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At least 19 recordsLinked to original sources
PubMed1992

Calcium depletion blocks proteolytic cleavages of plasma protein precursors which occur at the Golgi and/or trans-Golgi network. Possible involvement of Ca(2+)-dependent Golgi endoproteases.

The effects of calcium depletion on the proteolytic cleavage and secretion of plasma protein precursors were investigated in primary cultured rat hepatocytes and HepG2 cells. When the cells were incubated with A23187, the calcium-specific ionophore, in a medium lacking CaCl2, precursors of serum albumin and the third and fourth components of complement, C3 and C4, respectively, were found to be released into the medium. The addition of ionomycin or EGTA to the medium inhibited the processing of pro-C3 as well. Blocking the secretory pathway either at the mixed endoplasmic reticulum/Golgi in the presence of brefeldin A or at the endoplasmic reticulum/tubular-vesicular structure at a reduced temperature caused accumulation of pro-C3 within hepatocytes or HepG2 cells, indicating that the cleavage of the precursor occurs at a later stage of the secretory pathway. Once the blockade was released by incubating the cells either in the brefeldin A-free medium or at 37 degrees C, the secretion of plasma proteins resumed, irrespective of the presence of A23187. However, the processing of pro-C3 was almost completely inhibited in the presence of A23187, with only the precursor being released into the medium, implying that a decline in Ca2+ levels within the cell modulates the activity of a Golgi endoprotease responsible for the cleavage of pro-C3. When incubated with isolated Golgi membranes, pro-C3 secreted from Ca(2+)-depleted cells was cleaved in vitro into their subunits in the presence of Ca2+ but not in its absence, pointing to the involvement of a Ca(2+)-dependent Golgi endoprotease in the processing of pro-C3. These results collectively suggest that calcium depletion blocks the proteolytic cleavages of plasma protein precursors presumably by exhausting a Ca2+ pool available to the Ca(2+)-dependent processing enzyme(s) located at the Golgi and/or trans-Golgi network.

Animals↗
PubMed1992

Three-dimensional structure of cytidine monophosphatase reactive trans-Golgi elements in spinal ganglion cells of the rat.

In order to analyse, at the electron microscope level, the three-dimensional configuration of the trans compartment of the Golgi apparatus rat dorsal root ganglia were treated to demonstrate cytidine monophosphatase (CMPase) activity. The localization of enzymatic activity in the Golgi apparatus varied according to cell types. In type A and C cells, CMPase was exclusively located in the transmost sacculotubular element, whereas in type B cells all the saccules of the stacks forming the Golgi ribbon and the trans-Golgi networks were impregnated. Numerous dense bodies seen at proximity were also CMPase positive. In 3 microns thick sections of type A cells examined at low magnification, the impregnated element was scattered throughout the cytoplasm and never formed a continuous structure. In type B cells, the strongly reactive trans-Golgi networks did not follow the entire length of the impregnated Golgi ribbon but were preferentially located in the concavity of its arched portions. At higher magnification and in all cell types some tubular portions of the trans-Golgi networks took the appearance of spheroidal cage-like structures, the CMPase positive anastomotic tubules forming the bars of the cage. Anastomotic tubules separated from the trans-Golgi networks formed fenestrated spheres, while nearby CMPase-reactive dense bodies exhibited a paler hilus. These observations were taken to indicate that in ganglion cells, some CMPase positive dense bodies, presumably lysosomes, formed by fragmentation of the trans-Golgi networks.

Animals↗
PubMed2026

CRISPR/Cas9 loss-of-function screen in a neuronal model of AP-4 deficiency identifies ATG9A trafficking modulators.

