Surgical procedure of intratympanic injection and inner ear pharmacokinetics simulation in domestic pigs.

Introduction: One major obstacle in validating drugs for the treatment or prevention of hearing loss is the limited data available on the distribution and concentration of drugs in the human inner ear. Although small animal models offer some insights into inner ear pharmacokinetics, their smaller organ size and different barrier (round window membrane) permeabilities compared to humans can complicate study interpretation. Therefore, developing a reliable large animal model for inner ear drug delivery is crucial. The inner and middle ear anatomy of domestic pigs closely resembles that of humans, making them promising candidates for studying inner ear pharmacokinetics. However, unlike humans, the anatomical orientation and tortuosity of the porcine external ear canal frustrates local drug delivery to the inner ear. Methods: In this study, we developed a surgical technique to access the tympanic membrane of pigs. To assess hearing pre- and post-surgery, auditory brainstem responses to click and pure tones were measured. Additionally, we performed 3D segmentation of the porcine inner ear images and used this data to simulate the diffusion of dexamethasone within the inner ear through fluid simulation software (FluidSim). Results: We have successfully delivered dexamethasone and dexamethasone sodium phosphate to the porcine inner ear via the intratympanic injection. The recorded auditory brainstem measurements revealed no adverse effects on hearing thresholds attributable to the surgery. We have also simulated the diffusion rates for dexamethasone and dexamethasone sodium phosphate into the porcine inner ear and confirmed the accuracy of the simulations using in-vivo data. Discussion: We have developed and characterized a method for conducting pharmacokinetic studies of the inner ear using pigs. This animal model closely mirrors the size of the human cochlea and the thickness of its barriers. The diffusion time and drug concentrations we reported align closely with the limited data available from human studies. Therefore, we have demonstrated the potential of using pigs as a large animal model for studying inner ear pharmacokinetics.

Cerebrospinal Fluid Flow Extends to Peripheral Nerves.

Cerebrospinal fluid (CSF) is an aqueous solution responsible for nutrient delivery and waste removal for the central nervous system (CNS). The three-layer meningeal coverings of the CNS support CSF flow. Peripheral nerves have an analogous three-layer covering consisting of the epineurium, perineurium, and endoneurium. Peripheral axons, located in the inner endoneurium, are bathed in "endoneurial fluid" similar to CSF but of undefined origin. CSF flow in the peripheral nervous system has not been demonstrated. Here we show CSF flow extends beyond the CNS to peripheral nerves in a contiguous flowing system. Utilizing gold nanoparticles, we identified that CSF is continuous with the endoneurial fluid and reveal the endoneurial space as the likely site of CSF flow in the periphery. Nanogold distribution along entire peripheral nerves and within their axoplasm suggests CSF plays a role in nutrient delivery and waste clearance, fundamental aspects of peripheral nerve health and disease. One Sentence Summary: Cerebrospinal fluid unites the nervous system by extending beyond the central nervous system into peripheral nerves.

Immunologic Alterations Associated With Oral Delivery of Anti-CD3 (OKT3) Monoclonal Antibodies in Patients With Moderate-to-Severe Ulcerative Colitis.

AIM: The aim of this study was to determine the immunologic effects and safety of oral anti-CD3 in patients with ulcerative colitis (UC). METHODS: An open-label pilot study of orally delivered anti-CD3 was performed in patients with moderate-to-severe UC. The primary end points were changes in immunologic parameters and evaluation for safety. RESULTS: Six subjects received oral OKT3. Biologic effects of oral anti-CD3 included significantly increased proliferation in response to anti-CD3 and anti-inflammatory gene expression profile in peripheral blood mononuclear cells. No serious treatment-related adverse events occurred. CONCLUSION: Orally delivered anti-CD3 resulted in immunologic changes in patients with UC.

