Identification of the key immune-related genes and immune cell infiltration changes in renal interstitial fibrosis.

Background: Chronic kidney disease (CKD) is the third-leading cause of premature mortality worldwide. It is characterized by rapid deterioration due to renal interstitial fibrosis (RIF) via excessive inflammatory infiltration. The aim of this study was to discover key immune-related genes (IRGs) to provide valuable insights and therapeutic targets for RIF in CKD. Materials and methods: We screened differentially expressed genes (DEGs) between RIF samples from CKD patients and healthy controls from a public database. Least absolute shrinkage and selection operator regression analysis and receiver operating characteristic curve analysis were applied to identify significant key biomarkers. The single-sample Gene Set Enrichment Analysis (ssGSEA) algorithm was used to analyze the infiltration of immune cells between the RIF and control samples. The correlation between biomarkers and immune cell composition was assessed. Results: A total of 928 DEGs between CKD and control samples from six microarray datasets were found, 17 overlapping immune-correlated DEGs were identified by integration with the ImmPort database, and six IRGs were finally identified in the model: apolipoprotein H (APOH), epidermal growth factor (EGF), lactotransferrin (LTF), lysozyme (LYZ), phospholipid transfer protein (PLTP), and secretory leukocyte peptidase inhibitor (SLPI). Two additional datasets and in vivo experiments indicated that the expression levels of APOH and EGF in the fibrosis group were significantly lower than those in the control group, while the expression levels of LTF, LYZ, PLTP, and SLPI were higher (all P < 0.05). These IRGs also showed a significant correlation with renal function impairment. Moreover, four upregulated IRGs were positively associated with various T cell populations, which were enriched in RIF tissues, whereas two downregulated IRGs had opposite results. Several signaling pathways, such as the "T cell receptor signaling pathway" and "positive regulation of NF-κB signaling pathway", were discovered to be associated not only with immune cell infiltration, but also with the expression levels of six IRGs. Conclusion: In summary, six IRGs were identified as key biomarkers for RIF, and exhibited a strong correlation with various T cells and with the NF-κB signaling pathway. All these IRGs and their signaling pathways may evolve as valuable therapeutic targets for RIF in CKD.

Compatibility and durability of the gel stent material.

INTRODUCTION: The XEN Gel Stent (AbbVie Pharmaceuticals) is a device made from Gelatin; a well-known material in the medical field that is firm enough to hold its shape and soft enough to conform to tissues and reduce the risk of erosion. The Gel Stent creates a permanent outflow connection between the anterior chamber and subconjunctival space. AREAS COVERED: Validation testing done on the Gel Stent to evaluate biocompatibility and durability of the material as well as real-world experience are included and discussed in this paper. EXPERT OPINION: Correlating the results of the preclinical testing, study outcomes available in the published literature, and the surgeons' experiences, the device and materials have shown to have an acceptable biocompatibility and durability profile, with a stable, nondegradable, and permanent implant.

Association Between Geographic Distribution of Eye Care Clinicians and Visual Impairment in California.

Importance: The association between availability of eye care clinicians and visual impairment, a condition presenting with increased morbidity and health care costs, has not been thoroughly studied. Objective: To examine associations between the geographic distribution of eye care clinicians and visual impairment in California. Design, Setting, and Participants: This survey-based cross-sectional study included ophthalmologists and optometrists licensed in California in 2018 and 2020 as well as respondents to the 2014 to 2018 American Community Survey (ACS) by California counties and Medical Service Study Areas (MSSAs). Data were analyzed from August 2020 to December 2021. Main Outcomes and Measures: Prevalence of visual impairment by county and MSSA. Exposures: The number of eye care clinicians was determined based on the number of member ophthalmologists of the American Academy of Ophthalmology in 2018 and optometrists listed in the 2020 Blue Book of Optometrists in California. The prevalence of visual impairment was determined using questionnaire data from the American Community Survey. Linear regression was used to assess multivariable associations between number of eye care clinicians and visual impairment by MSSA. Results: A total of 30 068 581 California residents were included; 15 253 655 (50.7%) were female, and 5 314 389 (17.7%) were 65 years and older. The overall number of eye care clinicians was 22.18 clinicians per 100 000 residents. The overall prevalence of visual impairment was 2411.07 residents with visual impairment per 100 000 residents. San Francisco County had the highest number of eye care clinicians per 100 000 residents (39.24 clinicians per 100 000 residents). Four counties had no eye care clinicians (Alpine, Mariposa, Inyo, and Sierra counties). For every increase of 1 eye care clinician per 100 000 residents, there was a mean (SE) decrease of 3.90 (1.39) persons with visual impairment per 100 000 residents in adjusted analyses. Conclusions and Relevance: In this cross-sectional study, a higher number of eye care clinicians was potentially associated with lower prevalence of visual impairment in California. Additional studies are needed to assess eye care clinician availability on a national and global scale and strategies to improve access to eye care.