Biallelic loss-of-function variants in adaptor protein complex 4 (AP-4) disrupt trafficking of transmembrane proteins at the trans-Golgi network, including autophagy-related protein 9A (ATG9A), leading to childhood-onset hereditary spastic paraplegia (AP-4-HSP). AP-4-HSP is characterized by features of both a neurodevelopmental and a degenerative neurological disease. To investigate the molecular mechanisms underlying AP-4-HSP and identify potential therapeutic targets, we conducted an arrayed CRISPR/Cas9 loss-of-function screen of 8,478 genes, targeting the "druggable genome," in a human neuronal model of AP-4 deficiency. Through this phenotypic screen and subsequent experiments, key modulators of ATG9A trafficking were identified, and complementary pathway analyses provided insights into the regulatory landscape of ATG9A transport. Knockdown of ANPEP and NPM1 enhanced ATG9A availability outside the trans-Golgi network, suggesting that they regulate ATG9A localization. These findings deepen our understanding of ATG9A trafficking in the context of AP-4 deficiency and offer a framework for the development of targeted interventions for AP-4-HSP.

Humans↗
PubMedDate not supplied

Specific interactions of pancreatic amylase at acidic pH. Amylase and the major protein of the zymogen granule membrane (GP-2) bind to immobilized or polymerized amylase.

Regulated secretory proteins are thought to be sorted in the trans-Golgi network towards the secretory granule via acidic aggregation. In the exocrine pancreas, amylase is one of the major zymogens. It is a basic protein of pI 8.6 and does not precipitate in acidic conditions. To identify the mechanism by which amylase aggregates in the acidic cisternae of the pancreatic trans-Golgi network, we have developed an in vitro model in which amylase was fixed to plastic microtiter plates. The fixed amylase was probed with two ligands: amylase itself and GP-2, the major protein of the zymogen granule membrane. Biotinylated amylase bound to fixed amylase in a strict pH-dependent manner with optimal binding between pH 5.0 and 5.7. The affinity of binding was in the nanogram range (Kd approximately 20.0 ng/mL) at pH 5.5. Acid binding of amylase was not reversible by incubation at neutral pH, nor could it be displaced by native amylase. GP-2 binding to fixed amylase was also pH dependent with optimal binding between pH 5.0 and 5.7. As for amylase, it was not reversible by incubation at neutral pH. GP-2 binding sites on fixed amylase appeared to be different from those of biotinylated amylase. While native and biotinylated amylase did not bind to GP-2, polymerized amylase precipitated GP-2 at acidic pH. Taken together these data suggest that slight modifications are sufficient to reveal on the amylase molecule binding sites for GP-2 and for amylase itself. These new binding capacities acquired at acidic pH could be involved in the cascade of reactions that lead to the in vivo formation of the immature secretory granule.

Amylases↗
PubMed1992

Autophagy, cathepsin L transport, and acidification in cultured rat fibroblasts.

The mechanisms of enzyme delivery to and acidification of early autophagic vacuoles in cultured fibroblasts were elucidated by cryoimmunoelectron microscopic methods. The cation-independent mannose-6-phosphate receptor (MPR) was used as a marker of the pre-lysosomal compartment, and cathepsin L and an acidotropic amine (3-(2,4-dinitroanilino)-3'-amino-N-methyl-dipropylamine (DAMP), a cytochemical probe for low-pH organelles) as markers of both pre-lysosomal and lysosomal compartments. In addition, cationized ferritin was used as an endocytic marker. In ultrastructural double labeling experiments, the bulk of all the antigens was found in vesicles containing tightly packed membrane material. These vesicles also contained small amounts of endocytosed ferritin and probably correspond to the MPR-enriched pre-lysosomal compartment. Some immunolabeling was also visible in the trans-Golgi network. In addition, cathepsin L, DAMP, and large amounts of ferritin were found in smaller vesicles which can be classified as mature lysosomes. Early autophagic vacuoles were defined as vesicles containing recognizable cytoplasm. MPR, cathepsin L, and DAMP, but not ferritin, were detected in the early vacuoles. Inhibition of the acidification in the early vacuoles by monensin did not prevent the delivery of MPR and cathepsin L. The presence of MPR in the vacuoles suggests that cathepsin L is not delivered to early autophagic vacuoles solely by fusion with mature, MPR-deficient lysosomes. Furthermore, although lysosomes were loaded with endocytosed ferritin, it was not detected in autophagic vacuoles. Either the trans-Golgi network or the MPR-enriched pre-lysosomes may be the main source of enzymes and acidification machinery for the autophagic vacuoles in fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Structural and histochemical studies of Golgi complex differentiation in salivary gland cells during Drosophila development.

