Mortality and neurodevelopmental outcomes at 2 years' corrected age of very preterm infants with necrotising enterocolitis or spontaneous intestinal perforation: The EPIPAGE-2 cohort study.

PURPOSE: The primary objective was to evaluate the impact of necrotising enterocolitis (NEC) and spontaneous intestinal perforation (SIP) on mortality and neurodevelopmental outcomes at 2 years' corrected age (CA) in infants born before 32 weeks' gestation (WG). METHODS: We studied neurodevelopment at 2 years' CA of infants with NEC or SIP who were born before 32 WG from the EPIPAGE-2 cohort study. The primary outcome was death or the presence of moderate-to-severe motor or sensory disability defined by moderate-to-severe cerebral palsy or hearing or visual disability. The secondary outcome was developmental delay defined by a score < 2 SDs below the mean for any of the five domains of the Ages and Stages Questionnaire. RESULTS: At 2 years' CA, 46% of infants with SIP, 34% of infants with NEC, and 14% of control infants died or had a moderate-to-severe sensorimotor disability (p < 0.01). This difference was mainly due to an increase in in-hospital mortality in the infants with SIP or NEC. Developmental delay at 2 years' CA was more frequent for infants with SIP than controls (70.8% vs 44.0%, p = 0.02) but was similar for infants with NEC and controls (49.3% vs 44.0%, p = 0.5). On multivariate analysis, the likelihood of developmental delay was associated with SIP (adjusted odds ratio = 3.0, 95% CI 1.0-9.1) but not NEC as compared with controls. CONCLUSION: NEC and SIP significantly increased the risk of death or sensorimotor disability at 2 years' CA. SIP was also associated with risk of developmental delay at 2 years' CA.

Long-term weight gain in children with craniopharyngioma.

OBJECTIVE: Adamantinomatous craniopharyngioma mainly affects children. Excessive weight gain is a major long-term complication. The primary objective of this study was to assess long-term weight changes in children treated for craniopharyngioma. The secondary objectives were to identify risk factors for excessive weight gain and to look for associations with hypothalamic damage by the tumour or treatment. DESIGN: Single-centre retrospective cohort study. METHOD: Children managed for craniopharyngioma at our centre between 1990 and 2019 were included. The body mass index (BMI) standard deviation scores (SDS) at baseline and at last follow-up were compared. Univariate and multivariate analyses were performed in order to identify variables associated with the long-term BMI-SDS variation. RESULTS: The 108 patients had a mean follow-up of 10.4 years. The mean BMI-SDS increase over time was 2.11 (P < .001) overall, 1.21 (P < .001) in the group without hypothalamic involvement by the tumour, and 1.95 (P < .001) in the group managed using intended hypothalamus-sparing surgery. The absence of hypothalamic involvement by the tumour or treatment was significantly associated with less weight gain (P = .046 and P < .01, respectively). After adjustment, factors associated with a BMI-SDS change greater than 2 were female sex (P = .023), tumour involving the hypothalamus (P = .04), and higher baseline BMI (P < .001). CONCLUSION: Clinically significant weight gain occurred in nearly all children treated for craniopharyngioma, including those whose hypothalamus was spared by the tumour and intentionally by treatment. However, hypothalamus integrity was associated with less weight gain. Despite hypothalamus-sparing strategies, hypothalamic obesity remains a major concern, indicating a need for novel treatment approaches.

Outcomes after aortic coarctation repair in neonates weighing less than 2000 g.