Oxidative stress-mediated NFκB phosphorylation upregulates p62/SQSTM1 and promotes retinal pigmented epithelial cell survival through increased autophagy.

p62 is a scaffolding adaptor implicated in the clearance of protein aggregates by autophagy. Reactive oxygen species (ROS) can either stimulate or inhibit NFκB-mediated gene expression influencing cellular fate. We studied the effect of hydrogen peroxide (H2O2)-mediated oxidative stress and NFκB signaling on p62 expression in the retinal pigment epithelium (RPE) and investigated its role in regulation of autophagy and RPE survival against oxidative damage. Cultured human RPE cell line ARPE-19 and primary human adult and fetal RPE cells were exposed to H2O2-induced oxidative stress. The human apolipoprotein E4 targeted-replacement (APOE4) mouse model of AMD was used to study expression of p62 and other autophagy proteins in the retina. p62, NFκB p65 (total, phosphorylated, nuclear and cytoplasmic) and ATG10 expression was assessed by mRNA and protein analyses. Cellular ROS and mitochondrial superoxide were measured by CM-H2DCFDA and MitoSOX staining respectively. Mitochondrial viability was determined using MTT activity. qPCR-array system was used to investigate autophagic genes affected by p62. Nuclear and cytoplasmic levels of NFκB p65 were evaluated after cellular fractionation by Western blotting. We report that p62 is up-regulated in RPE cells under H2O2-induced oxidative stress and promotes autophagic activity. Depletion of endogenous p62 reduces autophagy by downregulation of ATG10 rendering RPE more susceptible to oxidative damage. NFκB p65 phosphorylation at Ser-536 was found to be critical for p62 upregulation in response to oxidative stress. Proteasome inhibition by H2O2 causes p62-NFκB signaling as antioxidant pre-treatment reversed p62 expression and p65 phosphorylation when RPE was challenged by H2O2 but not when by Lactacystin. p62 protein but not RNA levels are elevated in APOE4-HFC AMD mouse model, suggesting reduction of autophagic flux in disease conditions. Our findings suggest that p62 is necessary for RPE cytoprotection under oxidative stress and functions, in part, by modulating ATG10 expression. NFκB p65 activity may be a critical upstream initiator of p62 expression in RPE cells under oxidative stress.

"Bath Salts" the New York City Medical Examiner Experience: A 3-Year Retrospective Review.

"Bath salts" are synthetic derivatives of cathinones, compounds found in the leaves of Catha edulis, which possesses amphetamine-like properties. At the New York City Office of Chief Medical Examiner, we conducted a 3-year retrospective analysis of deaths in which cathinones were detected. Two categories emerged; those in which cathinones were a contributory cause of death (15 cases) and those in which they were an incidental finding (15 cases). Of the former group, 13 were associated with additional intoxicants; two deaths were attributed solely to cathinone intoxication, both survived 10 h: a man whose postmortem blood methylone concentration was 0.71 mg/L and a woman whose postmortem blood ethylone concentration was 1.7 mg/L. In the latter category, there were several individuals who had higher concentrations of cathinones than the above two, the highest being a blood methylone of 4.8 mg/L. Based upon our data and the literature presented, lethal concentrations of cathinones cannot be established.

A novel ATG4B antagonist inhibits autophagy and has a negative impact on osteosarcoma tumors.

Autophagy has been implicated in the progression and chemoresistance of various cancers. In this study, we have shown that osteosarcoma Saos-2 cells lacking ATG4B, a cysteine proteinase that activates LC3B, are defective in autophagy and fail to form tumors in mouse models. By combining in silico docking with in vitro and cell-based assays, we identified small compounds that suppressed starvation-induced protein degradation, LC3B lipidation, and formation of autophagic vacuoles. NSC185058 effectively inhibited ATG4B activity in vitro and in cells while having no effect on MTOR and PtdIns3K activities. In addition, this ATG4B antagonist had a negative impact on the development of Saos-2 osteosarcoma tumors in vivo. We concluded that tumor suppression was due to a reduction in ATG4B activity, since we found autophagy suppressed within treated tumors and the compound had no effects on oncogenic protein kinases. Our findings demonstrate that ATG4B is a suitable anti-autophagy target and a promising therapeutic target to treat osteosarcoma.

Rapamycin improves peripheral nerve myelination while it fails to benefit neuromuscular performance in neuropathic mice.