Morphological alterations in the Golgi complex (GC) and changes in the distribution of acid phosphatase (AcPase), thiamine pyrophosphatase (TPPase), complex carbohydrates and reduced osmium tetroxide compounds in this organelle were studied in the salivary gland cells of Drosophila during larval and prepupal development. The morphology and the AcPase, TPPase and complex carbohydrates cytochemical patterns of the Golgi complex varied characteristically during cell differentiation. At the early 3rd instar period the Golgi complex consisted mainly of vesiculated cisternae, and AcPase activity was observed in all cisternae but not in the secretory granules. As development proceeded to the late 3rd instar the Golgi complex displayed its typical appearance, consisting of four to six cisternae, and only the two to three cisternae towards the trans-face as well as the trans-Golgi network and some of the immature secretory granules exhibited AcPase reactivity. In the course of a 'wave' of production of the 'glue' secretory granules proceeding proximally through the gland, the number of AcPase positive cisternae changed correspondingly. After secretion of the 'glue' secretory granules, the size of the Golgi complex decreased and almost all cisternae displayed AcPase reactivity. The detection of TPPase activity presented some specificity problems, since staining was observed not only in the GC cisternae but in the endoplasmic reticulum (ER) and microvilli. The reaction products were seen in a few GC vesicles during the early 3rd instar and in the trans side of the organelle at the end of the 3rd instar. During production of the secretory granules, every GC cisterna was intensely stained. These results agree with previous findings suggesting that AcPase and TPPase in secretory cells may be primarily involved in the processing of exportable proteins. The vicinal (vic)-glycol groups of the complex carbohydrates were detected using the periodic acid/thiocarbohydrazide/silver proteinate (PA-TCH-SP) technique. During synthesis of the 'glue' secretory granules, the reaction products were observed over the GC cisternae and the trans-Golgi network, with increasing intensity from the cis to the trans side of the organelle. No PA-TCH-SP staining was observed over the GC cisternae during the early 3rd instar. Following discharge of the 'glue' secretory granules, all GC cisternae displayed uniform PA-TCH-SP staining. After OsO4 impregnation, the reaction products were observed mainly in ER and mitochondria and rarely in the GC. In numerous cells, only the mitochondria were stained, while in many cases the ER of neighboring cells exhibited differential staining.(ABSTRACT TRUNCATED AT 400 WORDS)

Acid Phosphatase↗
PubMed1992

The morphology but not the function of endosomes and lysosomes is altered by brefeldin A.

Brefeldin A (BFA) induces the formation of an extensively fused network of membranes derived from the trans-Golgi network (TGN) and early endosomes (EE). We describe in detail here the unaffected passage of endocytosed material through the fused TGN/EE compartments to lysosomes in BFA-treated cells. We also confirmed that BFA caused the formation of tubular lysosomes, although the kinetics and extent of tubulation varied greatly between different cell types. The BFA-induced tubular lysosomes were often seen to form simple networks. Formation of tubular lysosomes was microtubule-mediated and energy-dependent; interestingly, however, maintenance of the tubulated lysosomes only required microtubules and was insensitive to energy poisons. Upon removal of BFA, the tubular lysosomes rapidly recovered in an energy-dependent process. In most cell types examined, the extensive TGN/EE network is ephemeral, eventually collapsing into a compact cluster of tubulo-vesicular membranes in a process that precedes the formation of tubular lysosomes. However, in primary bovine testicular cells, the BFA-induced TGN/EE network was remarkably stable (for > 12 h). During this time, the TGN/EE network coexisted with tubular lysosomes, however, the two compartments remained completely separate. These results show that BFA has multiple, profound effects on the morphology of various compartments of the endosome-lysosome system. In spite of these changes, endocytic traffic can continue through the altered compartments suggesting that transport occurs through noncoated vesicles or through vesicles that are insensitive to BFA.