BACKGROUND: Preterm birth is common in children with congenital heart disease. However, data on how to manage low-birth-weight infants with aortic coarctation are scarce and outcomes are poorly reported. Surgery is often delayed in these infants because gaining weight is supposed to improve mortality and to reduce the risk for recoarctation. METHODS: All infants weighing less than 2000 g who underwent repair for aortic coarctation at our institution between January 2017 and December 2020 were included in a retrospective study. Baseline characteristics, medical and surgical management, and outcomes, including recoarctation, death, and complications of preterm birth, were analyzed. RESULTS: A total of 15 patients had coarctation repair at a median age of 15 days and at a median weight of 1585 g. Infants with a birth weight <1200 g were operated on later and did not have higher recoarctation rates compared to those with a birth weight >1200 g. The recoarctation rate was 26.6% and one infant died of an extracardiac cause. Concerning prematurity-related complications, we observed 40% of bronchopulmonary dysplasia, 40% of intraventricular hemorrhage, and 27% of retinopathy of prematurity. These complications were more prevalent in children with a birth weight of <1200 g. CONCLUSION: Delaying surgery beyond 15 days to gain weight does not appear to decrease the risk of recoarctation and may be deleterious in low-birth-weight infants who are exposed for a longer period to risk factors of prematurity-related complications.

Lifespan-increasing drug nordihydroguaiaretic acid inhibits p300 and activates autophagy.

Aging is characterized by the progressive loss of physiological function in all organisms. Remarkably, the aging process can be modulated by environmental modifications, including diet and small molecules. The natural compound nordihydroguaiaretic acid (NDGA) robustly increases lifespan in flies and mice, but its mechanism of action remains unclear. Here, we report that NDGA is an inhibitor of the epigenetic regulator p300. We find that NDGA inhibits p300 acetyltransferase activity in vitro and suppresses acetylation of a key p300 target in histones (i.e., H3K27) in cells. We use the cellular thermal shift assay to uniquely demonstrate NDGA binding to p300 in cells. Finally, in agreement with recent findings indicating that p300 is a potent blocker of autophagy, we show that NDGA treatment induces autophagy. These findings identify p300 as a target of NDGA and provide mechanistic insight into its role in longevity.

Age- and stress-associated C. elegans granulins impair lysosomal function and induce a compensatory HLH-30/TFEB transcriptional response.

The progressive failure of protein homeostasis is a hallmark of aging and a common feature in neurodegenerative disease. As the enzymes executing the final stages of autophagy, lysosomal proteases are key contributors to the maintenance of protein homeostasis with age. We previously reported that expression of granulin peptides, the cleavage products of the neurodegenerative disease protein progranulin, enhance the accumulation and toxicity of TAR DNA binding protein 43 (TDP-43) in Caenorhabditis elegans (C. elegans). In this study we show that C. elegans granulins are produced in an age- and stress-dependent manner. Granulins localize to the endolysosomal compartment where they impair lysosomal protease expression and activity. Consequently, protein homeostasis is disrupted, promoting the nuclear translocation of the lysosomal transcription factor HLH-30/TFEB, and prompting cells to activate a compensatory transcriptional program. The three C. elegans granulin peptides exhibited distinct but overlapping functional effects in our assays, which may be due to amino acid composition that results in distinct electrostatic and hydrophobicity profiles. Our results support a model in which granulin production modulates a critical transition between the normal, physiological regulation of protease activity and the impairment of lysosomal function that can occur with age and disease.

Multi-Granulin Domain Peptides Bind to Pro-Cathepsin D and Stimulate Its Enzymatic Activity More Effectively Than Progranulin in Vitro.

Progranulin (PGRN) is an evolutionarily conserved glycoprotein associated with several disease states, including neurodegeneration, cancer, and autoimmune disorders. This protein has recently been implicated in the regulation of lysosome function, whereby PGRN may bind to and promote the maturation and activity of the aspartyl protease cathepsin D (proCTSD, inactive precursor; matCTSD, mature, enzymatically active form). As the full-length PGRN protein can be cleaved into smaller peptides, called granulins, we assessed the function of these granulin peptides in binding to proCTSD and stimulating matCTSD enzyme activity in vitro. Here, we report that full-length PGRN and multi-granulin domain peptides bound to proCTSD with low to submicromolar binding affinities. This binding promoted proCTSD destabilization, the magnitude of which was greater for multi-granulin domain peptides than for full-length PGRN. Such destabilization correlated with enhanced matCTSD activity at acidic pH. The presence and function of multi-granulin domain peptides have typically been overlooked in previous studies. This work provides the first in vitro quantification of their binding and activity on proCTSD. Our study highlights the significance of multi-granulin domain peptides in the regulation of proCTSD maturation and enzymatic activity and suggests that attention to PGRN processing will be essential for the future understanding of the molecular mechanisms leading to neurodegenerative disease states with loss-of-function mutations in PGRN.