Study on the Effect of Fracturing Fluid on the Structure and Mechanical Properties of Igneous Rock.

Igneous rock oil and gas reservoirs have great development potential. Hydraulic fracturing is an important means for the development of these reservoirs. In the process of fracturing and increasing production, fracturing fluid is prone to a hydration reaction with clay minerals in igneous rock, and then, the structure and mechanical properties of the igneous rock are changed, affecting increased production. Therefore, it is necessary to establish a systematic water-rock reaction experiment method to understand the influence of fracturing fluid on the structure and mechanical properties of igneous rocks and to optimize the fracturing fluid system of igneous rock reservoirs. In this experiment, four solutions were used: slickwater, guar fracturing fluid, 2% KCl aqueous solution, and 4% KCl aqueous solution. Acoustic testing, porosity and permeability testing, XRD analysis, micro-CT scanning, and displacement experiments were performed. The influence of different fracturing fluids on the structure and mechanical properties of igneous rocks was studied. Igneous rock samples with a permeability of 0.05-0.1 mD and average porosity of 7-14% were used. The results show that all four liquid systems will reduce the permeability, Young's modulus, and brittleness index and increase the porosity and Poisson's ratio of the rock after fracturing. Among them, the permeability damage rate is as high as 37.37%, which may be related to the plugging of pores with solid residues in the gel breaking liquid; CT results show that there are microcracks in the rock, which increase over time, up to 13.54%. The brittleness index decreases. Among the fluids, the influence of slickwater on the rock brittleness index is the smallest, no more than 5%. Guar gum had the greatest effect on the Gel breaking liquid, up to 58%. One of the reasons for the increase in porosity is that adding a clay stabilizer composed of inorganic salts and organic cationic polymers to the slickwater fracturing fluid can effectively reduce the damage caused by the fracturing fluid to the rock during the fracturing process and can reduce the maximum by 50%. This paper can clarify the damage law of fracturing fluid systems to igneous rock reservoirs and provide the theoretical basis for the hydraulic fracturing of igneous rock reservoirs.

SS-31 ameliorates hepatic injury in rats subjected to severe burns plus delayed resuscitation via inhibiting the mtDNA/STING pathway in Kupffer cells.

Hepatic injury is common in patients who suffer from severe burns plus delayed resuscitation (B + DR). Stimulator of interferon genes (STING) is primarily expressed in Kupffer cells (KCs). We demonstrated that B + DR caused hepatic injury and oxidative stress. Reactive oxygen species (ROS) damage mitochondrial membranes in hepatocytes, leading to the release of mitochondrial DNA (mtDNA) into the hepatocyte cytosol and the circulation. The damaged hepatocytes then activate the mtDNA/STING pathway in KCs and trigger KCs polarization towards pro-inflammatory phenotype. SS-31 is a strong antioxidant that specifically concentrates in the inner mitochondrial membrane. SS-31 prevented hepatic injury by neutralizing ROS, inhibiting the release of mtDNA, protecting hepatocyte mitochondria, suppressing the activation of the mtDNA/STING pathway and inhibiting KCs polarization into pro-inflammatory phenotype.