Adenosine Triphosphate↗
PubMed2026

Host ESCRT machinery orchestrates the assembly of tomato spotted wilt virus ribonucleoproteins.

The genomic RNA of negative-strand RNA viruses is encapsidated by nucleocapsid proteins and associates with RNA polymerase to form a ribonucleoprotein (RNP) complex. Lacking both a 5' cap and a 3' poly (A) tail, viral RNAs are highly unstable and prone to degradation by cellular nucleases. Therefore, newly synthesized genomic and complementary-strand RNAs must be rapidly protected through RNP formation. However, the molecular mechanisms governing RNP assembly in cytoplasm-replicating negative-strand RNA viruses remain largely unknown. Here, we screened a yeast knockout library and isolated mutants in several components of the endosomal sorting complexes required for transport (ESCRT) genes that affected RNA replication of tomato spotted wilt virus (TSWV). In wild-type (WT) yeast cells, TSWV nucleocapsid (N) and RNA polymerase (L) proteins colocalize at the trans-Golgi network (TGN) in a replicon-RNA-dependent manner, suggesting that TSWV RNPs accumulate at the TGN. However, in the snf7Δ, bro1Δ, and doa4Δ mutant cells, N localization to TGN and RNP formation were impaired. Another RNA replication-defective mutant, vps36Δ, showed normal N localization, and SNF7, BRO1, and DOA4 were recruited to the TGN by TSWV N or L proteins, implying that the ESCRT components have additional roles in TSWV RNA replication beyond facilitating N transport. These findings suggest that ESCRT components play multifaceted roles in TSWV RNA replication, including the intracellular transport of N to the TGN-where RNA replication takes place-thereby ensuring accurate and efficient RNP assembly.

Tospovirus↗
PubMed1992

Investigation of the role of microtubules in protein secretion from lactating mouse mammary epithelial cells.

Disruption of microtubules has been shown to reduce protein secretion from lactating mammary epithelial cells. To investigate the involvement of microtubules in the secretory pathway in these cells we have examined the effect of nocodazole on protein secretion from mammary epithelial cells derived from the lactating mouse. Mouse mammary cells have extensive microtubule networks and 85% of their tubulin was in a polymeric form. Treatment with 1 micrograms/ml nocodazole converted most of the tubulin into a soluble form. In a continuous labelling protocol it was found that nocodazole did not interfere with protein synthesis but over a 5 h period secretion was markedly inhibited. To determine whether the inhibition was at the level of early or late stages of the secretory pathway mammary cells were pulse-labelled for 1 h to label protein throughout the secretory pathway before nocodazole treatment. When secretion was subsequently assayed it was found to be slower and only partially inhibited. These findings suggest that the major effect of nocodazole is on an early stage of the secretory pathway and that microtubules normally facilitate vesicle transport to the plasma membrane. An involvement of microtubules in vesicle transport to the plasma membrane is consistent with an observed accumulation of casein vesicles in nocodazole-treated cells. Exocytosis stimulated by the calcium ionophore ionomycin was unaffected by nocodazole treatment. We conclude from these results that the major effect of nocodazole is at an early stage of the secretory pathway, one possible target being casein vesicle biogenesis in the trans-Golgi network.

Animals↗
PubMed1992

Modulation of transcytotic and direct targeting pathways in a polarized thyroid cell line.

