Neutrophils Recruited by NKX2-1 Suppression via Activation of CXCLs/CXCR2 Axis Promote Lung Adenocarcinoma Progression.

NK2 Homeobox 1 (NKX2-1) is a well-characterized pathological marker that delineates lung adenocarcinoma (LUAD) progression. The advancement of LUAD is influenced by the immune tumor microenvironment through paracrine signaling. However, the involvement of NKX2-1 in modeling the tumor immune microenvironment is still unclear. Here, the downregulation of NKX2-1 is observed in high-grade LUAD. Meanwhile, single-cell RNA sequencing and Visium in situ capturing profiling revealed the recruitment and infiltration of neutrophils in orthotopic syngeneic tumors exhibiting strong cell-cell communication through the activation of CXCLs/CXCR2 signaling. The depletion of NKX2-1 triggered the expression and secretion of CXCL1, CXCL2, CXCL3, and CXCL5 in LUAD cells. Chemokine secretion is analyzed by chemokine array and validated by qRT-PCR. ATAC-seq revealed the restrictive regulation of NKX2-1 on the promoters of CXCL1, CXCL2, and CXCL5 genes. This phenomenon led to increased tumor growth, and conversely, tumor growth decreased when inhibited by the CXCR2 antagonist SB225002. This study unveils how NKX2-1 modulates the infiltration of tumor-promoting neutrophils by inhibiting CXCLs/CXCR2-dependent mechanisms. Hence, targeting CXCR2 in NKX2-1-low tumors is a potential antitumor therapy that may improve LUAD patient outcomes.

Ossicular Reconstruction With Various Degrees of Malleus Preservation in Patients With Chronic Otitis Media.

Objective: We examined the relationship between factors of middle ear conditions and the outcome of ossiculoplasty in chronic otitis media (COM) by measuring the improvement in the air-bone gap (ABG) and air conduction threshold (TAC). Methods: This retrospective study analyzed 76 patients (77 ears) who underwent ossiculoplasty from among 520 COM patients who underwent tympanoplasty based on the maximum preservation of the original ossicles. The reconstructed ossicular chain was performed by preserving or utilizing the remaining malleus in all cases with the presence of the malleus manubrium. Patients with eardrum adhesion, cholesteatoma, and cholesterol granuloma were defined as having a compromised middle ear condition (Group A), and those without as having an uncompromised middle ear condition (Group B). In each group, pure-tone audiometry was performed preoperatively and postoperatively, and improvements in the ABG and TAC were compared. The effects of the types of tympanoplasty and the method of ossiculoplasty (columella versus incus interposition) on postoperative ABG and TAC were also compared. Results: The postoperative ABG improvement in Group B was significantly higher than that in Group A [ß = 7.31, 95% confidence interval (CI) = 1.93-12.69, P < .05]. Type III minor columella tympanoplasty yielded significantly better results than type III major and type Vb tympanoplasty (ß = 11.42, 95% CI = 5.16-17.68, P < .01). There were no significant differences in the postoperative ABG or TAC between the reconstruction groups with and without preservation of malleus. Conclusions: Our results indicate that complex cases compromised by adhesions, cholesteatoma, and cholesterol granuloma have worse outcomes regarding hearing improvement and success rates, while those with intact stapes suprastructure have better outcomes. Malleus was maximally preserved in the patients of this study; however, this showed no significant prognostic benefit in hearing.

HLA-Homozygous iPSC-Derived Mesenchymal Stem Cells Rescue Rotenone-Induced Experimental Leber's Hereditary Optic Neuropathy-like Models In Vitro and In Vivo.

