Genetic Mapping and Phenotypic Analysis of GstE14 E.4.1 on Eye and Antennae Development in Drosophila melanogaster.

Genetic screens are valuable for identifying novel genes involved in the regulation of developmental processes. To identify genes associated with cell growth regulation in Drosophila melanogaster , a mutagenesis screen was performed. Undergraduate students participating in Fly-CURE phenotypically characterized the E.4.1 mutant which is associated with rough eyes and antennae overgrowth. Following complementation analysis and subsequent genomic sequencing, E.4.1 was identified as a novel mutant allele of GstE14 , a gene involved in ecdysone biosynthesis important for the timing of developmental events. The abnormal eye and antenna phenotypes observed resulting from the loss of GstE14 suggest its role in tissue growth.

c-Abl Phosphorylates MFN2 to Regulate Mitochondrial Morphology in Cells under Endoplasmic Reticulum and Oxidative Stress, Impacting Cell Survival and Neurodegeneration.

The endoplasmic reticulum is a subcellular organelle key in the control of synthesis, folding, and sorting of proteins. Under endoplasmic reticulum stress, an adaptative unfolded protein response is activated; however, if this activation is prolonged, cells can undergo cell death, in part due to oxidative stress and mitochondrial fragmentation. Here, we report that endoplasmic reticulum stress activates c-Abl tyrosine kinase, inducing its translocation to mitochondria. We found that endoplasmic reticulum stress-activated c-Abl interacts with and phosphorylates the mitochondrial fusion protein MFN2, resulting in mitochondrial fragmentation and apoptosis. Moreover, the pharmacological or genetic inhibition of c-Abl prevents MFN2 phosphorylation, mitochondrial fragmentation, and apoptosis in cells under endoplasmic reticulum stress. Finally, in the amyotrophic lateral sclerosis mouse model, where endoplasmic reticulum and oxidative stress has been linked to neuronal cell death, we demonstrated that the administration of c-Abl inhibitor neurotinib delays the onset of symptoms. Our results uncovered a function of c-Abl in the crosstalk between endoplasmic reticulum stress and mitochondrial dynamics via MFN2 phosphorylation.

Effects of the energy balance transition on bone mass and strength.

Chronic positive energy balance has surged among societies worldwide due to increasing dietary energy intake and decreasing physical activity, a phenomenon called the energy balance transition. Here, we investigate the effects of this transition on bone mass and strength. We focus on the Indigenous peoples of New Mexico in the United States, a rare case of a group for which data can be compared between individuals living before and after the start of the transition. We show that since the transition began, bone strength in the leg has markedly decreased, even though bone mass has apparently increased. Decreased bone strength, coupled with a high prevalence of obesity, has resulted in many people today having weaker bones that must sustain excessively heavy loads, potentially heightening their risk of a bone fracture. These findings may provide insight into more widespread upward trends in bone fragility and fracture risk among societies undergoing the energy balance transition.

Genomics of Re-Emergent Aeromonas salmonicida in Atlantic Salmon Outbreaks.

Furunculosis, caused by Aeromonas salmonicida, poses a significant threat to both salmonid and non-salmonid fish in diverse aquatic environments. This study explores the genomic intricacies of re-emergent A. salmonicida outbreaks in Atlantic salmon (Salmo salar). Previous clinical cases have exhibited pathological characteristics, such as periorbital hemorrhages and gastrointestinal abnormalities. Genomic sequencing of three Chilean isolates (ASA04, ASA05, and CIBA_5017) and 25 previously described genomes determined the pan-genome, phylogenomics, insertion sequences, and restriction-modification systems. Unique gene families have contributed to an improved understanding of the psychrophilic and mesophilic clades, while phylogenomic analysis has been used to identify mesophilic and psychrophilic strains, thereby further differentiating between typical and atypical psychrophilic isolates. Diverse insertion sequences and restriction-modification patterns have highlighted genomic structural differences, and virulence factor predictions can emphasize exotoxin disparities, especially between psychrophilic and mesophilic strains. Thus, a novel plasmid was characterized which emphasized the role of plasmids in virulence and antibiotic resistance. The analysis of antibiotic resistance factors revealed resistance against various drug classes in Chilean strains. Overall, this study elucidates the genomic dynamics of re-emergent A. salmonicida and provides novel insights into their virulence, antibiotic resistance, and population structure.