Charcot--Marie-Tooth disease type 1A (CMT1A) is a hereditary peripheral neuropathy characterized by progressive demyelination and distal muscle weakness. Abnormal expression of peripheral myelin protein 22 (PMP22) has been linked to CMT1A and is modeled by Trembler J (TrJ) mice, which carry the same leucine to proline substitution in PMP22 as affected pedigrees. Pharmacologic modulation of autophagy by rapamycin in neuron-Schwann cell explant cultures from neuropathic mice reduced PMP22 aggregate formation and improved myelination. Here we asked whether rapamycin administration by food supplementation, or intraperitoneal injection, could alleviate the neuropathic phenotype of affected mice and improve neuromuscular performance. Cohorts of male and female wild type (Wt) and TrJ mice were assigned to placebo or rapamycin treatment starting at 2 or 4months of age and tested monthly on the rotarod. While neither long-term feeding (8 or 10months) on rapamycin-enriched diet, or short-term injection (2months) of rapamycin improved locomotor performance of the neuropathic mice, both regimen benefited peripheral nerve myelination. Together, these results indicate that while treatment with rapamycin benefits the myelination capacity of neuropathic Schwann cells, this intervention does not improve neuromuscular function. The observed outcome might be the result of the differential response of nerve and skeletal muscle tissue to rapamycin.

Suicidal carbon monoxide poisoning by combining formic acid and sulfuric acid within a confined space.

Suicide by inhalation of carbon monoxide produced by mixing formic acid and sulfuric acid within a confined space is a rare method of suicide. This method is similar to the so-called "detergent suicide" method where an acid-based detergent is mixed with a sulfur source to produce hydrogen sulfide. Both methods produce a toxic gas that poses significant hazards for death investigators, first responders and bystanders. Carbon monoxide is an odorless gas, while hydrogen sulfide has a characteristic rotten eggs odor, so the risks associated with carbon monoxide are potentially greater due to lack of an important warning signal. While detergent suicides have become increasingly common in the USA, suicide with formic acid and sulfuric acid is rare with only three prior cases being reported. Greater awareness of this method among death investigators is warranted because of the special risks of accidental intoxication by toxic gas and the possibility that this method of suicide will become more common in the future.

Deletion of lipoprotein PG0717 in Porphyromonas gingivalis W83 reduces gingipain activity and alters trafficking in and response by host cells.

P. gingivalis (Pg), a causative agent of chronic generalized periodontitis, has been implicated in promoting cardiovascular disease. Expression of lipoprotein gene PG0717 of Pg strain W83 was found to be transiently upregulated during invasion of human coronary artery endothelial cells (HCAEC), suggesting this protein may be involved in virulence. We characterized the virulence phenotype of a PG0717 deletion mutant of pg W83. There were no differences in the ability of W83Δ717 to adhere and invade HCAEC. However, the increased proportion of internalized W83 at 24 hours post-inoculation was not observed with W83∆717. Deletion of PG0717 also impaired the ability of W83 to usurp the autophagic pathway in HCAEC and to induce autophagy in Saos-2 sarcoma cells. HCAEC infected with W83Δ717 also secreted significantly greater amounts of MCP-1, IL-8, IL-6, GM-CSF, and soluble ICAM-1, VCAM-1, and E-selectin when compared to W83. Further characterization of W83Δ717 revealed that neither capsule nor lipid A structure was affected by deletion of PG0717. Interestingly, the activity of both arginine (Rgp) and lysine (Kgp) gingipains was reduced in whole-cell extracts and culture supernatant of W83Δ717. RT-PCR revealed a corresponding decrease in transcription of rgpB but not rgpA or kgp. Quantitative proteome studies of the two strains revealed that both RgpA and RgpB, along with putative virulence factors peptidylarginine deiminase and Clp protease were significantly decreased in the W83Δ717. Our results suggest that PG0717 has pleiotropic effects on W83 that affect microbial induced manipulation of host responses important for microbial clearance and infection control.

Upregulated autophagy protects cardiomyocytes from oxidative stress-induced toxicity.