BACKGROUND: Mesenchymal stem cells (MSCs) hold promise for cell-based therapy, yet the sourcing, quality, and invasive methods of MSCs impede their mass production and quality control. Induced pluripotent stem cell (iPSC)-derived MSCs (iMSCs) can be infinitely expanded, providing advantages over conventional MSCs in terms of meeting unmet clinical demands. METHODS: The potential of MSC therapy for Leber's hereditary optic neuropathy (LHON) remains uncertain. In this study, we used HLA-homozygous induced pluripotent stem cells to generate iMSCs using a defined protocol, and we examined their therapeutic potential in rotenone-induced LHON-like models in vitro and in vivo. RESULTS: The iMSCs did not cause any tumorigenic incidence or inflammation-related lesions after intravitreal transplantation, and they remained viable for at least nine days in the mouse recipient's eyes. In addition, iMSCs exhibited significant efficacy in safeguarding retinal ganglion cells (RGCs) from rotenone-induced cytotoxicity in vitro, and they ameliorated CGL+IPL layer thinning and RGC loss in vivo. Optical coherence tomography (OCT) and an electroretinogram demonstrated that iMSCs not only prevented RGC loss and impairments to the retinal architecture, but they also improved retinal electrophysiology performance. CONCLUSION: The generation of iMSCs via the HLA homozygosity of iPSCs offers a compelling avenue for overcoming the current limitations of MSC-based therapies. The results underscore the potential of iMSCs when addressing retinal disorders, and they highlight their clinical significance, offering renewed hope for individuals affected by LHON and other inherited retinal conditions.

Galnt17 loss-of-function leads to developmental delay and abnormal coordination, activity, and social interactions with cerebellar vermis pathology.

GALNT17 encodes a N-acetylgalactosaminyltransferase (GalNAc-T) protein specifically involved in mucin-type O-linked glycosylation of target proteins, a process important for cell adhesion, cell signaling, neurotransmitter activity, neurite outgrowth, and neurite sensing. GALNT17, also known as WBSCR17, is located at the edge of the Williams-Beuren Syndrome (WBS) critical region and adjacent to the AUTS2 locus, genomic regions associated with neurodevelopmental phenotypes that are thought to be co-regulated. Although previous data have implicated Galnt17 in neurodevelopment, the in vivo functions of this gene have not been investigated. In this study, we have analyzed behavioral, brain pathology, and molecular phenotypes exhibited by Galnt17 knockout (Galnt17-/-) mice. We show that Galnt17-/- mutants exhibit developmental neuropathology within the cerebellar vermis, along with abnormal activity, coordination, and social interaction deficits. Transcriptomic and protein analysis revealed reductions in both mucin type O-glycosylation and heparan sulfate synthesis in the developing mutant cerebellum along with disruption of pathways central to neuron differentiation, axon pathfinding, and synaptic signaling, consistent with the mutant neuropathology. These brain and behavioral phenotypes and molecular data confirm a specific role for Galnt17 in brain development and suggest new clues to factors that could contribute to phenotypes in certain WBS and AUTS2 syndrome patients.

Intra-auricular modification of facelift incision decreased the risk of Frey syndrome.

OBJECTIVE: Frey syndrome is a complication followed by parotidectomy which caused gustatory sweating and facial flush. There were several methods for the prevention of Frey syndrome, but most of them had no obvious effects. In this study, we compare the intra-auricular modification of facelift incision with the traditional lazy-S incision to see if it can decrease the risk of Frey syndrome. MATERIALS AND METHODS: This is a retrospective study. From 2003 to 2009, a total of 61 patients with benign parotid tumor who received parotidectomy at Hualien Tzu Chi Hospital and were followed at outpatient department for at least 5 years were enrolled. Patients were divided into two groups according to the type of incisions during operation: (1) Group M: intra-auricular modification of facelift incision or (2) Group S: traditional lazy-S incision. All patients received the partial thickness sternocleidomastoid muscle flap. Clinical data including age, gender, pathologic result, presentation of Frey syndrome, size of tumor, length of operation, blood loss from surgery, length of placement of drain, total amount of drainage, and length of stay were collected and analyzed. RESULTS: Sixty-one patients were enrolled. Eighteen patients were in Group M and forty-three were in Group S. There was no significant difference of age, gender, and size of tumor between the two groups. The pathologic results included parotitis, pleomorphic adenoma, Warthin's tumor, and others. No significant difference of pathologic results, blood loss from surgery, length of placement of drain, total amount of drainage, and length of stay between two groups was obtained. The length of operation was longer in Group M (P = 0.001) and the incidence of Frey syndrome was lower in Group M than Group S (P < 0.05). CONCLUSIONS: The use of intra-auricular modification of facelift incision can decrease the incidence of Frey syndrome.

