Comparison of biological behavior of lacrimal gland adenoid cystic carcinoma with high-grade transformation cells.

AIM: To evaluate the differences between human lacrimal gland adenoid cystic carcinoma with high-grade transformation (LACC-HGT) primary cells cultured by high-grade transformation tissue and non-high-grade transformation (non-HGT) primary cells cultured by non-high-grade transformation tissue in proliferation, metastasis, drug susceptibility, and genes. METHODS: LACC-HGT primary cells were established by tissue block culture, and the 4th to 10th generation primary cells were selected as research objects. The cells were preliminarily identified by immunofluorescent staining. The differences between non-HGT and LACC-HGT primary cells in terms of proliferation, metastasis, and drug susceptibility were compared by cell counting kit-8 (CCK-8) assay, wound healing, and drug sensitivity experiments. Differentially expressed genes were screened using mRNA array. Gene expression was analyzed using real-time quantitative polymerase chain reaction (RT-qPCR). RESULTS: LACC-HGT primary cells were successfully cultured by tissue block culture. Immunofluorescence staining results showed that cytokeratin (CK) and CK7 expression levels were positive in LACC-HGT primary cells. CCK-8 results showed that the proliferation ability of LACC-HGT cells was significantly higher than that of non-HGT cells. Wound healing experiment showed that the migration ability of LACC-HGT cells was significantly higher than that of non-HGT cells. LACC-HGT cells were also less sensitive to cisplatin and paclitaxel than non-HGT cells. Compared with non-HGT cells, 9566 differentially expressed genes were found in LACC-HGT primary cells, of which 5162 were up-regulated and 4404 were down-regulated. The expression of N-acetylneuraminate pyruvate lyase (NPL), MARVEL domain containing 3 (MARVELD3), syntabulin (SYBU), and allograft inflammatory factor 1 (AIF1) was higher in LACC-HGT cells than in non-HGT cells, whereas that of periostin (POSTN) was lower. CONCLUSION: LACC-HGT primary cells have faster proliferation, stronger migration ability, and poorer sensitivity to chemotherapy drugs than non-HGT primary cells. The expression of mRNAs in non-HGT and LACC-HGT primary cells are significantly different. These features are speculated to be the reasons why high-grade transformation tissues exhibit higher malignant degree and poorer prognosis than their counterparts.

Tripartite Crosstalk between Cytokine IL-1ß, NMDA-R and Misplaced Mitochondrial Anchor in Neuronal Dendrites is a Novel Pathway for Neurodegeneration in Inflammatory Diseases.

The mitochondrial anchor syntaphilin (SNPH) is a key mitochondrial protein normally expressed in axons to maintain neuronal health by positioning mitochondria along axons for metabolic needs. However, in 2019 we discovered a novel form of excitotoxicity that results when SNPH is misplaced into neuronal dendrites in disease models. A key unanswered question about this SNPH excitotoxicity is the pathologic molecules that trigger misplacement or intrusion of SNPH into dendrites. Here, we identified two different classes of pathologic molecules that interact to trigger dendritic SNPH intrusion. Using primary hippocampal neuronal cultures from mice of either sex, we demonstrated that the pro-inflammatory cytokine IL-1ß interacts with NMDA to trigger SNPH intrusion into dendrites. First, IL-1ß and NMDA each individually triggers dendritic SNPH intrusion. Second, IL-1ß and NMDA do not act independently but interact. Thus, blocking NMDAR by the antagonist MK-801 blocks IL-1ß from triggering dendritic SNPH intrusion. Further, de-coupling the known interaction between IL-1ß and NMDAR by tyrosine inhibitors prevents either IL-1ß or NMDA from triggering dendritic SNPH intrusion. Third, neuronal toxicity caused by IL-1ß or NMDA are strongly ameliorated in SNPH-/- neurons. Taken together, we hypothesize that the known bipartite IL-1ß/NMDAR crosstalk converges to trigger misplacement of SNPH in dendrites as a final common pathway to cause neurodegeneration. Targeting dendritic SNPH in this novel tripartite IL-1ß/NMDAR/SNPH interaction could be a strategic downstream locus for ameliorating neurotoxicity in inflammatory diseases.SIGNIFICANCE STATEMENTThe mitochondrial anchor Syntaphilin (SNPH) is a key mitochondrial protein normally expressed specifically in healthy axons to help position mitochondria along axons to match metabolic needs. In 2019, we discovered that misplacement of SNPH into neuronal dendrites causes a novel form of excitotoxicity in rodent models of multiple sclerosis. A key unanswered question about this new form of dendritic SNPH toxicity concerns pathologic molecules that trigger toxic misplacement of SNPH into dendrites. Here we identified two major categories of pathologic molecules, the pro-inflammatory cytokines and NMDA, that interact and converge to trigger toxic misplacement of SNPH into dendrites. We propose that dendritic mitochondrial anchor provides a novel, single common target for ameliorating diverse inflammatory and excitatory injuries in neurodegenerative diseases.