Autophagy is a cellular self-digestion process that mediates protein quality control and serves to protect against neurodegenerative disorders, infections, inflammatory diseases and cancer. Current evidence suggests that autophagy can selectively remove damaged organelles such as the mitochondria. Mitochondria-induced oxidative stress has been shown to play a major role in a wide range of pathologies in several organs, including the heart. Few studies have investigated whether enhanced autophagy can offer protection against mitochondrially-generated oxidative stress. We induced mitochondrial stress in cardiomyocytes using antimycin A (AMA), which increased mitochondrial superoxide generation, decreased mitochondrial membrane potential and depressed cellular respiration. In addition, AMA augmented nuclear DNA oxidation and cell death in cardiomyocytes. Interestingly, although oxidative stress has been proposed to induce autophagy, treatment with AMA did not result in stimulation of autophagy or mitophagy in cardiomyocytes. Our results showed that the MTOR inhibitor rapamycin induced autophagy, promoted mitochondrial clearance and protected cardiomyocytes from the cytotoxic effects of AMA, as assessed by apoptotic marker activation and viability assays in both mouse atrial HL-1 cardiomyocytes and human ventricular AC16 cells. Importantly, rapamycin improved mitochondrial function, as determined by cellular respiration, mitochondrial membrane potential and morphology analysis. Furthermore, autophagy induction by rapamycin suppressed the accumulation of ubiquitinylated proteins induced by AMA. Inhibition of rapamycin-induced autophagy by pharmacological or genetic interventions attenuated the cytoprotective effects of rapamycin against AMA. We propose that rapamycin offers cytoprotection against oxidative stress by a combined approach of removing dysfunctional mitochondria as well as by degrading damaged, ubiquitinated proteins. We conclude that autophagy induction by rapamycin could be utilized as a potential therapeutic strategy against oxidative stress-mediated damage in cardiomyocytes.

Autophagy and leucine promote chronological longevity and respiration proficiency during calorie restriction in yeast.

We have previously shown that autophagy is required for chronological longevity in the budding yeast Saccharomyces cerevisiae. Here we examine the requirements for autophagy during extension of chronological life span (CLS) by calorie restriction (CR). We find that autophagy is upregulated by two CR interventions that extend CLS: water wash CR and low glucose CR. Autophagy is required for full extension of CLS during water wash CR under all growth conditions tested. In contrast, autophagy was not uniformly required for full extension of CLS during low glucose CR, depending on the atg allele and strain genetic background. Leucine status influenced CLS during CR. Eliminating the leucine requirement in yeast strains or adding supplemental leucine to growth media extended CLS during CR. In addition, we observed that both water wash and low glucose CR promote mitochondrial respiration proficiency during aging of autophagy-deficient yeast. In general, the extension of CLS by water wash or low glucose CR was inversely related to respiration deficiency in autophagy-deficient cells. Also, autophagy is required for full extension of CLS under non-CR conditions in buffered media, suggesting that extension of CLS during CR is not solely due to reduced medium acidity. Thus, our findings show that autophagy is: (1) induced by CR, (2) required for full extension of CLS by CR in most cases (depending on atg allele, strain, and leucine availability) and, (3) promotes mitochondrial respiration proficiency during aging under CR conditions.

Amino acid homeostasis and chronological longevity in Saccharomyces cerevisiae.

Understanding how non-dividing cells remain viable over long periods of time, which may be decades in humans, is of central importance in understanding mechanisms of aging and longevity. The long-term viability of non-dividing cells, known as chronological longevity, relies on cellular processes that degrade old components and replace them with new ones. Key among these processes is amino acid homeostasis. Amino acid homeostasis requires three principal functions: amino acid uptake, de novo synthesis, and recycling. Autophagy plays a key role in recycling amino acids and other metabolic building blocks, while at the same time removing damaged cellular components such as mitochondria and other organelles. Regulation of amino acid homeostasis and autophagy is accomplished by a complex web of pathways that interact because of the functional overlap at the level of recycling. It is becoming increasingly clear that amino acid homeostasis and autophagy play important roles in chronological longevity in yeast and higher organisms. Our goal in this chapter is to focus on mechanisms and pathways that link amino acid homeostasis, autophagy, and chronological longevity in yeast, and explore their relevance to aging and longevity in higher eukaryotes.

Autophagy suppresses age-dependent ischemia and reperfusion injury in livers of mice.