A Mouse Mutation That Dysregulates Neighboring Galnt17 and Auts2 Genes Is Associated with Phenotypes Related to the Human AUTS2 Syndrome.

AUTS2 was originally discovered as the gene disrupted by a translocation in human twins with Autism spectrum disorder, intellectual disability, and epilepsy. Since that initial finding, AUTS2-linked mutations and variants have been associated with a very broad array of neuropsychiatric disorders, sugg esting that AUTS2 is required for fundamental steps of neurodevelopment. However, genotype-phenotype correlations in this region are complicated, because most mutations could also involve neighboring genes. Of particular interest is the nearest downstream neighbor of AUTS2, GALNT17, which encodes a brain-expressed N-acetylgalactosaminyltransferase of unknown brain function. Here we describe a mouse (Mus musculus) mutation, T(5G2;8A1)GSO (abbreviated 16Gso), a reciprocal translocation that breaks between Auts2 and Galnt17 and dysregulates both genes. Despite this complex regulatory effect, 16Gso homozygotes model certain human AUTS2-linked phenotypes very well. In addition to abnormalities in growth, craniofacial structure, learning and memory, and behavior, 16Gso homozygotes display distinct pathologies of the cerebellum and hippocampus that are similar to those associated with autism and other types of AUTS2-linked neurological disease. Analyzing mutant cerebellar and hippocampal transcriptomes to explain this pathology, we identified disturbances in pathways related to neuron and synapse maturation, neurotransmitter signaling, and cellular stress, suggesting possible cellular mechanisms. These pathways, coupled with the translocation's selective effects on Auts2 isoforms and coordinated dysregulation of Galnt17, suggest novel hypotheses regarding the etiology of the human "AUTS2 syndrome" and the wide array of neurodevelopmental disorders linked to variance in this genomic region.

Acacetin from Traditionally Used Saussurea involucrata Kar. et Kir. Suppressed Adipogenesis in 3T3-L1 Adipocytes and Attenuated Lipid Accumulation in Obese Mice.

Acacetin, a flavone that can be isolated from the Saussurea involucrata plant, has anti-tumor and anti-inflammatory properties that ameliorate airway hyperresponsiveness in asthmatic mice. This study investigated whether acacetin has anti-adipogenic effects in 3T3-L1 adipocytes and whether it regulates the inflammatory response in adipocytes and macrophages. It also investigated whether acacetin ameliorates lipid accumulation in high-fat diet- (HFD) induced obese mice. Differentiated 3T3-L1 cells were treated with acacetin. The glycerol levels in the culture medium were measured, and the expression of proteins and genes involved in adipogenesis and lipolysis were assayed by Western blot and real-time PCR, respectively. Inflammatory cytokine signaling pathway activity was assessed in macrophages that were treated with acacetin and cultured with differentiated medium from 3T3-L1 cells. Intraperitoneal injections of acacetin were administered to HFD-induced obese mice twice a week for 10 weeks. Acacetin significantly increased the levels of glycerol in the culture medium and significantly inhibited lipid accumulation in adipocytes. Acacetin reduced the expression of adipogenesis-related transcription factors, including the expression of the CCAAT/enhancer-binding protein; it also increased sirtuin 1 expression and AMPK phosphorylation in adipocytes. In macrophages cultured with differentiated media from 3T3-L1 adipocytes, acacetin reduced the levels of inflammatory mediators and the activity of the mitogen-activated protein kinase and NF-κB pathways. In obese mice, acacetin reduced both body weight and visceral adipose tissue weight. These results demonstrate that acacetin inhibited adipogenesis in adipocytes and in obese mice. Acacetin also reduced the inflammatory response of macrophages that were stimulated with differentiated media from 3T3-L1 cells.