Changes in Corneal Biomechanics and Glaucomatous Visual Field Loss.

PRECIS: A lower baseline corneal hysteresis and a decrease in corneal resistance factor (CRF) over time are associated with higher risk of visual field progression in glaucomatous and glaucoma suspect eyes. PURPOSE: The aim was to investigate the longitudinal change in CRF and cornea hysteresis (CH) as risk factors for visual field progression. MATERIALS AND METHODS: In this prospective observational cohort study, 72 eyes of 48 glaucoma or glaucoma suspect patients were followed for an average of 4.5 years. Baseline and follow-up CH and CRF measurements were performed with the Ocular Response Analyzer (Reichert Ophthalmic Instruments Inc., Depew, N.Y.). Evaluation of rates of visual field change during follow-up was performed using visual field mean deviation. Univariable and multivariable linear mixed models assessed the relationship of visual field progression with baseline CRF and CH as well as with changes in CRF and CH. RESULTS: The mean baseline CH was 9.0 (95% confidence interval: 8.6-9.4) mm Hg and the mean baseline CRF was 9.3 (95% confidence interval: 8.8-9.9) mm Hg. There was no statistically significant difference in average CH and CRF measurements over time. In multivariable modeling adjusting for age, race, and mean intraocular pressure during follow-up, each 1 mm Hg lower in baseline CH and 1 mm Hg decrease in CRF over time were associated with a 0.12 (P=0.042) and 0.14 dB/year (P=0.007) faster rate of visual field mean deviation loss, respectively. Similar findings were found in glaucoma eyes but not found in glaucoma suspect eyes. CONCLUSION: Visual field progression was associated with a lower baseline CH and a decrease in CRF over time. Assessment of corneal resistance and elasticity at baseline and during follow-up examinations should be considered to identify those eyes at highest risk of visual field progression.

Progressive Thinning of Retinal Nerve Fiber Layer and Ganglion Cell-Inner Plexiform Layer in Glaucoma Eyes with Disc Hemorrhage.

PURPOSE: To evaluate the thinning of the circumpapillary retinal nerve fiber layer (cpRNFL) and macular ganglion cell-inner plexiform layer (mGCIPL) in primary open-angle glaucoma eyes with and without a history of disc hemorrhage (DH). DESIGN: Observational cohort study. PARTICIPANTS: Thirty-nine 39 eyes (34 participants) with DH and 117 eyes (104 participants) without DH from the Diagnostic Innovations in Glaucoma Study and the African Decent and Glaucoma Evaluation Study. METHODS: Participants had at least 1.5 years of follow-up, with a minimum of 3 visits with biannual spectral-domain OCT cpRNFL and mGCIPL thickness measurements and visual fields (VFs). The rates of cpRNFL and mGCIPL thinning were calculated using mixed-effects models. The dynamic range-based normalized rates of cpRNFL and mGCIPL thinning were calculated and compared between the DH and non-DH groups. MAIN OUTCOME MEASURES: Rates of cpRNFL and mGCIPL thinning. RESULTS: The rate of mGCIPL thinning was significantly faster in the DH group compared with the non-DH group (-0.62 µm/year vs. -0.38 µm/year; P = 0.024). The rate of cpRNFL thinning in the DH quadrant and rate of mGCIPL thinning in the inferotemporal sector in the DH group were faster than the corresponding regions in the non-DH group after adjusting for intraocular pressure (-1.33 µm/year vs. -0.58 µm/year; P = 0.053) and race (-0.82 µm/year vs. -0.44 µm/year; P = 0.048). In the DH group, percent rate of loss was significantly faster for the mGCIPL than the cpRNFL (-1.59 %/year vs. -1.31 %/year; P = 0.046). Rates of mGCIPL thinning were associated weakly with mean deviation slope, VF index slope, and guided progression analysis (GPA). The areas under the receiver operating characteristic curve for VF progression were 0.75 for mGCIPL and 0.56 for cpRNFL in the DH group. CONCLUSIONS: The rate of mGCIPL and cpRNFL thinning was faster in DH eyes than non-DH eyes. Compared with cpRNFL, mGCIPL showed higher proportional rates of thinning and greater association with functional progression. In addition to cpRNFL, clinicians should consider incorporating mGCIPL imaging to monitor glaucoma progression, especially in glaucoma eyes with DH.