Progranulin Stimulates the In Vitro Maturation of Pro-Cathepsin D at Acidic pH.

Single-copy loss-of-function mutations in the progranulin gene (PGRN) underlie the neurodegenerative disease frontotemporal lobar degeneration, while homozygous loss-of-function of PGRN results in the lysosomal storage disorder neuronal ceroid lipofuscinosis. Despite evidence that normal PGRN levels are critical for neuronal health, the function of this protein is not yet understood. Here, we show that PGRN stimulates the in vitro maturation of the lysosomal aspartyl protease cathepsin D (CTSD). CTSD is delivered to the endolysosomal system as an inactive precursor (proCTSD) and requires sequential cleavage steps via intermediate forms to achieve the mature state (matCTSD). In co-immunoprecipitation experiments, PGRN interacts predominantly with immature pro- and intermediate forms of CTSD. PGRN enhances in vitro conversion of proCTSD to matCTSD in a concentration-dependent manner. Differential scanning fluorimetry shows a destabilizing effect induced by PGRN on proCTSD folding (∆Tm = -1.7 °C at a 3:1 molar ratio). We propose a mechanism whereby PGRN binds to proCTSD, destabilizing the propeptide from the enzyme catalytic core and favoring conversion to mature forms of the enzyme. Further understanding of the role of PGRN in CTSD maturation will assist in the development of targeted therapies for neurodegenerative disease.

Tau/MAPT disease-associated variant A152T alters tau function and toxicity via impaired retrograde axonal transport.

Mutations in the microtubule-associated protein tau (MAPT) underlie multiple neurodegenerative disorders, yet the pathophysiological mechanisms are unclear. A novel variant in MAPT resulting in an alanine to threonine substitution at position 152 (A152T tau) has recently been described as a significant risk factor for both frontotemporal lobar degeneration and Alzheimer's disease. Here we use complementary computational, biochemical, molecular, genetic and imaging approaches in Caenorhabditis elegans and mouse models to interrogate the effects of the A152T variant on tau function. In silico analysis suggests that a threonine at position 152 of tau confers a new phosphorylation site. This finding is borne out by mass spectrometric survey of A152T tau phosphorylation in C. elegans and mouse. Optical pulse-chase experiments of Dendra2-tau demonstrate that A152T tau and phosphomimetic A152E tau exhibit increased diffusion kinetics and the ability to traverse across the axon initial segment more efficiently than wild-type (WT) tau. A C. elegans model of tauopathy reveals that A152T and A152E tau confer patterns of developmental toxicity distinct from WT tau, likely due to differential effects on retrograde axonal transport. These data support a role for phosphorylation of the variant threonine in A152T tau toxicity and suggest a mechanism involving impaired retrograde axonal transport contributing to human neurodegenerative disease.

The Use of Caenorhabditis elegans to Study Progranulin in the Regulation of Programmed Cell Death and Stress Response.

The nematode Caenorhabditis elegans (C. elegans) has proven to be a powerful model organism for the study of many biological processes, with major implications for human health and disease. As progranulin is a pleiotropic, secreted protein with both cell autonomous and non-autonomous roles, a multicellular organism such as C. elegans is ideal for the investigation of its normal function and pathological effects. The C. elegans genome contains a progranulin-like gene known as pgrn-1. The nematode pgrn-1 encodes a protein with three cysteine-rich granulin domains, compared to the seven and a half granulins in the human protein. We have shown that C. elegans mutants lacking pgrn-1 appear grossly normal, but exhibit accelerated apoptotic cell engulfment as well as a stress resistance phenotype (Kao et al., Proc Natl Acad Sci U S A 108:4441-4446, 2011; Judy et al., PLoS Genet 9:e1003714, 2013). In addition, the roles of individual granulins can also be dissected in C. elegans (Salazar et al., J Neurosci 35:9315-9328, 2015). Here, we describe methods for studying apoptosis and stress response in C. elegans.