Comparative Effectiveness of Carotid Artery Stenting Versus Carotid Endarterectomy Among Medicare Beneficiaries.

BACKGROUND: Effectiveness of carotid artery stenting (CAS) relative to carotid endarterectomy (CEA) among Medicare patients has not been established. We compared effectiveness of CAS versus CEA among Medicare beneficiaries. METHODS AND RESULTS: We linked Medicare data (2000-2009) to the Society for Vascular Surgery's Vascular Registry (2005-2008) and the National Cardiovascular Data Registry's (NCDR) Carotid Artery Revascularization and Endarterectomy Registry (2006-2008/2009). Medicare patients were followed up from procedure date until death, stroke/transient ischemic attack, periprocedural myocardial infarction, or a composite end point for these outcomes. We derived high-dimensional propensity scores using registry and Medicare data to control for patient factors and adjusted for provider factors in a Cox regression model comparing CAS with CEA. Among 5254 Society for Vascular Surgery's Vascular Registry (1999 CAS; 3255 CEA) and 4055 Carotid Artery Revascularization and Endarterectomy Registry (2824 CAS; 1231 CEA) Medicare patients, CAS patients had a higher comorbidity burden and were more likely to be at high surgical risk (Society for Vascular Surgery's Vascular Registry: 96.7% versus 44.5%; Carotid Artery Revascularization and Endarterectomy Registry: 71.3% versus 44.7%). Unadjusted outcome risks were higher for CAS. Mortality risks remained elevated for CAS after adjusting for patient-level factors (hazard ratio, 1.24; 95% confidence interval, 1.06-1.46). After further adjustment for provider factors, differences between CAS and CEA were attenuated or no longer present (hazard ratio for mortality, 1.13; 95% confidence interval, 0.94-1.37). Performance was comparable across subgroups defined by sex and degree of carotid stenosis, but there was a nonsignificant trend suggesting a higher risk of adverse outcomes in older (>80) and symptomatic patients undergoing CAS. CONCLUSIONS: Outcomes after CAS and CEA among Medicare beneficiaries were comparable after adjusting for both patient- and provider-level factors.

The antileukemia activity of natural product HQ17(3) is possibly associated with downregulation of miR-17-92 cluster.

The compound 10'(Z),13'(E),15'(E)-heptadecatrienylhydroquinone [HQ17(3)] was purified from the sap of the lacquer tree Rhus succedanea. HQ17(3) has cytotoxic effect on cancer cells and can inhibit topoisomerase (topo) IIα activity. We treated various cancer cells with different doses of HQ17(3) and found that leukemia cells were most sensitive to HQ17(3). After analysis of microRNA (miRNA) profiling, we found that treatment with HQ17(3) caused downregulation of miR-17-92 cluster in some leukemia cells. These changes partially restored the normal levels from leukemia-specific miRNA expression signature. Messenger RNAs of tumor suppressor proteins, such as pRB, PTEN, and Dicer, are targets of miR-17-92 cluster. Their protein levels were increased after the treatment. c-Myc is a regulatory protein for miR-17-92 gene. Similar to topo IIα, we found that c-Myc decreased its activity after the HQ17(3) treatment, which may explain the downregulation of miR-17-92 cluster. Combined with 5-fluorouracil, NaAsO2, or ABT-737, HQ17(3) elicited additive inhibitory effects on leukemia cells. In conclusion, the high sensitivity of leukemia cells to HQ17(3) may be associated with the reduction of topo IIα and c-Myc activities, as well as with the downregulation of the miR-17-92 cluster expression.

Coding region of adiponectin contains a silent intron reactivated by the adjacent intervening sequence of vector.