Inappropriate Intrusion of an Axonal Mitochondrial Anchor into Dendrites Causes Neurodegeneration.

Syntaphilin (SNPH) is a major mitochondrial anchoring protein targeted to axons and excluded from dendrites. In this study, we provide in vivo evidence that this spatial specificity is lost in Shiverer (Shi) mice, a model for progressive multiple sclerosis (MS), resulting in inappropriate intrusion of SNPH into dendrites of cerebellar Purkinje cells with neurodegenerative consequences. Thus, reconstituting dendritic SNPH intrusion in SNPH-KO mice by viral transduction greatly sensitizes Purkinje cells to excitotoxicity when the glutamatergic climbing fibers are stimulated. Finally, we demonstrate in vitro that overexpression of SNPH in dendrites compromises neuronal viability by inducing N-methyl-D-aspartate (NMDA) excitotoxicity, reducing mitochondrial calcium uptake, and interfering with quality control of mitochondria by blocking somal mitophagy. Collectively, we propose that inappropriate immobilization of dendritic mitochondria by SNPH intrusion produces excitotoxicity and suggest that interception of dendritic SNPH intrusion is a therapeutic strategy to combat neurodegeneration.

Chemoradiotherapy in combination with radical surgery is associated with better outcome in cervical cancer patients.

OBJECTIVES: To retrospectively assess the influence of radical surgery following concurrent chemoradiotherapy (CCRT) on outcomes in cervical cancer (CC) patients. METHODS: Patients diagnosed with cervical squamous cell carcinoma or adenocarcinoma (FIGO stages IB2 to IIB) at the Yinbin Second People's Hospital between September 2008 and September 2013, were included in this study. Patients were classified into 2 groups based on the treatment received: surgery group (CCRT plus radical surgery) and non-surgery groups (CCRT only). In addition to clinical information, inter-group differences with respect to local control rate (LCR), local recurrence rate (LRR), metastasis rate, overall survival (OS), progress free survival(PFS) and complications were assessed. RESULTS: A total of 314 patients were included in the analysis. Parametrial invasion, pelvic lymph node metastasis, tumor diameter > 4 cm and presence of residual disease were risk factors for recurrence in the non-surgery group. In patients with risk factors, radical surgery significantly improved their clinical outcome. The 3-year/5-year LCR in the surgery and non-surgery groups was 88.3%/87.4% and 82.3%/77.5%, respectively (P = 0.04). The 3-year/5-year OS rate in the two groups was 87.1%/81.7% and 72.8%/67.3%, respectively (P = 0.001). The 3-year/5-year LRR in the two groups were 11.7%/12.6% and 17.7%/22.5%, respectively (P = 0.04). The metastasis rates in the two groups were 19.9% and 24.8%, respectively (P = 0.09). CONCLUSIONS: Surgery following CCRT could improve overall survival and progressfree survival. Radical surgery following CCRT appears to confer significant benefits including an increase in LCRs and decrease in LRR in CC patients with risk factors.