Two biosynthetic pathways exist for delivery of membrane proteins to the apical surface of epithelial cells, direct transport from the trans-Golgi network (TGN) and transcytosis from the basolateral membrane. Different epithelial cells vary in the expression of these mechanisms. Two extremes are MDCK cells, that use predominantly the direct route and hepatocytes, which deliver all apical proteins via the basolateral membrane. To determine how epithelial cells establish a particular targeting phenotype, we studied the apical delivery of endogenous dipeptidyl peptidase IV (DPPIV) at early and late stages in the development of monolayers of a highly polarized epithelial cell line derived from Fischer rat thyroid (FRT). In 1 day old monolayers, surface delivery of DPPIV from the TGN was unpolarized (50%/50%) but a large basal to apical transcytotic component resulted in a polarized apical distribution. In contrast, after 7 days of culture, delivery of DPPIV was mainly direct (85%) with no transcytosis of the missorted component. A basolateral marker, Ag 35/40 kD, on the other hand, was directly targeted (90-98%) at all times. These results indicate that the sorting machinery for apical proteins develops independently from the sorting machinery for basolateral proteins and that the sorting site relocates progressively from the basal membrane to the TGN during development of the epithelium. The transient expression of the transcytotic pathway may serve as a salvage pathway for missorted apical proteins when the polarized phenotype is being established.

Animals↗
PubMed2024

Dysfunction of ATP7B Splicing Variant Caused by Enhanced Interaction With COMMD1 in Wilson Disease.

BACKGROUND & AIMS: The association between Wilson disease and various ATP7B mutations is well-established; however, the molecular mechanism underlying the functional consequence of these mutations, particularly the splicing mutations, remains unclear. This study focused on the ATP7B c.1543+1G>C variant, to reveal a universal pathogenic mechanism of the ATP7B mutants with altered N-terminus. METHODS: The splicing assay and RNA pull-down were performed to explore the mechanism of the aberrant splicing. The ATP7B knockout HuH-7 cell line and Atp7b-/- mice were created, and the functional consequence of the mutant ATP7B were evaluated in vitro and in vivo. RESULTS: The c.1543+1G>C mutation resulted in the skipping of ATP7B exon 3, and the mutant ATP7B showed a loss of trans-Golgi network localization and was degraded via the ubiquitin-proteasome pathway, facilitated by enhanced interactions with COMMD1. Elevated intercellular copper concentration and reduced survival rate were observed in HuH-7 cells expressing mutant ATP7B. Restoration of wild-type ATP7B in Atp7b-/- mice resulted in a substantial improvement in phenotype, whereas mice treated with mutant ATP7B did not demonstrate equivalent benefits. CONCLUSIONS: Our research investigated the pathogenicity and mechanism of ATP7B c.1543+1G>C variant, with particular focus on its enhanced interaction with COMMD1 as a potential universal mechanism contributing to the dysfunction of various ATP7B variants. These findings provide a foundation for the development of innovative therapeutic strategies that target abnormal splicing events in a range of hereditary diseases, including Wilson disease.

Copper-Transporting ATPases↗
PubMed1992

Selective inhibition of protein targeting to the apical domain of MDCK cells by brefeldin A.

Dipeptidyl peptidase IV (DPPIV) is mainly vectorially targeted to the apical surface in MDCK cells. BFA was found to abolish the apical targeting of DPPIV. This BFA effect could be achieved under conditions where the ER to Golgi transport and the total surface expression of DPPIV were essentially unaffected. BFA executed its effect during the transport from the trans-Golgi network (TGN) to the surface. The inhibition of apical targeting resulted in enhanced mis-targeting to the basolateral surface. The mistargeted DPPIV was transcytosed back to the apical domain only after BFA withdrawal. In contrast, the basolateral targeting of uvomorulin was unaffected by BFA. These results established that the apical targeting of DPPIV was selectively abolished by BFA.

Animals↗
PubMed1992

Domain-specific and cell type-specific localization of two types of cell wall matrix polysaccharides in the clover root tip.