An in-depth quantitative analysis of wind turbine blade tip wake flow based on the lattice Boltzmann method.

With the continuous increase in the total quantity and quality of wind energy used by society, the aerodynamic complexity of wind turbine impellers has also gradually increased. This requires a more accurate analysis and understanding of the aerodynamic performance of important parts of the wind turbine impeller. The blade tip vortex is undoubtedly one of the most important issues. This article used the state-of-the-art lattice Boltzmann method (LBM), combining large eddy simulation (LES) and wall-adapting local-eddy (WALE) model, to investigate the unsteady flow characteristics of the blade tip region due to impeller wake for a wind turbine. The trend of this calculation result is consistent with the experimental data, both for the axial force and the torque on the impeller. Therefore, it indicates the calculation model of LBM is reasonable and effective. The relevant in-depth results clearly showed the dynamic stability of a spiral structure will increase as operating conditions continue to advance and it is also synchronously affected by the tower wake flow structures. The fluctuation amplitude of the static pressure value on both sides of the impeller gradually decreases with time. The distribution of static pressure near the leading edge of the tip is very different for each blade due to the influences of turbulence intensity and the tower shadow effect. The random minor fluctuations of the axial force and torque both exhibit similar characteristics, and the overall torque is more sensitive to random changes in the tip vortex on the impeller surface.

MitoQ protects against liver injury induced by severe burn plus delayed resuscitation by suppressing the mtDNA-NLRP3 axis.

INTRODUCTION: Liver injury induced by burn plus delayed resuscitation (B + DR) is life threatening in clinical settings. Mitochondrial damage and oxidative stress may account for the liver injury. MitoQ is a mitochondria-targeted antioxidant. We aimed to evaluate whether MitoQ protects against B + DR-induced liver injury. METHODS: Rats were randomly divided into three groups: (1) the sham group; (2) the B + DR group, which was characterized by third-degree burn of 30% of the total body surface area plus delayed resuscitation, and (3) the treatment group, in which rats from the B + DR model received the target treatment. MitoQ was injected intraperitoneally (i.p) at 15 min before resuscitation and shortly after resuscitation. In the vitro experiments, Kupffer cells (KCs) were subjected to hypoxia/reoxygenation (H/R) injury to simulate the B + DR model. Mitochondrial characteristics, oxidative stress, liver function, KCs apoptosis and activation of the NLRP3 inflammasome in KCs were measured. RESULTS: B + DR caused liver injury and oxidative stress. Excessive ROS lead to liver injury by damaging mitochondrial integrity and activating the mitochondrial DNA (mtDNA)-NLRP3 axis in KCs. The oxidized mtDNA, which was released into the cytosol during KCs apoptosis, directly bound and activated the NLRP3 inflammasome. MitoQ protected against liver injury by scavenging intracellular and mitochondrial ROS, preserving mitochondrial integrity and function, reducing KCs apoptosis, inhibiting the release of mtDNA, and suppressing the mtDNA-NLRP3 axis in KCs. CONCLUSION: MitoQ protected against B + DR-induced liver injury by suppressing the mtDNA-NLRP3 axis.

Combined Ab Externo Cyclopexy and Cryopexy in Cyclodialysis Cleft Repair.