WBSCR16 Is a Guanine Nucleotide Exchange Factor Important for Mitochondrial Fusion.

Regulated inter-mitochondrial fusion/fission is essential for maintaining optimal mitochondrial respiration and control of apoptosis and autophagy. In mammals, mitochondrial fusion is controlled by outer membrane GTPases MFN1 and MFN2 and by inner membrane (IM) GTPase OPA1. Disordered mitochondrial fusion/fission contributes to various pathologies, and MFN2 or OPA1 mutations underlie neurodegenerative diseases. Here, we show that the WBSCR16 protein is primarily associated with the outer face of the inner mitochondrial membrane and is important for mitochondrial fusion. We provide evidence of a WBSCR16/OPA1 physical interaction in the intact cell and of a WBSCR16 function as an OPA1-specific guanine nucleotide exchange factor (GEF). Homozygosity for a Wbscr16 mutation causes early embryonic lethality, whereas neurons of mice heterozygous for the mutation have mitochondria with reduced membrane potential and increased susceptibility to fragmentation upon exposure to stress, suggesting roles for WBSCR16 deficits in neuronal pathologies.

Deletion of mitochondrial anchoring protects dysmyelinating shiverer: implications for progressive MS.

The demyelinating disease multiple sclerosis (MS) has an early inflammatory phase followed by an incurable progressive phase with subdued inflammation and poorly understood neurodegenerative mechanism. In this study, we identified various parallelisms between progressive MS and the dysmyelinating mouse model Shiverer and then genetically deleted a major neuron-specific mitochondrial anchoring protein Syntaphilin (SNPH) from the mouse. Prevailing evidence suggests that deletion of SNPH is harmful in demyelination. Surprisingly, SNPH deletion produces striking benefits in the Shiverer by prolonging survival, reducing cerebellar damage, suppressing oxidative stress, and improving mitochondrial health. In contrast, SNPH deletion does not benefit clinical symptoms in experimental autoimmune encephalomyelitis (EAE), a model for early-phase MS. We propose that deleting mitochondrial anchoring is a novel, specific treatment for progressive MS.

[Near infrared spectroscopy on-line and real-time monitoring of alcohol precipitation process of reduning injection].

Near infrared (NIR) spectroscopy as a kind of rapid process analysis technology has been successfully applied in Chinese medicine pharmaceutical process. In this research, the technology was adopted to establish the rapid quantitative analysis models of main indicators from the Lonicera japonica and Artemisia annua alcohol precipitation process of Reduning injection. On-line NIR spectra of 142 samples from alcohol precipitation process were collected and the content of main indicators for each sample were detected through off-line HPLC. With eliminating outliers, determination of spectra pretreatment method and selecting optimal band, the NIR quantitative calibration model for each indicator was established using partial least squares (PLS). These models were used to predict the unknown samples from precipitation process of Reduning injection to achieve the goal of rapid detection. The results showed that the models were ideal. The correlation coefficients of models for neochlorogenic acid, chlorogenic acid, 4-O-caffeoylquinic acid and secoxyloganin were 0.973 872, 0.985 449, 0.975 509 and 0.979 790, respectively and their relative standard errors of prediction (RSEP) were 2.922 49%, 2.341 37%, 2.930 40% and 2.184 60%, respectively. This study indicated that the NIR quantitative calibration model showed good stability and precision, and it can be used in rapid quantitative detection of main indicators of efficacy in order to on-line monitor the alcohol precipitation process of Reduning injection.

Spontaneous optic nerve compression in the osteopetrotic (op/op) mouse: a novel model of myelination failure.