Precise splicing pre-mRNA into correct mRNA is a tightly orchestrated process involving both cis and trans factors. However, the regulatory mechanism underlying alternative splicing remain elusive. An alternative splicing was revealed by comparing RT-PCR products (cDNA) of human adiponectin gene (ADPN) genes and sequencing the corresponding cDNA recovered from CHO-K1 cells transfected with a pIRES-neo vector carrying the cDNA. We determined that an 88-nt sequence in the original cDNA was missing from the adiponectin mRNA isolated from the transfected cells. After analyzing the flanking sequences and context of the 88-nt fragment, we discovered that it does have a typical intron configuration containing the splicing donor and acceptor, polypyrimidine tract, and branch site. A point mutation at the acceptor site (AG→TG) abolishes this splicing site indicating that it is a bona fide intron. The intron splicing defaulted again when the adjacent intervening sequence (IVS) of pIRES-neo was deleted or adiponectin 3'-UTR was present. We found that 3'-UTR segment contained several splicing silencers and IVS contained high density of splicing enhancers. It explained the reactivation of this silent intron. Our results elicited the possibility that a 3'-UTR-free cDNA may reactivate an otherwise silent intron in the coding region as it is cloned for expression in mammalian cells.

Clathrin promotes centrosome integrity in early mitosis through stabilization of centrosomal ch-TOG.

Clathrin depletion by ribonucleic acid interference (RNAi) impairs mitotic spindle stability and cytokinesis. Depletion of several clathrin-associated proteins affects centrosome integrity, suggesting a further cell cycle function for clathrin. In this paper, we report that RNAi depletion of CHC17 (clathrin heavy chain 17) clathrin, but not the CHC22 clathrin isoform, induced centrosome amplification and multipolar spindles. To stage clathrin function within the cell cycle, a cell line expressing SNAP-tagged clathrin light chains was generated. Acute clathrin inactivation by chemical dimerization of the SNAP-tag during S phase caused reduction of both clathrin and ch-TOG (colonic, hepatic tumor overexpressed gene) at metaphase centrosomes, which became fragmented. This was phenocopied by treatment with Aurora A kinase inhibitor, suggesting a centrosomal role for the Aurora A-dependent complex of clathrin, ch-TOG, and TACC3 (transforming acidic coiled-coil protein 3). Clathrin inactivation in S phase also reduced total cellular levels of ch-TOG by metaphase. Live-cell imaging showed dynamic clathrin recruitment during centrosome maturation. Therefore, we propose that clathrin promotes centrosome maturation by stabilizing the microtubule-binding protein ch-TOG, defining a novel role for the clathrin-ch-TOG-TACC3 complex.

Systemic comparison of repression activity for miRNA and siRNA associated with different types of target sequences.

Most miRNA target sites are determined by Watson-Crick pairing via the seed region (positions ∼2-7nt of the mature miRNA). The binding sites of target mRNAs are categorized primarily as 7mer-m8, the 7mer-1A and the 8mer sites. This study analyzed post-transcriptional repression as a function of associations among various seed/target sequences. The target sequence of miR-155 from TP53INP1 was modified such that their various monomers and dimers could be inserted into the 3'UTR of a reporter gene for monitoring repression activity of miR-155. Results revealed that the level of repression could be ordered as follows: perfect-matched target≫dimeric targets≫monomeric targets. For dimeric targets, the order is 2×8mer>2×7mer-m8>2×7mer-1A. Fold repression of 8mer+7mer-1A lay between 2×8mer and 2×7mer-1A. A mismatch in one seed dramatically decreasing repressive activity of the dimer. This indicated that the degree of repression could be synergistically enhanced through the cooperation of the two miRISC-loaded monomers. The siRNA-155 (siRNA carrying miR-155 sequence) elicited higher repressive activity than miR-155, as they bound to the perfectly matched target. However, strong repression of miR-155 and siRNA-155 with a perfectly matched target was due primarily to translational attenuation. Cleavage/degradation of the target mRNA was not a major cause of the observed repression.