Examining the Relationship between the Lethality Assessment/Domestic Violence High-risk Team Monitoring (LAP/DVHRT) Program and Prosecution Outcomes.

Lethality assessment (LAP) and team monitoring of high-risk offenders (DVHRT) are recent U.S. policy innovations designed to identify domestic violence offenders who are at high risk for perpetrating serious or lethal violence against their intimate partners. One goal of LAP/DVHRT is to increase offenders' accountability for domestic violence within the legal system. This study examines associations between LAP/DVHRT and prosecution and sentencing outcomes using data on domestic violence offenses (n = 88) involving 37 offenders monitored by a DVHRT and 51 nonmonitored comparison offenders who were identified as high risk on the LAP. We use logistic and OLS regression to estimate models of six prosecution and sentencing outcomes for the full sample and for a sample matched using the coarsened exact matching technique (n = 73). Multivariate results indicate that when the treatment and comparison samples are matched and control variables are included in regression models, the LAP/DVHRT program is not associated with prosecution or conviction rates, number of charges, or bail amount. DVHRT monitoring is positively associated with sentence length in multivariate analysis and in models using the matched sample. Findings suggest that the LAP/DVHRT program increases offender accountability in the form of incapacitation at the sentencing stages.

Whole-Genome sequencing and comparative genomics of Mycobacterium spp. from farmed Atlantic and coho salmon in Chile.

Several members of the Mycobacterium genus cause invasive infections in humans and animals. According to a recent phylogenetic analysis, some strains of Mycobacterium salmoniphilum (Msal), which are the main culprit in bacterial outbreaks in freshwater fish aquaculture, have been assigned to a separate branch containing Mycobacterium franklinii (Mfra), another species that causes infections in humans. However, this genus is little studied in an aquaculture context. Here, we isolated four Mycobacterium spp. strains from freshwater cultures of Atlantic and coho salmon in Chile and performed whole-genome sequencing for deep genomic characterization. In addition, we described the gross pathology and histopathology of the outbreaks. Several bioinformatic analyses were performed using the genomes of these four Mycobacterium isolates in conjunction with those of Msal strains, four Msal-like strains, and one Mfra strains, plus 17 other publicly available Mycobacterium genomes. We found that three isolates are clustered into the Msal branch, whereas one isolate clustered with the Mfra/Msal-like strains. We further evaluated the presence of virulence and antimicrobial resistance genes and observed that the four isolates were closely related to the Msal and Msal-like taxa and carried several antimicrobial resistance and virulence genes that are similar to those of other pathogenic members of the Mycobacterium clade. Altogether, our characterization Msal and Msal-like presented here shed new light on the basis of mycobacteriosis provides quantitative evidence that Mycobacterium strains are a potential risk for aquaculture asetiological agents of emerging diseases, and highlight their biological scopes in the aquaculture industry.

Enforced dimerization between XBP1s and ATF6f enhances the protective effects of the UPR in models of neurodegeneration.

Alteration to endoplasmic reticulum (ER) proteostasis is observed in a variety of neurodegenerative diseases associated with abnormal protein aggregation. Activation of the unfolded protein response (UPR) enables an adaptive reaction to recover ER proteostasis and cell function. The UPR is initiated by specialized stress sensors that engage gene expression programs through the concerted action of the transcription factors ATF4, ATF6f, and XBP1s. Although UPR signaling is generally studied as unique linear signaling branches, correlative evidence suggests that ATF6f and XBP1s may physically interact to regulate a subset of UPR target genes. In this study, we designed an ATF6f/XBP1s fusion protein termed UPRplus that behaves as a heterodimer in terms of its selective transcriptional activity. Cell-based studies demonstrated that UPRplus has a stronger effect in reducing the abnormal aggregation of mutant huntingtin and α-synuclein when compared to XBP1s or ATF6 alone. We developed a gene transfer approach to deliver UPRplus into the brain using adeno-associated viruses (AAVs) and demonstrated potent neuroprotection in vivo in preclinical models of Parkinson's disease and Huntington's disease. These results support the concept in which directing UPR-mediated gene expression toward specific adaptive programs may serve as a possible strategy to optimize the beneficial effects of the pathway in different disease conditions.

