A series of clinical trials targeting the tumor stroma have been launched continually. In this review, we introduce and discuss current advances in the understanding of various stromal elements and their roles in cancers. We also elaborate on potential novel approaches for tumor-stroma-based therapeutic targeting, with the aim to promote the leap from bench to bedside.

Targeting TGF-ß signal transduction for fibrosis and cancer therapy.

Transforming growth factor ß (TGF-ß) has long been identified with its intensive involvement in early embryonic development and organogenesis, immune supervision, tissue repair, and adult homeostasis. The role of TGF-ß in fibrosis and cancer is complex and sometimes even contradictory, exhibiting either inhibitory or promoting effects depending on the stage of the disease. Under pathological conditions, overexpressed TGF-ß causes epithelial-mesenchymal transition (EMT), extracellular matrix (ECM) deposition, cancer-associated fibroblast (CAF) formation, which leads to fibrotic disease, and cancer. Given the critical role of TGF-ß and its downstream molecules in the progression of fibrosis and cancers, therapeutics targeting TGF-ß signaling appears to be a promising strategy. However, due to potential systemic cytotoxicity, the development of TGF-ß therapeutics has lagged. In this review, we summarized the biological process of TGF-ß, with its dual role in fibrosis and tumorigenesis, and the clinical application of TGF-ß-targeting therapies.

CDC6 is a prognostic biomarker and correlated with immune infiltrates in glioma.

BACKGROUND: Cell division cycle 6 (CDC6) has been proven to be associated with the initiation and progression of human multiple tumors. However, it's role in glioma, which is ranked as one of the common primary malignant tumor in the central nervous system and is associated with high morbidity and mortality, is unclear. METHODS: In this study, we explored CDC6 gene expression level in pan-cancer. Furthermore, we focused on the relationships between CDC6 expression, its prognostic value, potential biological functions, and immune infiltrates in glioma patients. We also performed vitro experiments to assess the effect of CDC6 expression on proliferative, apoptotic, migrant and invasive abilities of glioma cells. RESULTS: As a result, CDC6 expression was upregulated in multiple types of cancer, including glioma. Moreover, high expression of CDC6 was significantly associated with age, IDH status, 1p/19q codeletion status, WHO grade and histological type in glioma (all p < 0.05). Meanwhile, high CDC6 expression was associated with poor overall survival (OS) in glioma patients, especially in different clinical subgroups. Furthermore, a univariate Cox analysis showed that high CDC6 expression was correlated with poor OS in glioma patients. Functional enrichment analysis indicated that CDC6 was mainly involved in pathways related to DNA transcription and cytokine activity, and Gene Set Enrichment Analysis (GSEA) revealed that MAPK pathway, P53 pathway and NF-κB pathway in cancer were differentially enriched in glioma patients with high CDC6 expression. Single-sample gene set enrichment analysis (ssGSEA) showed CDC6 expression in glioma was positively correlated with Th2 cells, Macrophages and Eosinophils, and negative correlations with plasmacytoid dendritic cells, CD8 T cells and NK CD56bright cells, suggesting its role in regulating tumor immunity. Finally, CCK8 assay, flow cytometry and transwell assays showed that silencing CDC6 could significantly inhibit proliferation, migration, invasion, and promoted apoptosis of U87 cells and U251 cells (p < 0.05). CONCLUSION: In conclusion, high CDC6 expression may serve as a promising biomarker for prognosis and correlated with immune infiltrates, presenting to be a potential immune therapy target in glioma.

mRNA Vaccines Against SARS-CoV-2 Variants Delivered by Lipid Nanoparticles Based on Novel Ionizable Lipids.

SARS-CoV-2 variants are now still challenging all the approved vaccines, including mRNA vaccines. There is an urgent need to develop new generation mRNA vaccines with more powerful efficacy and better safety against SARS-CoV-2 variants. In this study, a new set of ionizable lipids named 4N4T are constructed and applied to form novel lipid nanoparticles called 4N4T-LNPs. Leading 4N4T-LNPs exhibit much higher mRNA translation efficiency than the approved SM-102-LNPs. To test the effectiveness of the novel delivery system, the DS mRNA encoding the full-length S protein of the SARS-CoV-2 variant is synthesized and loaded in 4N4T-LNPs. The obtained 4N4T-DS mRNA vaccines successfully trigger robust and durable humoral immune responses against SARS-CoV-2 and its variants including Delta and Omicron. Importantly, the novel vaccines have higher RBD-specific IgG titers and neutralizing antibody titers than SM-102-based DS mRNA vaccine. Besides, for the first time, the types of mRNA vaccine-induced neutralizing antibodies are found to be influenced by the chemical structure of ionizable lipids. 4N4T-DS mRNA vaccines also induce strong Th1-skewed T cell responses and have good safety. This work provides a novel vehicle for mRNA delivery that is more effective than the approved LNPs and shows its application in vaccines against SARS-CoV-2 variants.

Discovery and preclinical evaluations of JBD0131, a novel nitrodihydro-imidazooxazole anti-tuberculosis agent.

Multidrug-resistant pulmonary tuberculosis (MDR-TB) is a major health problem worldwide. The treatment for MDR-TB requires medications for a long duration (up to 20-24 months) with second-line drugs resulting in unfavorable outcomes. Nitroimidazoles are promising antimycobacterial agents known to inhibit both aerobic and anaerobic mycobacterial activity. Delamanid and pretomanid are two nitroimidazoles approved by the regulatory agencies for MDR-TB treatment. However, both agents possess unsatisfactory absorption and QTc prolongation. In our search for a safer nitroimidazole, we discovered JBD0131 (2). It exhibited excellent anti-mycobacterial activity against M. tuberculosis H37Rv in vitro and in vivo, improved PK and absorption, reduced QT prolongation potential of delamanid. JBD0131 is currently in clinical development in China for pulmonary tuberculosis (CTR20202308).

Structural and functional studies of TBC1D23 C-terminal domain provide a link between endosomal trafficking and PCH.

Pontocerebellar hypoplasia (PCH) is a group of neurological disorders that affect the development of the brain, in particular, the pons and cerebellum. Homozygous mutations of TBC1D23 have been found recently to lead to PCH; however, the underlying molecular mechanisms remain unclear. Here, we show that the crystal structure of the TBC1D23 C-terminal domain adopts a Pleckstrin homology domain fold and selectively binds to phosphoinositides, in particular, PtdIns(4)P, through one surface while binding FAM21 via the opposite surface. Mutation of key residues of TBC1D23 or FAM21 selectively disrupts the endosomal vesicular trafficking toward the Trans-Golgi Network. Finally, using the zebrafish model, we show that PCH patient-derived mutants, impacting either phosphoinositide binding or FAM21 binding, lead to abnormal neuronal growth and brain development. Taken together, our data provide a molecular basis for the interaction between TBC1D23 and FAM21, and suggest a plausible role for PtdIns(4)P in the TBC1D23-mediating endosome-to-TGN trafficking pathway. Defects in this trafficking pathway are, at least partially, responsible for the pathogenesis of certain types of PCH.