PURPOSE: To evaluate outcomes of an ab externo surgical technique combining cyclopexy with partial-thickness scleral flap dissection and suture reattachment of ciliary muscle and cryopexy through partial-scleral bed for cyclodialysis cleft repair. MATERIALS AND METHODS: Consecutive patients in a tertiary academic practice with cyclodialysis cleft confirmed by gonioscopy or ultrasound biomicroscopy and had received the combined procedure were reviewed. Primary outcomes included differences between the preoperative and postoperative best-corrected visual acuity (BCVA) and intraocular pressure (IOP). Secondary outcomes included complications and additional surgeries. RESULTS: Six consecutive patients (eyes) from October 2006 to November 2012 (6 y) were enrolled. No patient had received prior laser or surgical treatment for cyclodialysis cleft. Patient's age ranged from 14 to 81 years (median=37 y). Follow-up ranged from 1 to 72 months (median=12 mo). The cyclodialysis clefts of 3 patients (50%) were caused by blunt injuries and the other 3 (50%) from complicated intraocular surgery. Preoperative BCVA ranged from 20/40 to counting fingers with mean IOP of 2.3±2.1 mm Hg (range, 0 to 6 mm Hg). Final postoperative BCVA ranged from 20/20 to hand motions with mean IOP of 11.3±5.7 mm Hg (range, 3 to 18 mm Hg). Five patients (83%) had an increase in IOP and recovery of vision to 20/50 or better at the final visit (including further surgeries). No complication was noted and no additional cyclopexy was required. CONCLUSIONS: Combined ab externo cyclopexy with partial-thickness scleral flap dissection and suture reattachment of the ciliary muscle and cryopexy delivered on the partial-thickness scleral bed is safe and effective as primary surgical repair for cyclodialysis cleft.

Autologous Partial-thickness Scleral Flap and Donor Corneal Graft in Management of Tube Erosion of Glaucoma Drainage Device.

PURPOSE: To evaluate the outcomes of autologous limbus-hinged partial-thickness scleral flap and half-moon-shaped split-thickness donor corneal allograft in management of tube erosion of glaucoma drainage device. METHODS: Consecutive patients who had undergone a limbus-hinged partial-thickness scleral flap and half-moon-shaped split-thickness corneal graft were reviewed. Primary outcome was surgical success of coverage of the exposed tube without complications or additional surgeries. Secondary outcomes were visual acuity (VA), intraocular pressure, number of glaucoma medications, surgical complications, and additional surgeries. RESULTS: From March 1, 2016 to August 30, 2017 (18 mo), 8 consecutive cases (7 patients) of tube erosions were included for analysis. Mean follow-up and age were 16.6±5.4 months and 74.4±15.2 years, respectively. Mean number of intraocular surgeries was 4.8±1.6 (range: 3 to 6 surgeries) and intraocular glaucoma surgeries was 2.9±1.7 (range: 1 to 6 surgeries) before tube erosion. There were no intraoperative or postoperative complications or recurrence of tube erosion in any patient. Differences between the preoperative and postoperative number of medications (2.6±1.8, 2.9±1.8, respectively; P=0.171) and intraocular pressure (12.9±6.9, 10.8±3.5 mm Hg, respectively; P=0.209) were not statistically significant. Preoperative and 3-month postoperative VA were identical in all eyes except in 1 eye that the VA improved from 20/300 to 20/70 after repair. CONCLUSIONS: Combination of limbus-hinged partial-thickness scleral flap and half-moon-shaped split-thickness corneal allograft is a viable surgical option to repair tube erosion.

Serotonin receptor 2C regulates neurite growth and is necessary for normal retinal processing of visual information.