c-Abl Inhibition Activates TFEB and Promotes Cellular Clearance in a Lysosomal Disorder.

The transcription factor EB (TFEB) has emerged as a master regulator of lysosomal biogenesis, exocytosis, and autophagy, promoting the clearance of substrates stored in cells. c-Abl is a tyrosine kinase that participates in cellular signaling in physiological and pathophysiological conditions. In this study, we explored the connection between c-Abl and TFEB. Here, we show that under pharmacological and genetic c-Abl inhibition, TFEB translocates into the nucleus promoting the expression of its target genes independently of its well-known regulator, mammalian target of rapamycin complex 1. Active c-Abl induces TFEB phosphorylation on tyrosine and the inhibition of this kinase promotes lysosomal biogenesis, autophagy, and exocytosis. c-Abl inhibition in Niemann-Pick type C (NPC) models, a neurodegenerative disease characterized by cholesterol accumulation in lysosomes, promotes a cholesterol-lowering effect in a TFEB-dependent manner. Thus, c-Abl is a TFEB regulator that mediates its tyrosine phosphorylation, and the inhibition of c-Abl activates TFEB promoting cholesterol clearance in NPC models.

Correction: The necroptosis machinery mediates axonal degeneration in a model of Parkinson disease.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The necroptosis machinery mediates axonal degeneration in a model of Parkinson disease.

Parkinson's disease (PD) is the second most common neurodegenerative condition, characterized by motor impairment due to the progressive degeneration of dopaminergic neurons in the substantia nigra and depletion of dopamine release in the striatum. Accumulating evidence suggest that degeneration of axons is an early event in the disease, involving destruction programs that are independent of the survival of the cell soma. Necroptosis, a programmed cell death process, is emerging as a mediator of neuronal loss in models of neurodegenerative diseases. Here, we demonstrate activation of necroptosis in postmortem brain tissue from PD patients and in a toxin-based mouse model of the disease. Inhibition of key components of the necroptotic pathway resulted in a significant delay of 6-hydroxydopamine-dependent axonal degeneration of dopaminergic and cortical neurons in vitro. Genetic ablation of necroptosis mediators MLKL and RIPK3, as well as pharmacological inhibition of RIPK1 in preclinical models of PD, decreased dopaminergic neuron degeneration, improving motor performance. Together, these findings suggest that axonal degeneration in PD is mediated by the necroptosis machinery, a process here referred to as necroaxoptosis, a druggable pathway to target dopaminergic neuronal loss.

Targeting of the unfolded protein response (UPR) as therapy for Parkinson's disease.

Parkinson's disease is the second most common neurodegenerative disorder, leading to the progressive decline of motor control due to the loss of dopaminergic neurons in the substantia nigra pars compacta. At the molecular level, Parkinson's disease share common molecular signatures with most neurodegenerative diseases including the accumulation of misfolded proteins in the brain. Alteration in the buffering capacity of the proteostasis network during aging is proposed as one of the triggering steps leading to abnormal protein aggregation in this disease, highlighting disturbances in the function of the endoplasmic reticulum (ER). The ER is the main subcellular compartment involved in protein folding and quality control. ER stress triggers a signalling reaction known as the unfolded protein response (UPR), which aims restoring proteostasis through the induction of adaptive programs or the activation of cell death pathways when damage is chronic and cannot be repaired. Here, we overview most evidence linking ER stress to Parkinson's disease. Strategies to alleviate ER stress by targeting specific components of the UPR using small molecules and gene therapy are highlighted.