The Progranulin Cleavage Products, Granulins, Exacerbate TDP-43 Toxicity and Increase TDP-43 Levels.

Mutations in the human progranulin gene resulting in protein haploinsufficiency cause frontotemporal lobar degeneration with TDP-43 inclusions. Although progress has been made in understanding the normal functions of progranulin and TDP-43, the molecular interactions between these proteins remain unclear. Progranulin is proteolytically processed into granulins, but the role of granulins in the pathogenesis of neurodegenerative disease is unknown. We used a Caenorhabditis elegans model of neuronal TDP-43 proteinopathy to specifically interrogate the contribution of granulins to the neurodegenerative process. Complete loss of the progranulin gene did not worsen TDP-43 toxicity, whereas progranulin heterozygosity did. Interestingly, expression of individual granulins alone had little effect on behavior. In contrast, when granulins were coexpressed with TDP-43, they exacerbated its toxicity in a variety of behaviors including motor coordination. These same granulins increased TDP-43 levels via a post-translational mechanism. We further found that in human neurodegenerative disease subjects, granulin fragments accumulated specifically in diseased regions of brain. To our knowledge, this is the first demonstration of a toxic role for granulin fragments in a neurodegenerative disease model. These studies suggest that presence of cleaved granulins, rather than or in addition to loss of full-length progranulin, may contribute to disease in TDP-43 proteinopathies.

A consistent muscle activation strategy underlies crawling and swimming in Caenorhabditis elegans.

Although undulatory swimming is observed in many organisms, the neuromuscular basis for undulatory movement patterns is not well understood. To better understand the basis for the generation of these movement patterns, we studied muscle activity in the nematode Caenorhabditis elegans. Caenorhabditis elegans exhibits a range of locomotion patterns: in low viscosity fluids the undulation has a wavelength longer than the body and propagates rapidly, while in high viscosity fluids or on agar media the undulatory waves are shorter and slower. Theoretical treatment of observed behaviour has suggested a large change in force-posture relationships at different viscosities, but analysis of bend propagation suggests that short-range proprioceptive feedback is used to control and generate body bends. How muscles could be activated in a way consistent with both these results is unclear. We therefore combined automated worm tracking with calcium imaging to determine muscle activation strategy in a variety of external substrates. Remarkably, we observed that across locomotion patterns spanning a threefold change in wavelength, peak muscle activation occurs approximately 45° (1/8th of a cycle) ahead of peak midline curvature. Although the location of peak force is predicted to vary widely, the activation pattern is consistent with required force in a model incorporating putative length- and velocity-dependence of muscle strength. Furthermore, a linear combination of local curvature and velocity can match the pattern of activation. This suggests that proprioception can enable the worm to swim effectively while working within the limitations of muscle biomechanics and neural control.

Virtual finger boosts three-dimensional imaging and microsurgery as well as terabyte volume image visualization and analysis.

Three-dimensional (3D) bioimaging, visualization and data analysis are in strong need of powerful 3D exploration techniques. We develop virtual finger (VF) to generate 3D curves, points and regions-of-interest in the 3D space of a volumetric image with a single finger operation, such as a computer mouse stroke, or click or zoom from the 2D-projection plane of an image as visualized with a computer. VF provides efficient methods for acquisition, visualization and analysis of 3D images for roundworm, fruitfly, dragonfly, mouse, rat and human. Specifically, VF enables instant 3D optical zoom-in imaging, 3D free-form optical microsurgery, and 3D visualization and annotation of terabytes of whole-brain image volumes. VF also leads to orders of magnitude better efficiency of automated 3D reconstruction of neurons and similar biostructures over our previous systems. We use VF to generate from images of 1,107 Drosophila GAL4 lines a projectome of a Drosophila brain.