Serotonin (5HT) is present in a subpopulation of amacrine cells, which form synapses with retinal ganglion cells (RGCs), but little is known about the physiological role of retinal serotonergic circuitry. We found that the 5HT receptor 2C (5HTR2C) is upregulated in RGCs after birth. Amacrine cells generate 5HT and about half of RGCs respond to 5HTR2C agonism with calcium elevation. We found that there are on average 83 5HT+ amacrine cells randomly distributed across the adult mouse retina, all negative for choline acetyltransferase and 90% positive for tyrosine hydroxylase. We also investigated whether 5HTR2C and 5HTR5A affect RGC neurite growth. We found that both suppress neurite growth, and that RGCs from the 5HTR2C knockout (KO) mice grow longer neurites. Furthermore, 5HTR2C is subject to post-transcriptional editing, and we found that only the edited isoform's suppressive effect on neurite growth could be reversed by a 5HTR2C inverse agonist. Next, we investigated the physiological role of 5HTR2C in the retina, and found that 5HTR2C KO mice showed increased amplitude on pattern electroretinogram. Finally, RGC transcriptional profiling and pathways analysis suggested partial developmental compensation for 5HTR2C absence. Taken together, our findings demonstrate that 5HTR2C regulates neurite growth and RGC activity and is necessary for normal amplitude of RGC response to physiologic stimuli, and raise the hypothesis that these functions are modulated by a subset of 5HT+/ChAT-/TH+ amacrine cells as part of retinal serotonergic circuitry. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419-437, 2017.

[High Resolution Remote Sensing Monitoring and Assessment of Secondary Geological Disasters Triggered by the Lushan Earthquake].

The secondary geological disasters triggered by the Lushan earthquake on April 20, 2013, such as landslides, collapses, debris flows, etc., had caused great casualties and losses. We monitored the number and spatial distribution of the secondary geological disasters in the earthquake-hit area from airborne remote sensing images, which covered areas about 3 100 km2. The results showed that Lushan County, Baoxing County and Tianquan County were most severely affected; there were 164, 126 and 71 secondary geological disasters in these regions. Moreover, we analyzed the relationship between the distribution of the secondary geological disasters, geological structure and intensity. The results indicate that there were 4 high-hazard zones in the monitored area, one focused within six kilometers from the epicenter, and others are distributed along the two main fault zones of the Longmen Mountain. More than 97% secondary geological disasters occurred in zones with a seismic intensity of VII to IX degrees, a slope between 25 A degrees and 50 A degrees, and an altitude of between 800 and 2 000 m. At last, preliminary suggestions were proposed for the rehabilitation and reconstruction planning of Lushan earthquake. According to the analysis result, airborne and space borne remote sensing can be used accurately and effectively in almost real-time to monitor and assess secondary geological disasters, providing a scientific basis and decision making support for government emergency command and post-disaster reconstruction.

Combining 2-deoxy-D-glucose with fenofibrate leads to tumor cell death mediated by simultaneous induction of energy and ER stress.

Unregulated growth and replication as well as an abnormal microenvironment, leads to elevated levels of stress which is a common trait of cancer. By inducing both energy and endoplasmic reticulum (ER) stress, 2-Deoxy-glucose (2-DG) is particularly well-suited to take advantage of the therapeutic window that heightened stress in tumors provides. Under hypoxia, blocking glycolysis with 2-DG leads to significant lowering of ATP resulting in energy stress and cell death in numerous carcinoma cell types. In contrast, under normoxia, 2-DG at a low-concentration is not toxic in most carcinomas tested, but induces growth inhibition, which is primarily due to ER stress. Here we find a synergistic toxic effect in several tumor cell lines in vitro combining 2-DG with fenofibrate (FF), a drug that has been safely used for over 40 years to lower cholesterol in patients. This combination induces much greater energy stress than either agent alone, as measured by ATP reduction, increased p-AMPK and downregulation of mTOR. Inhibition of mTOR results in blockage of GRP78 a critical component of the unfolded protein response which we speculate leads to greater ER stress as observed by increased p-eIF2α. Moreover, to avoid an insulin response and adsorption by the liver, 2-DG is delivered by slow-release pump yielding significant anti-tumor control when combined with FF. Our results provide promise for developing this combination clinically and others that combine 2-DG with agents that act synergistically to selectively increase energy and ER stress to a level that is toxic to numerous tumor cell types.

The N-terminal Set-ß Protein Isoform Induces Neuronal Death.