Multilocus sequence typing detects new Piscirickettsia salmonis hybrid genogroup in Chilean fish farms: Evidence for genetic diversity and population structure.

Piscirickettsia salmonisis the causative bacterial pathogen of piscirickettsiosis, a salmonid disease that causes notable mortalities in the worldwide aquaculture industry. Published research describes the phenotypic traits, virulence factors, pathogenicity and antibiotic-resistance potential for various P. salmonisstrains. However, evolutionary and genetic information is scarce for P. salmonis. The present study used multilocus sequence typing (MLST) to gain insight into the population structure and evolution of P. salmonis. Forty-two Chilean P. salmonisisolates, as well as the type strain LF-89T , were recovered from diseased Salmo salar, Oncorhynchus kisutchand Oncorhynchus mykissfrom two Chilean Regions. MLST assessed the loci sequences of dnaK, efp, fumC, glyA, murG, rpoD and trpB. Bioinformatics analyses established the genetic diversity among P. salmonis isolates (H = 0.5810). A total of 23 sequence types (ST) were identified, 53.48% of which were represented by ST1, ST5 and ST2. Population structure analysis through polymorphism patterns showed few polymorphic sites (218 nucleotides from 4,010 bp), while dN/dS ratio analysis indicated purifying selection for dnaK, epf, fumC, murG, and rpoD but neutral selection for the trpB loci. The standardized index of association indicated strong linkage disequilibrium, suggesting clonal population structure. However, recombination events were detected in a group of seven isolates. Findings included genogroups homologous to the LF-89T and EM-90 strains, as well as a seven-isolate hybrid genogroup recovered from both assessed regions (three O. mykiss and four S. salar isolates). The presented MLST scheme has comparative potential, with promising applications in studying distinct P. salmonis isolates (e.g., from different hosts, farms, geographical areas) and in understanding the epidemiology of this pathogen.

New Insights into the Spontaneous Human Alzheimer's Disease-Like Model Octodon degus: Unraveling Amyloid-ß Peptide Aggregation and Age-Related Amyloid Pathology.

Alzheimer's disease (AD) is the most common cause of dementia worldwide. Despite advances in our understanding of the molecular milieu driving AD pathophysiology, no effective therapy is currently available. Moreover, various clinical trials have continued to fail, suggesting that our approach to AD must be revised. Accordingly, the development and validation of new models are highly desirable. Over the last decade, we have been working with Octodon degus (degu), a Chilean rodent, which spontaneously develops AD-like neuropathology, including increased amyloid-ß (Aß) aggregates, tau hyperphosphorylation, and postsynaptic dysfunction. However, for proper validation of degu as an AD model, the aggregation properties of its Aß peptide must be analyzed. Thus, in this study, we examined the capacity of the degu Aß peptide to aggregate in vitro. Then, we analyzed the age-dependent variation in soluble Aß levels in the hippocampus and cortex of third- to fifth-generation captive-born degu. We also assessed the appearance and spatial distribution of amyloid plaques in O. degus and compared them with the plaques in two AD transgenic mouse models. In agreement with our previous studies, degu Aß was able to aggregate, forming fibrillar species in vitro. Furthermore, amyloid plaques appeared in the anterior brain structures of O. degus at approximately 32 months of age and in the whole brain at 56 months, along with concomitant increases in Aß levels and the Aß42/Aß40 ratio, indicating that O. degus spontaneously develops AD-like pathology earlier than other spontaneous models. Based on these results, we can confirm that O. degus constitutes a valuable model to improve AD research.

A new model to study cell-to-cell transfer of αSynuclein in vivo.