Applying science and strategy to operating room workforce management.

The traditional means of planning nurse staffing for operating rooms are either poorly translated to the setting or do not provide decision makers with a platform to defend their needs, especially in an era of health care reform. The surgical operations department of the Cleveland Clinic initiated a quality improvement project aimed at applying a scientific method to operating room staffing. One goal was to provide a defensible plan for allocating direct caregiver positions. A second goal was to provide a quick and easy way for nurse managers and directors to track positions and graphically depict the effect of vacancies and orientation on their staffing budgets. Using an objective, scientific method allows position requests to be approved quickly and allows managers to feel much more comfortable functioning in a "lean" mode because they know needed positions will be approved quickly. Managers and directors also have found that graphically depicting numbers of vacant positions, as well as staff in orientation, could quickly relate a story visually rather than getting "bogged down" in narrative (often losing finance administrators along the way).

Proprioceptive coupling within motor neurons drives C. elegans forward locomotion.

Locomotion requires coordinated motor activity throughout an animal's body. In both vertebrates and invertebrates, chains of coupled central pattern generators (CPGs) are commonly evoked to explain local rhythmic behaviors. In C. elegans, we report that proprioception within the motor circuit is responsible for propagating and coordinating rhythmic undulatory waves from head to tail during forward movement. Proprioceptive coupling between adjacent body regions transduces rhythmic movement initiated near the head into bending waves driven along the body by a chain of reflexes. Using optogenetics and calcium imaging to manipulate and monitor motor circuit activity of moving C. elegans held in microfluidic devices, we found that the B-type cholinergic motor neurons transduce the proprioceptive signal. In C. elegans, a sensorimotor feedback loop operating within a specific type of motor neuron both drives and organizes body movement.

The transcriptional response of Caenorhabditis elegans to Ivermectin exposure identifies novel genes involved in the response to reduced food intake.

We have examined the transcriptional response of Caenorhabditis elegans following exposure to the anthelmintic drug ivermectin (IVM) using whole genome microarrays and real-time QPCR. Our original aim was to identify candidate molecules involved in IVM metabolism and/or excretion. For this reason the IVM tolerant strain, DA1316, was used to minimise transcriptomic changes related to the phenotype of drug exposure. However, unlike equivalent work with benzimidazole drugs, very few of the induced genes were members of xenobiotic metabolising enzyme families. Instead, the transcriptional response was dominated by genes associated with fat mobilization and fatty acid metabolism including catalase, esterase, and fatty acid CoA synthetase genes. This is consistent with the reduction in pharyngeal pumping, and consequential reduction in food intake, upon exposure of DA1316 worms to IVM. Genes with the highest fold change in response to IVM exposure, cyp-37B1, mtl-1 and scl-2, were comparably up-regulated in response to short-term food withdrawal (4 hr) independent of IVM exposure, and GFP reporter constructs confirm their expression in tissues associated with fat storage (intestine and hypodermis). These experiments have serendipitously identified novel genes involved in an early response of C. elegans to reduced food intake and may provide insight into similar processes in higher organisms.

Characterization of the xenobiotic response of Caenorhabditis elegans to the anthelmintic drug albendazole and the identification of novel drug glucoside metabolites.

Knowledge of how anthelmintics are metabolized and excreted in nematodes is an integral part of understanding the factors that determine their potency, spectrum of activity and for investigating mechanisms of resistance. Although there is remarkably little information on these processes in nematodes, it is often suggested that they are of minimal importance for the major anthelmintic drugs. Consequently, we have investigated how the model nematode Caenorhabditis elegans responds to and metabolizes albendazole, one of the most important anthelmintic drugs for human and animal use. Using a mutant strain lacking the ß-tubulin drug target to minimize generalized stress responses, we show that the transcriptional response is dominated by genes encoding XMEs (xenobiotic-metabolizing enzymes), particularly cytochrome P450s and UGTs (UDP-glucuronosyl transferases). The most highly induced genes are predominantly expressed in the worm intestine, supporting their role in drug metabolism. HPLC-MS/MS revealed the production of two novel glucoside metabolites in C. elegans identifying a major difference in the biotransformation of this drug between nematodes and mammals. This is the first demonstration of metabolism of a therapeutic anthelmintic in C. elegans and provides a framework for its use to functionally investigate nematode anthelmintic metabolism.