We report a focal disturbance in myelination of the optic nerve in the osteopetrotic (op/op) mouse, which results from a spontaneous compression of the nerve resulting from stenosis of the optic canal. The growth of the op/op optic nerve was significantly affected, being maximally suppressed at postnatal day 30 (P30; 33% of age matched control). Myelination of the nerve in the optic canal was significantly delayed at P15, and myelin was almost completely absent at P30. The size of nerves and myelination were conserved both in the intracranial and intraorbital segments at P30, suggesting that the axons in the compressed site are spared in all animals at P30. Interestingly, we observed recovery both in the nerve size and the density of myelinated axons at 7 months in almost half of the optic nerves examined, although some nerves lost axons and became atrophic. In vivo and ex vivo electrophysiological examinations of P30 op/op mice showed that nerve conduction was significantly delayed but not blocked with partial recovery in some mice by 7 months. Transcardial perfusion of FITC-labeled albumin suggested that local ischemia was at least in part the cause of this myelination failure. These results suggest that the primary abnormality is dysmyelination of the optic nerve in early development. This noninvasive model system will be a valuable tool to study the effects of nerve compression on the function and survival of oligodendrocyte progenitor cells/oligodendrocytes and axons and to explore the mechanism of redistribution of oligodendrocyte progenitor cells with compensatory myelination.

A novel method to study the local mitochondrial fusion in myelinated axons in vivo.

Mitochondrial remodeling (replication, fission/fusion) is a dynamically regulated process with diverse functions in neurons. A myelinated axon is an extension from the cell soma of a fully differentiated neuron. Mitochondria, once synthesized in the cell body, enter the axon displaying robust trafficking and accumulation at nodes of Ranvier to match metabolic needs. This long-distance deployment of mitochondria to axons raises the issue of whether myelinated axons can function independently of the cell body to execute mitochondrial remodeling to match local demands. Mitochondrial fusion has been suggested to occur in axons in simple neuronal cultures in vitro. However, whether such events occur in vivo in an intact nervous system remains unanswered. Here we describe a novel technique which allows monitoring of mitochondrial fusion in intact sciatic nerve of frog (Xenopus laevis). Mitochondrial population was labeled by injecting two different MitoTracker dyes (Red and Green), spatially apart along sciatic nerves surgically and then allow to "meet"in vivo. At 24h post-surgery, the sciatic nerves were taken out for mitochondrial imaging at the half-way point. During the post-injection periods, the anterograde-directed Green mitochondria meet with the retrograde-directed Red mitochondria. If fusion occurs, the merged of Green and Red fluorophores in the same mitochondrion will produce a Yellow color in merged images. The labeled mitochondria were observed with a Nikon A1 confocal microscope. Our new mitochondrial imaging method opens an avenue to separately assess the role of local axonal mitochondrial fusion, independent of the cell body of nerve fibers.

Activity-dependent regulation of mitochondrial motility by calcium and Na/K-ATPase at nodes of Ranvier of myelinated nerves.

The node of Ranvier is a tiny segment of a myelinated fiber with various types of specializations adapted for generation of high-speed nerve impulses. It is ionically specialized with respect to ion channel segregation and ionic fluxes, and metabolically specialized in ionic pump expression and mitochondrial density augmentation. This report examines the interplay of three important parameters (calcium fluxes, Na pumps, mitochondrial motility) at nodes of Ranvier in frog during normal nerve activity. First, we used calcium dyes to resolve a highly localized elevation in axonal calcium at a node of Ranvier during action potentials, and showed that this calcium elevation retards mitochondrial motility during nerve impulses. Second, we found, surprisingly, that physiologic activation of the Na pumps retards mitochondrial motility. Blocking Na pumps alone greatly prevents action potentials from retarding mitochondrial motility, which reveals that mitochondrial motility is coupled to Na/K-ATPase. In conclusion, we suggest that during normal nerve activity, Ca elevation and activation of Na/K-ATPase act, possibly in a synergistic manner, to recruit mitochondria to a node of Ranvier to match metabolic needs.