Set-ß protein plays different roles in neurons, but the diversity of Set-ß neuronal isoforms and their functions have not been characterized. The expression and subcellular localization of Set-ß are altered in Alzheimer disease, cleavage of Set-ß leads to neuronal death after stroke, and the full-length Set-ß regulates retinal ganglion cell (RGC) and hippocampal neuron axon growth and regeneration in a subcellular localization-dependent manner. Here we used various biochemical approaches to investigate Set-ß isoforms and their role in the CNS, using the same type of neurons, RGCs, across studies. We found multiple alternatively spliced isoforms expressed from the Set locus in purified RGCs. Set transcripts containing the Set-ß-specific exon were the most highly expressed isoforms. We also identified a novel, alternatively spliced Set-ß transcript lacking the nuclear localization signal and demonstrated that the full-length (∼39-kDa) Set-ß is localized predominantly in the nucleus, whereas a shorter (∼25-kDa) Set-ß isoform is localized predominantly in the cytoplasm. Finally, we show that an N-terminal Set-ß cleavage product can induce neuronal death.

Regulating Set-ß's Subcellular Localization Toggles Its Function between Inhibiting and Promoting Axon Growth and Regeneration.

The failure of the CNS neurons to regenerate axons after injury or stroke is a major clinical problem. Transcriptional regulators like Set-ß are well positioned to regulate intrinsic axon regeneration capacity, which declines developmentally in maturing CNS neurons. Set-ß also functions at cellular membranes and its subcellular localization is disrupted in Alzheimer's disease, but many of its biological mechanisms have not been explored in neurons. We found that Set-ß was upregulated postnatally in CNS neurons, and was primarily localized to the nucleus but was also detected in the cytoplasm and adjacent to the plasma membrane. Remarkably, nuclear Set-ß suppressed, whereas Set-ß localized to cytoplasmic membranes promoted neurite growth in rodent retinal ganglion cells and hippocampal neurons. Mimicking serine 9 phosphorylation, as found in Alzheimer's disease brains, delayed nuclear import and furthermore blocked the ability of nuclear Set-ß to suppress neurite growth. We also present data on gene regulation and protein binding partner recruitment by Set-ß in primary neurons, raising the hypothesis that nuclear Set-ß may preferentially regulate gene expression whereas Set-ß at cytoplasmic membranes may regulate unique cofactors, including PP2A, which we show also regulates axon growth in vitro. Finally, increasing recruitment of Set-ß to cellular membranes promoted adult rat optic nerve axon regeneration after injury in vivo. Thus, Set-ß differentially regulates axon growth and regeneration depending on subcellular localization and phosphorylation.

MicroRNA 375 mediates the signaling pathway of corticotropin-releasing factor (CRF) regulating pro-opiomelanocortin (POMC) expression by targeting mitogen-activated protein kinase 8.

Pro-opiomelanocortin (POMC) is a common precursor of melanocortin-related peptides in the pituitary and primarily regulated by corticotropin- releasing factor (CRF). Our results show that miR-375 is highly expressed in the mouse pituitary gland and located specifically in the intermediate lobe of pituitary. The functional studies show that the forced inhibition of endogenous miR-375 in AtT-20 mouse pituitary tumor cells and in the intermediate lobe of the pituitary gland significantly increases POMC expression, whereas miR-375 overexpression down-regulates POMC expression and ACTH secretion stimulated by CRF. This function of miR-375 is accomplished by its binding to the 3'-UTR of mitogen-activated protein kinase kinase kinase-8. Our results here have demonstrated that miR-375 acts as a negative regulating molecule mediating the signaling pathway of CRF and affecting POMC expression by targeting mitogen-activated protein kinase kinase kinase-8, which subsequently down-regulates ERK1/2 phosphorylation and nerve growth factor-induced clone B (NGFI-B) transcription activity. Taken together, our results show that miR-375 is a novel negative regulator of POMC expression and related hormone secretion.

Premature translational termination products are rapidly degraded substrates for MHC class I presentation.