Parkinson's disease (PD) compromises motor control due to the loss of dopaminergic neurons in the substantia nigra pars compacta. At the histopathological level, PD is characterized by the accumulation of Lewy bodies, large protein inclusions containing aggregated αSynuclein (αSyn). The progression of PD involves the spreading of αSyn misfolding through the brain mediated by a prion-like mechanism, where the protein is transferred between cells. Here we report that αSyn internalization is a dynamic process, where the protein transits through different sub-cellular compartments. Importantly, cells incorporating αSyn develop larger protein-like inclusions when compared to αSyn producing cells. We developed a new tool to monitor cell-to-cell transfer of αSyn in vivo using an adeno-associated viral (AAV) vector expressing αSyn fused to a red fluorescent protein in addition to soluble EGFP to label donor cells. Intra-nigral delivery of this reporter AAV construct allowed the visualization of αSyn incorporation into surrounding neurons. This work provides a new tool to study αSyn cell-to-cell transfer in vivo and may open new opportunities to study PD pathogenesis.

MLN64 induces mitochondrial dysfunction associated with increased mitochondrial cholesterol content.

MLN64 is a late endosomal cholesterol-binding membrane protein that has been implicated in cholesterol transport from endosomal membranes to the plasma membrane and/or mitochondria, in toxin-induced resistance, and in mitochondrial dysfunction. Down-regulation of MLN64 in Niemann-Pick C1 deficient cells decreased mitochondrial cholesterol content, suggesting that MLN64 functions independently of NPC1. However, the role of MLN64 in the maintenance of endosomal cholesterol flow and intracellular cholesterol homeostasis remains unclear. We have previously described that hepatic MLN64 overexpression increases liver cholesterol content and induces liver damage. Here, we studied the function of MLN64 in normal and NPC1-deficient cells and we evaluated whether MLN64 overexpressing cells exhibit alterations in mitochondrial function. We used recombinant-adenovirus-mediated MLN64 gene transfer to overexpress MLN64 in mouse liver and hepatic cells; and RNA interference to down-regulate MLN64 in NPC1-deficient cells. In MLN64-overexpressing cells, we found increased mitochondrial cholesterol content and decreased glutathione (GSH) levels and ATPase activity. Furthermore, we found decreased mitochondrial membrane potential and mitochondrial fragmentation and increased mitochondrial superoxide levels in MLN64-overexpressing cells and in NPC1-deficient cells. Consequently, MLN64 expression was increased in NPC1-deficient cells and reduction of its expression restore mitochondrial membrane potential and mitochondrial superoxide levels. Our findings suggest that MLN64 overexpression induces an increase in mitochondrial cholesterol content and consequently a decrease in mitochondrial GSH content leading to mitochondrial dysfunction. In addition, we demonstrate that MLN64 expression is increased in NPC cells and plays a key role in cholesterol transport into the mitochondria.

Reactive oxygen species trigger motoneuron death in non-cell-autonomous models of ALS through activation of c-Abl signaling.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which pathogenesis and death of motor neurons are triggered by non-cell-autonomous mechanisms. We showed earlier that exposing primary rat spinal cord cultures to conditioned media derived from primary mouse astrocyte conditioned media (ACM) that express human SOD1(G93A) (ACM-hSOD1(G93A)) quickly enhances Nav channel-mediated excitability and calcium influx, generates intracellular reactive oxygen species (ROS), and leads to death of motoneurons within days. Here we examined the role of mitochondrial structure and physiology and of the activation of c-Abl, a tyrosine kinase that induces apoptosis. We show that ACM-hSOD1(G93A), but not ACM-hSOD1(WT), increases c-Abl activity in motoneurons, interneurons and glial cells, starting at 60 min; the c-Abl inhibitor STI571 (imatinib) prevents this ACM-hSOD1(G93A)-mediated motoneuron death. Interestingly, similar results were obtained with ACM derived from astrocytes expressing SOD1(G86R) or TDP43(A315T). We further find that co-application of ACM-SOD1(G93A) with blockers of Nav channels (spermidine, mexiletine, or riluzole) or anti-oxidants (Trolox, esculetin, or tiron) effectively prevent c-Abl activation and motoneuron death. In addition, ACM-SOD1(G93A) induces alterations in the morphology of neuronal mitochondria that are related with their membrane depolarization. Finally, we find that blocking the opening of the mitochondrial permeability transition pore with cyclosporine A, or inhibiting mitochondrial calcium uptake with Ru360, reduces ROS production and c-Abl activation. Together, our data point to a sequence of events in which a toxic factor(s) released by ALS-expressing astrocytes rapidly induces hyper-excitability, which in turn increases calcium influx and affects mitochondrial structure and physiology. ROS production, mediated at least in part through mitochondrial alterations, trigger c-Abl signaling and lead to motoneuron death.