Using immunocytochemical techniques and antibodies that specifically recognize xyloglucan (anti-XG), polygalacturonic acid/rhamnogalacturonan I (anti-PGA/RG-I), and methylesterified pectins (JIM 7), we have shown that these polysaccharides are differentially synthesized and localized during cell development and differentiation in the clover root tip. In cortical cells XG epitopes are present at a threefold greater density in the newly formed cross walls than in the older longitudinal walls, and PGA/RG-I epitopes are detected solely in the expanded middle lamella of cortical cell corners, even after pretreatment of sections with pectinmethylesterase to uncover masked epitopes. These results suggest that in cortical cells XG and PGA/RG-I are differentially localized not only to particular wall domains, but also to particular cell walls. In contrast to their nonoverlapping distribution in cortical cells, XG epitopes and PGA/RG-I epitopes largely colocalize in the epidermal cell walls. The results also demonstrate that the middle lamella of the longitudinal walls shared by epidermal cells and by epidermal and cortical cells constitutes a barrier to the diffusion of cell wall and mucilage molecules. Synthesis of XG and PGA/RG-I epitope-containing polysaccharides also varies during cellular differentiation in the root cap. The differentiation of gravitropic columella cells into mucilage-secreting peripheral cells is marked by a dramatic increase in the synthesis and secretion of molecules containing XG and PGA/RG-I epitopes. In contrast, JIM 7 epitopes are present at abundant levels in columella cell walls, but are not detectable in peripheral cell walls or in secreted mucilage. There were also changes in the cisternal labeling of the Golgi stacks during cellular differentiation in the root tip. Whereas PGA/RG-I epitopes are detected primarily in cis- and medial Golgi cisternae in cortical cells (Moore, P. J., K. M. M. Swords, M. A. Lynch, and L. A. Staehelin. 1991. J. Cell Biol. 112:589-602), they are localized predominantly in the trans-Golgi cisternae and the trans-Golgi network in epidermal and peripheral root cap cells. These observations suggest that during cellular differentiation the plant Golgi apparatus can be both structurally and functionally reorganized.

Antibodies, Monoclonal↗
PubMed1992

Overexpression of human alpha-galactosidase A results in its intracellular aggregation, crystallization in lysosomes, and selective secretion.

Human lysosomal alpha-galactosidase A (alpha-Gal A) was stably overexpressed in CHO cells and its biosynthesis and targeting were investigated. Clone AGA5.3-1000Mx, which was the highest enzyme overexpressor, produced intracellular alpha-Gal A levels of 20,900 U/mg (approximately 100 micrograms of enzyme/10(7) cells) and secreted approximately 13,000 U (or 75 micrograms/10(7) cells) per day. Ultrastructural examination of these cells revealed numerous 0.25-1.5 microns crystalline structures in dilated trans-Golgi network (TGN) and in lysosomes which stained with immunogold particles using affinity-purified anti-human alpha-Gal A antibodies. Pulse-chase studies revealed that approximately 65% of the total enzyme synthesized was secreted, while endogenous CHO lysosomal enzymes were not, indicating that the alpha-Gal A secretion was specific. The recombinant intracellular and secreted enzyme forms were normally processed and phosphorylated; the secreted enzyme had mannose-6-phosphate moieties and bound the immobilized 215-kD mannose-6-phosphate receptor (M6PR). Thus, the overexpressed enzyme's selective secretion did not result from oversaturation of the M6PR-mediated pathway or abnormal binding to the M6PR. Of note, the secreted alpha-Gal A was sulfated and the percent of enzyme sulfation decreased with increasing amplification, presumably due to the inaccessibility of the enzyme's tyrosine residues for the sulfotransferase in the TGN. Overexpression of human lysosomal alpha-N-acetylgalactosaminidase and acid sphingomyelinase in CHO cell lines also resulted in their respective selective secretion. In vitro studies revealed that purified secreted alpha-Gal A was precipitated as a function of enzyme concentration and pH, with 30% of the soluble enzyme being precipitated when 10 mg/ml of enzyme was incubated at pH 5.0. Thus, it is hypothesized that these overexpressed lysosomal enzymes are normally modified until they reach the TGN where the more acidic environment of this compartment causes the formation of soluble and particulate enzyme aggregates. A significant proportion of these enzyme aggregates are unable to bind the M6PR and are selectively secreted via the constitutive secretory pathway, while endogenous lysosomal enzymes bind the M6PRs and are transported to lysosomes.

Animals↗
PubMed1992

Renal epithelial cell polarity.