BACKGROUND & AIMS: As life expectancy increases, there are greater numbers of patients with liver diseases who require surgery or transplantation. Livers of older patients have significantly less reparative capacity following ischemia and reperfusion (I/R) injury, which occurs during these operations. There are no strategies to reduce the age-dependent I/R injury. We investigated the role of autophagy in the age dependence of sensitivity to I/R injury. METHODS: Hepatocytes and livers from 3- and 26-month-old mice were subjected to in vitro and in vivo I/R, respectively. We analyzed changes in autophagy-related proteins (Atg). Mitochondrial dysfunction was visualized using confocal and intravital multi-photon microscopy of isolated hepatocytes and livers from anesthetized mice, respectively. RESULTS: Immunoblot, autophagic flux, genetic, and imaging analyses all associated the increase in sensitivity to I/R injury with age with decreased autophagy and subsequent mitochondrial dysfunction due to calpain-mediated loss of Atg4B. Overexpression of either Atg4B or Beclin-1 recovered Atg4B, increased autophagy, blocked the onset of the mitochondrial permeability transition, and suppressed cell death after I/R in old hepatocytes. Coimmunoprecipitation analysis of hepatocytes and Atg3-knockout cells showed an interaction between Beclin-1 and Atg3, a protein required for autophagosome formation. Intravital multi-photon imaging revealed that overexpression of Beclin-1 or Atg4B attenuated autophagic defects and mitochondrial dysfunction in livers of older mice after I/R. CONCLUSIONS: Loss of Atg4B in livers of old mice increases their sensitivity to I/R injury. Increasing autophagy might ameliorate liver damage and restore mitochondrial function after I/R.

aeGEPUCI: a database of gene expression in the dengue vector mosquito, Aedes aegypti.

BACKGROUND: Aedes aegypti is the principal vector of dengue and yellow fever viruses. The availability of the sequenced and annotated genome enables genome-wide analyses of gene expression in this mosquito. The large amount of data resulting from these analyses requires efficient cataloguing before it becomes useful as the basis for new insights into gene expression patterns and studies of the underlying molecular mechanisms for generating these patterns. FINDINGS: We provide a publicly-accessible database and data-mining tool, aeGEPUCI, that integrates 1) microarray analyses of sex- and stage-specific gene expression in Ae. aegypti, 2) functional gene annotation, 3) genomic sequence data, and 4) computational sequence analysis tools. The database can be used to identify genes expressed in particular stages and patterns of interest, and to analyze putative cis-regulatory elements (CREs) that may play a role in coordinating these patterns. The database is accessible from the address http://www.aegep.bio.uci.edu. CONCLUSIONS: The combination of gene expression, function and sequence data coupled with integrated sequence analysis tools allows for identification of expression patterns and streamlines the development of CRE predictions and experiments to assess how patterns of expression are coordinated at the molecular level.

Rapamycin activates autophagy and improves myelination in explant cultures from neuropathic mice.

Misexpression and cytosolic retention of peripheral myelin protein 22 (PMP22) within Schwann cells (SCs) is associated with a genetically heterogeneous group of demyelinating peripheral neuropathies. PMP22 overproducer C22 and spontaneous mutant Trembler J (TrJ) mice display neuropathic phenotypes and affected nerves contain abnormally localized PMP22. Nutrient deprivation-induced autophagy is able to suppress the formation of PMP22 aggregates in a toxin-induced cellular model, and improve locomotor performance and myelination in TrJ mice. As a step toward therapies, we assessed whether pharmacological activation of autophagy by rapamycin (RM) could facilitate the processing of PMP22 within neuropathic SCs and enhance their capacity to myelinate peripheral axons. Exposure of mouse SCs to RM induced autophagy in a dose- and time-dependent manner and decreased the accumulation of poly-ubiquitinated substrates. The treatment of myelinating dorsal root ganglion (DRG) explant cultures from neuropathic mice with RM (25 nm) improved the processing of PMP22 and increased the abundance and length of myelin internodes, as well as the expression of myelin proteins. Notably, RM is similarly effective in both the C22 and TrJ model, signifying that the benefit overlaps among distinct genetic models of PMP22 neuropathies. Furthermore, lentivirus-mediated shRNA knockdown of the autophagy-related gene 12 (Atg12) abolished the activation of autophagy and the increase in myelin proteins, demonstrating that autophagy is critical for the observed improvement. Together, these results support the potential use of RM and other autophagy-enhancing compounds as therapeutic agents for PMP22-associated demyelinating neuropathies.