Butyl benzyl phthalate suppresses the ATP-induced cell proliferation in human osteosarcoma HOS cells.

Butyl benzyl phthalate (BBP), an endocrine disruptor present in the environment, exerts its genomic effects via intracellular steroid receptors and elicits non-genomic effects by interfering with membrane ion-channel receptors. We previously found that BBP blocks the calcium signaling coupled with P2X receptors in PC12 cells (Liu & Chen, 2006). Osteoblast P2X receptors were recently reported to play a role in cell proliferation and bone remodeling. In this present study, the effects of BBP on ATP-induced responses were investigated in human osteosarcoma HOS cells. These receptors mRNA had been detected, named P2X4, P2X7, P2Y2, P2Y4, P2Y5, P2Y9, and P2Y11, in human osteosarcoma HOS cells by RT-PCR. The enhancement of cell proliferation and the decrease of cytoviability had both been shown to be coupled to stimulation via different concentrations of ATP. BBP suppressed the ATP-induced calcium influx (mainly coupled with P2X) and cell proliferation but not the ATP-induced intracellular calcium release (mainly coupled with P2Y) and cytotoxicity in human osteosarcoma HOS cells. Suramin, a common P2 receptor's antagonist, blocked the ATP-induced calcium signaling, cell proliferation, and cytotoxicity. We suggest that P2X is mainly responsible for cell proliferation, and P2Y might be partially responsible for the observed cytotoxicity. BBP suppressed the calcium signaling coupled with P2X, suppressing cell proliferation. Since the importance of P2X receptors during bone metastasis has recently become apparent, the possible toxic risk of environmental BBP during bone remodeling is a public problem of concern.

A multisampling reporter system for monitoring microRNA activity in the same population of cells.

MicroRNAs (miRNAs) downregulate gene expression by binding to the partially complementary sites in the 3' untranslated region (UTR) of target mRNAs. Several methods, such as Northern blot analysis, quantitative real-time RT-PCR, microarray, and the luciferase reporter system, are commonly used to quantify the relative level or activity of miRNAs. The disadvantage of these methods is the requirement for cell lysis, which means that several sets of wells/dishes of cells must be prepared to monitor changes in miRNA activity in time-course studies. In this study, we developed a multisampling reporter system in which two secretable bioluminescence-generating enzymes are employed, one as a reporter and the other as an internal control. The reporters consist of a pair of vectors containing the Metridia luciferase gene, one with and one without a duplicated miRNA targeting sequence at their 3'UTR, while the other vector coding for the secreted alkaline phosphatase gene is used as an internal control. This method allows miRNA activity to be monitored within the same population of cells over time by withdrawing aliquots of the culture medium. The practicability and benefits of this system are addressed in this report.

TIM-2 is expressed on B cells and in liver and kidney and is a receptor for H-ferritin endocytosis.

T cell immunoglobulin-domain and mucin-domain (TIM) proteins constitute a receptor family that was identified first on kidney and liver cells; recently it was also shown to be expressed on T cells. TIM-1 and -3 receptors denote different subsets of T cells and have distinct regulatory effects on T cell function. Ferritin is a spherical protein complex that is formed by 24 subunits of H- and L-ferritin. Ferritin stores iron atoms intracellularly, but it also circulates. H-ferritin, but not L-ferritin, shows saturable binding to subsets of human T and B cells, and its expression is increased in response to inflammation. We demonstrate that mouse TIM-2 is expressed on all splenic B cells, with increased levels on germinal center B cells. TIM-2 also is expressed in the liver, especially in bile duct epithelial cells, and in renal tubule cells. We further demonstrate that TIM-2 is a receptor for H-ferritin, but not for L-ferritin, and expression of TIM-2 permits the cellular uptake of H-ferritin into endosomes. This is the first identification of a receptor for ferritin and reveals a new role for TIM-2.