Recent cell biologic studies of protein trafficking, sorting, and distribution in polarized renal epithelial cells have begun to provide important new insights into the mechanisms involved in generating and maintaining cell surface polarity. Advances in this field have been rapid in the last year, due in part to the development of new approaches to analyzing protein delivery and distribution in polarized renal cells grown in vitro. Sorting signals within apical and basal-lateral membrane proteins have been described that may be involved in the segregation of proteins into different populations of transport vesicles in the trans-Golgi network; the nature of these signals has provided insight into the mechanisms involved. Elements of the cytoskeleton appear to be involved in the delivery of these transport vesicles to the appropriate membrane domain (microtubules) and in the retention of specific proteins in the correct membrane domain (membrane skeleton). Finally, detailed analysis of two prominent renal diseases, ischemia and polycystic kidney disease, indicates that abnormalities in the regulation of membrane protein distribution may be a contributing factor in generating the disease state.

Animals↗
PubMed1992

Inhibition of human cytomegalovirus maturation by brefeldin A.

Brefeldin A (BFA) was found to interfere with specific events of human cytomegalovirus (HCMV) maturation in human fibroblasts. Ultrastructural as well as biochemical studies suggested that short-term exposure of infected cultures to BFA during the late infectious cycle primarily prevented Golgi-dependent processes, e.g. envelopment of naked cytoplasmic nucleocapsids in the trans-Golgi network (TGN) and normal processing of glycoprotein B. In contrast, the nuclear phase of viral morphogenesis, e.g. transport budding at the nuclear envelope, was not impaired. These observations were compatible with the interpretation that HCMV morphogenesis may involve sequential budding events at the nuclear envelope and at cisternae of the TGN. BFA treatment during the early infectious cycle efficiently inhibited HCMV-DNA synthesis and thus late viral functions, preventing production of viral progeny. Cytotoxicity was excluded as a cause for these findings.

Antiviral Agents↗
PubMed2026

Comprehensive bioinformatics analysis identifies candidate ciliogenesis-related genes preferentially associated with N0-stage lung squamous cell carcinoma.

PURPOSE: There is few research on which genes play an important role in tumors without lymph metastasis. This study aimed to identify candidate molecular alterations preferentially associated with N0-stage LUSC. METHODS: we conducted a comprehensive bioinformatics analysis using publicly available The Cancer Genome Atlas (TCGA) data. Differentially expressed genes (DEGs) were identified separately by comparing N0 tumors and N+ tumors with normal lung tissues. Genes dysregulated in both N0 and N+ tumors were excluded to identify candidate N0-associated genes PPI networks were constructed using STRING and Cytoscape, with module analysis performed via MCODE. Hub genes were identified using multiple Cytohubba algorithms. Functional enrichment analyses were conducted using GO, and KEGG pathways using DAVID. Gene interaction networks were further explored using GeneMANIA. Immune cell infiltration was evaluated with TIMER. Associations with pathological stage and patient survival were assessed using GEPIA and other relevant tools. RESULTS: A total of 1103 candidate N0-associated DEGs were identified, including 748 upregulated and 355 downregulated genes. The PPI network contained five major MCODE clusters. One cluster (MCODE 4) included TTC30A, TTC30B, BBS7, and KIF3B genes implicated in ciliogenesis. TTC30B showed significant differential expression across pathological stages in the overall LUSC cohort. Seven consensus hub genes (ERBB2, CHUK, CASP8, NOTCH1, HNF4A, CREBBP, and IRS1) were identified based on their consistent ranking across multiple CytoHubba algorithms. Upregulated candidate N0-associated genes were primarily enriched in immune-related processes, including B-cell-mediated immunity and humoral responses, whereas downregulated genes were enriched in lysosomal and trans-Golgi network-related pathways. Exploratory immune infiltration analyses identified associations between the four ciliogenesis-related genes and several immune cell populations. CONCLUSIONS: This study identified candidate molecular signatures preferentially associated with N0-stage LUSC, including ciliogenesis-related genes and consensus hub genes. These findings provide hypotheses regarding molecular features of N0-stage LUSC and warrant further validation in independent cohorts and experimental studies.

Humans↗