Using LC3 to monitor autophagy flux in the retinal pigment epithelium.

Autophagy is a highly conserved housekeeping pathway that plays a critical role in the removal of aged or damaged intracellular organelles and their delivery to lysosomes for degradation. Autophagy begins with the formation of membranes, arising in part from the endoplasmic reticulum, that elongate and fuse engulfing cytoplasmic constituents into a classic double-membrane bound nascent autophagosome. These early autophagosomes undergo a stepwise maturation process to form the late autophagosome or amphisome that ultimately fuses with a lysosome. Efficient autophagy is dependent on an equilibrium between the formation and elimination of autophagosomes; thus, a deficit in any part of this pathway will cause autophagic dysfunction. Autophagy plays a role in aging and age-related diseases. However, few studies of autophagy in retinal disease have been reported.

Autophagy is required for extension of yeast chronological life span by rapamycin.

Rapamycin is an antibiotic that stimulates autophagy in a wide variety of eukaryotes, including the budding yeast Saccharomyces cerevisiae. Low concentrations of rapamycin extend yeast chronological life span (CLS). We have recently shown that autophagy is required for chronological longevity in yeast, which is attributable in part to a role for autophagy in amino acid homeostasis. We report herein that low concentrations of rapamycin stimulate macroautophagy during chronological aging and extend CLS.

Autophagy and amino acid homeostasis are required for chronological longevity in Saccharomyces cerevisiae.

Following cessation of growth, yeast cells remain viable in a nondividing state for a period of time known as the chronological lifespan (CLS). Autophagy is a degradative process responsible for amino acid recycling in response to nitrogen starvation and amino acid limitation. We have investigated the role of autophagy during chronological aging of yeast grown in glucose minimal media containing different supplemental essential and nonessential amino acids. Deletion of ATG1 or ATG7, both of which are required for autophagy, reduced CLS, whereas deletion of ATG11, which is required for selective targeting of cellular components to the vacuole for degradation, did not reduce CLS. The nonessential amino acids isoleucine and valine, and the essential amino acid leucine, extended CLS in autophagy-deficient as well as autophagy-competent yeast. This extension was suppressed by constitutive expression of GCN4, which encodes a transcriptional regulator of general amino acid control (GAAC). Consistent with this, GCN4 expression was reduced by isoleucine and valine. Furthermore, elimination of the leucine requirement extended CLS and prevented the effects of constitutive expression of GCN4. Interestingly, deletion of LEU3, a GAAC target gene encoding a transcriptional regulator of branched side chain amino acid synthesis, dramatically increased CLS in the absence of amino acid supplements. In general, this indicates that activation of GAAC reduces CLS whereas suppression of GAAC extends CLS in minimal medium. These findings demonstrate important roles for autophagy and amino acid homeostasis in determining CLS in yeast.

Intermittent fasting alleviates the neuropathic phenotype in a mouse model of Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth type 1A (CMT1A) neuropathies linked to the misexpression of peripheral myelin protein 22 (PMP22) are progressive demyelinating disorders of the peripheral nervous system. In this study we asked whether dietary restriction by intermittent fasting (IF) could alleviate the neuropathic phenotype in the Trembler J (TrJ) mouse model of CMT1A. Our results show that neuropathic mice kept on a five month long IF regimen had improved locomotor performance compared to ad libitum (AL) fed littermates. The functional benefits of this dietary intervention are associated with an increased expression of myelin proteins combined with a thicker myelin sheath, less redundant basal lamina, and a reduction in aberrant Schwann cell proliferation. These morphological improvements are accompanied by a decrease in PMP22 protein aggregates, and enhanced expression of cytosolic chaperones and constituents of the autophagy-lysosomal pathway. These results indicate that dietary restriction is beneficial for peripheral nerve function in TrJ neuropathic mice, as it promotes the maintenance of locomotor performance.