Action potentials induce uniform calcium influx in mammalian myelinated optic nerves.

The myelin sheath enables saltatory conduction by demarcating the axon into a narrow nodal region for excitation and an extended, insulated internodal region for efficient spread of passive current. This anatomical demarcation produces a dramatic heterogeneity in ionic fluxes during excitation, a classical example being the restriction of Na influx at the node. Recent studies have revealed that action potentials also induce calcium influx into myelinated axons of mammalian optic nerves. Does calcium influx in myelinated axons show spatial heterogeneity during nerve excitation? To address this, we analyzed spatial profiles of axonal calcium transients during action potentials by selectively staining axons with calcium indicators and subjected the data to theoretical analysis with parameters for axial calcium diffusion empirically determined using photolysis of caged compounds. The results show surprisingly that during action potentials, calcium influx occurs uniformly along an axon of a fully myelinated mouse optic nerve.

Nicotinic acetylcholine receptors in mouse and rat optic nerves.

Receptor-mediated calcium signaling in axons of mouse and rat optic nerves was examined by selectively staining the axonal population with a calcium indicator. Nicotine (1-50 microM) induced an axonal calcium elevation that was eliminated when calcium was removed from the bath, suggesting that nicotine induces calcium influx into axons. The nicotine response was blocked by d-tubocurarine and mecamylamine but not alpha-bungarotoxin, indicating the presence of calcium permeable, non-alpha7 nicotinic acetylcholine receptor (nAChR) subtype. Agonist efficacy order for eliciting the axonal nAChR calcium response was cytisine approximately nicotine >> acetylcholine. The nicotine-mediated calcium response was attenuated during the process of normal myelination, decreasing by approximately 10-fold from P1 (premyelinated) to P30 (myelinated). Nicotine also caused a rapid reduction in the compound action potential in neonatal optic nerves, consistent with a shunting of the membrane after opening of the nonspecific cationic nicotinic channels. Voltagegated calcium channels contributed little to the axonal calcium elevation during nAChR activation. During repetitive stimulations, the compound action potential in neonatal mouse optic nerves underwent a gradual reduction in amplitude that could be partially prevented by d-tubocurarine, suggesting an activity-dependent release of acetylcholine that activates axonal AChRs. We conclude that mammalian optic nerve axons express nAChRs and suggest that these receptors are activated in an activity-dependent fashion during optic nerve development to modulate axon excitability and biology.

Coupling of calcium homeostasis to axonal sodium in axons of mouse optic nerve.

Axonal populations in neonatal and mature optic nerves were selectively stained with calcium dyes for analysis of calcium homeostasis and its possible coupling to axonal Na. Repetitive nerve stimulation causes a rise in axonal [Ca(2+)](i) the posttetanus recovery of which is impeded by increasing the number of action potentials in the tetanus. This effect is augmented in 4-aminopyridine (4-AP; 1 mM), which dramatically increases the calcium and presumably sodium load during the tetanus. Increasing axonal [Na](i) with the Na-ionophore monensin (4-50 microM) and ouabain (30 microM) retards posttetanus calcium decline, suggesting that efficient calcium clearance depends on a low level of axonal [Na](i). Posttetanus calcium clearance is not affected by K-mediated depolarization. To further examine coupling between axonal [Na](i) and [Ca(2+)](i), the resting axonal [Ca(2+)](i) was monitored as axonal [Na(+)](i) was elevated with ouabain, veratridine, and monensin. In all cases, elevation of axonal [Na(+)](i) evokes a calcium influx into axons. This influx is unrelated to activation of calcium channels but is consistent with calcium influx via reversal of the Na/Ca exchanger expected as a consequence of axonal [Na(+)](i) elevation. In conclusion, this study demonstrates that calcium homeostasis in the axons of the optic nerve is strongly coupled to axonal [Na(+)](i) in a manner consistent with the Na/Ca exchanger playing a major role in extruding calcium following nerve activity.