Selective chemical intervention in the proteome of Caenorhabditis elegans.

We present the first study of protein regulation by ligands in Caenorhabditis elegans. The ligands were peptidyl-prolyl isomerase inhibitors of cyclophilins. Up-regulation is observed for several heat shock proteins and one ligand in particular caused a greater than 2-fold enhancement of cyclophilin CYN-5. Additionally, several metabolic enzymes display elevated levels. This approach, using label-free relative quantification, provides an extremely attractive way of measuring the effect of ligands on an entire proteome, with minimal sample pretreatment, which could be applicable to large-scale studies. In this initial study, which compares the effect of three ligands, 54 unique proteins have been identified that are up- (51) or down- (3) regulated in the presence of a given ligand. A total of 431 C. elegans proteins were identified. Our methodology provides an intriguing new direction for in vivo screening of the effects of novel and untested ligands at the whole organism level.

D-cycloserine potentiates the reconsolidation of cocaine-associated memories.

Conditioned cue-induced relapse to drug seeking is a major challenge to the treatment of drug addiction. It has been proposed that D-cycloserine might be useful in the prevention of relapse by reducing the conditioned reinforcing properties of drug-associated stimuli through facilitation of extinction. Here we show that intrabasolateral amygdala infusions of D-cycloserine in fact potentiate the reconsolidation of stimulus-cocaine memories to increase cue-induced relapse to drug seeking in rats with an extensive drug self-administration history. This elevation of cocaine seeking was correlated with an increase in the expression of the reconsolidation-associated gene zif268.

Intra-amygdala and systemic antagonism of NMDA receptors prevents the reconsolidation of drug-associated memory and impairs subsequently both novel and previously acquired drug-seeking behaviors.

The amygdala has long been considered a primary locus in mediating the effects of previously drug-associated stimuli on subsequent drug-seeking behavior, and the NMDA subtype of glutamate receptor within the amygdala is important for the consolidation of associations between environmental conditioned stimuli and the effects of addictive drugs. Here we demonstrate that amygdala NMDA receptors are also necessary for the reconsolidation of drug-associated memories. Using a behavioral task that specifically measures the conditioned reinforcing properties of a previously drug-paired stimulus, we show that infusion of the NMDA receptor antagonist D(-)-2-amino-5-phosphonopentanoic acid (D-APV) into the basolateral amygdala before a memory reactivation session disrupted the drug-associated memory and abolished subsequent instrumental responding for conditioned reinforcement. This effect was memory reactivation dependent, and the memory deficit persisted for at least 4 weeks. In contrast, infusion of d-APV immediately after the memory reactivation session had no effect on subsequent responding for conditioned reinforcement, indicating that NMDA receptors have a temporally limited role in the reconsolidation process. Furthermore, in molecular studies, we show that the reconsolidation-impairing effect of D-APV is correlated with downstream reductions in expression of the plasticity-related immediate early gene, zif268. We also demonstrate that systemic antagonism of NMDA receptors with MK-801 [(+)-5-methyl-10,11-dihydro-SH-dibenzo[a,d]cyclohepten-5,10-imine maleate] before memory reactivation subsequently reduced previously acquired instrumental drug-seeking behavior that depends on drug-associated cues acting as conditioned reinforcers. These data suggest that drugs modulating glutamatergic transmission at the NMDA receptor may be useful in the future treatment of relapse prevention in drug addiction through memory reconsolidation blockade.