Drug-related deaths and the sales of needles through pharmacies.

BACKGROUND: Providing needles to people who inject drugs is a well-proven public health response to the transmission of HIV and other blood borne viruses. Despite over a quarter of a century of research, new concerns about potential unintended negative consequences of needle distribution continue to emerge. Specifically, a claim was recently made that the introduction of pharmacy sales of needles was followed by an increase in overdoses in pharmacy parking lots. If true, this would have serious implications for the design of needle access programs, particularly those involving pharmacy sales of needles. METHODS: We examine spatial relationships between drug-related deaths and pharmacies in Los Angeles County (population 9.8 million) before and after the 2007 enactment of a California law allowing pharmacy sales of needles without a prescription. Seven thousand and forty-nine drugs related deaths occurred in Los Angeles county from 2000 to 2009 inclusive. Four thousand two hundred and seventy-five of these deaths could be geocoded, and were found to be clustered at the census tract level. RESULTS: We used three methods to examine spatial relationships between overdose death locations and pharmacy locations for two years on either side of the enactment of the pharmacy sales law, and found no statistically significant changes. Among the 711 geocodable deaths occurring in the two years following the change in law, no death was found to occur within 50m of a pharmacy which sold needles. CONCLUSION: These results are consistent with prior studies which suggest pharmacy sales of needles improve access to needles without causing increased harms to the surrounding community.

Relationship among the physiologic channel interactions, spectral-ripple discrimination, and vowel identification in cochlear implant users.

The hypothesis of this study was that broader patterns of physiological channel interactions in the local region of the cochlea are associated with poorer spectral resolution in the same region. Electrically evoked compound action potentials (ECAPs) were measured for three to six probe electrodes per subject to examine the channel interactions in different regions across the electrode array. To evaluate spectral resolution at a confined location within the cochlea, spectral-ripple discrimination (SRD) was measured using narrowband ripple stimuli with the bandwidth spanning five electrodes: Two electrodes apical and basal to the ECAP probe electrode. The relationship between the physiological channel interactions, spectral resolution in the local cochlear region, and vowel identification was evaluated. Results showed that (1) there was within- and across-subject variability in the widths of ECAP channel interaction functions and in narrowband SRD performance, (2) significant correlations were found between the widths of the ECAP functions and narrowband SRD thresholds, and between mean bandwidths of ECAP functions averaged across multiple probe electrodes and broadband SRD performance across subjects, and (3) the global spectral resolution reflecting the entire electrode array, not the local region, predicts vowel identification.

Control of dopaminergic neuron survival by the unfolded protein response transcription factor XBP1.

Parkinson disease (PD) is characterized by the selective loss of dopaminergic neurons of the substantia nigra pars compacta (SNpc). Although growing evidence indicates that endoplasmic reticulum (ER) stress is a hallmark of PD, its exact contribution to the disease process is not well understood. Here we report that developmental ablation of X-Box binding protein 1 (XBP1) in the nervous system, a key regulator of the unfolded protein response (UPR), protects dopaminergic neurons against a PD-inducing neurotoxin. This survival effect was associated with a preconditioning condition that resulted from induction of an adaptive ER stress response in dopaminergic neurons of the SNpc, but not in other brain regions. In contrast, silencing XBP1 in adult animals triggered chronic ER stress and dopaminergic neuron degeneration. Supporting this finding, gene therapy to deliver an active form of XBP1 provided neuroprotection and reduced striatal denervation in animals injected with 6-hydroxydopamine. Our results reveal a physiological role of the UPR in the maintenance of protein homeostasis in dopaminergic neurons that may help explain the differential neuronal vulnerability observed in PD.