Nearly thirty percent of all newly synthesized polypeptides are targeted for rapid proteasome-mediated degradation. These rapidly degraded polypeptides (RDPs) are a source of antigenic substrates for the MHC class I presentation pathway, allowing for immunosurveillance of newly synthesized proteins by cytotoxic T lymphocytes. Despite the recognized role of RDPs in MHC I presentation, it remains unclear what molecular characteristics distinguish RDPs from their more stable counterparts. It has been proposed that premature translational termination products may constitute a form of RDP; indeed, in prokaryotes translational drop-off products are normal by-products of protein synthesis and are subsequently rapidly degraded. To study the cellular fate of premature termination products, we used the antibiotic puromycin as a means to experimentally manipulate prematurely terminated polypeptide production in human cells. At low concentrations, puromycin enhanced flux into rapidly degraded polypeptide pools, with small polypeptides being markedly more labile then high molecular weight puromycin adducts. Immunoprecipitation experiments using anti-puromycin antisera demonstrated that the majority of peptidyl-puromycins are rapidly degraded in a proteasome-dependent manner. Low concentrations of puromycin increased the recovery of cell surface MHC I-peptide complexes, indicating that prematurely terminated polypeptides can be processed for presentation via the MHC I pathway. In the continued presence of puromycin, however, MHC I export to the cell surface was inhibited, coincident with the accumulation of polyubiquitinated proteins. The time- and dose-dependent effects of puromycin suggest that the pool of peptidyl-puromycin adducts differ in their targeting to various proteolytic pathways that, in turn, differ in the efficiency with which they access the MHC I presentation machinery. These studies highlight the diversity of cellular proteolytic pathways necessary for the metabolism and immunosurveillance of prematurely terminated polypeptides that are, by their nature, highly heterogeneous.

2-Deoxy-D-glucose activates autophagy via endoplasmic reticulum stress rather than ATP depletion.

PURPOSE: The glucose analog and glycolytic inhibitor 2-deoxy-D-glucose (2-DG), which is currently under clinical evaluation for targeting cancer cells, not only blocks glycolysis thereby reducing cellular ATP, but also interferes with N-linked glycosylation, which leads to endoplasmic reticulum (ER) stress and an unfolded protein response (UPR). Both bioenergetic challenge and ER stress have been shown to activate autophagy, a bulk cellular degradation process that plays either a pro- or anti-death role. Here, we investigate which pathway 2-DG interferes with that activates autophagy and the role of this process in modulating 2-DG-induced toxicity. METHODS: Pancreatic cancer cell line 1420, melanoma cell line MDA-MB-435 and breast cancer cell line SKBR3 were used to investigate the relationship between induction by 2-DG treatment of ER stress/UPR, ATP reduction and activation of autophagy. ER stress/UPR (Grp78 and CHOP) and autophagy (LC3B II) markers were assayed by immunoblotting, while ATP levels were measured using the CellTiter-Glo Luminescent Cell Viability Assay. Autophagy was also measured by immunofluorescence utilizing LC3B antibody. Cell death was detected with a Vi-Cell cell viability analyzer using trypan blue exclusion. RESULTS: In the three different cancer cell lines described earlier, we find that 2-DG upregulates autophagy, increases ER stress and lowers ATP levels. Addition of exogenous mannose reverses 2-DG-induced autophagy and ER stress but does not recover the lowered levels of ATP. Moreover, under anaerobic conditions where 2-DG severely depletes ATP, autophagy is diminished rather than activated, which correlates with lowered levels of the ER stress marker Grp78. Additionally, when autophagy is blocked by siRNA, cell sensitivity to 2-DG is increased corresponding with upregulation of ER stress-mediated apoptosis. Similar increased toxicity is observed with 3-methyladenine, a known autophagy inhibitor. In contrast, rapamycin which enhances autophagy reduces 2-DG-induced toxicity. CONCLUSIONS: Overall, these results indicate that the major mechanism by which 2-DG stimulates autophagy is through ER stress/UPR and not by lowering ATP levels. Furthermore, autophagy plays a protective role against 2-DG-elicited cell death apparently by relieving ER stress. These data suggest that combining autophagy inhibitors with 2-DG may be useful clinically.