Assessment of nitrification process in a sequencing batch reactor: Modelling and genomic approach.

Nitrification of ammoniacal nitrogen (N-NH4+) to nitrate (N-NO3-) was investigated in a lab-scale sequencing batch reactor (SBR) to evaluate its efficiency. During the nitrification process the removal of N-NH4+ reached 96%, resulting in 73% formation of N-NO3-. A lineal correlation (r2 = 0.9978) was obtained between the concentration of volatile suspended solids (VSS) and the maximal N-NO3- concentration at the end of each batch cycle under stationary state. The bacterial taxons in the initial inoculum were identified, revealing a complex diverse community mainly in the two major bacterial phyla Proteobacteria and Actinobacteria. The FAPROTAX algorithm predicted the presence in the inoculum of taxa involved in relevant processes of the nitrogen metabolism, highlighting the bacterial genera Nitrospira and Nitrosomonas that are both involved in the nitrification process. A kinetic model was formulated for predicting and validating the transformation of N-NH4+, N-NO2- and N-NO3- and the removal of organic and inorganic carbon (TOC and IC, respectively). The results showed how the increase in biomass concentration slowed down the transformation to oxidised forms of nitrogen and increased denitrification in the settling and filling stages under free aeration conditions.

Cocaine use disorder criteria in a clinical sample: an analysis using Item Response Theory, factor analysis, and network analysis.

Background: The conceptualization of substance use disorders (SUDs) was modified in successive editions of the DSM. Dimensionality and inclusion/exclusion of several criteria was studied using various analytic approaches.Objective: The study aimed to deepen our knowledge of the interrelationships between the diagnostic criteria for cocaine use disorder (CUD), applying three different analytical techniques: factor analysis, Item Response Theory (IRT) models, and network analysis.Methods: 425 (85.4% male) outpatients were evaluated for CUD using the Substance Dependence Severity Scale. Confirmatory Factor Analysis, 2-parameter logistic model (IRT) and network analysis were applied to analyze the relationships between the diagnostic criteria.Results: The results show that "legal problems" criterion is not congruent with the CUD measure on three analyses. Also, network analysis suggests the usefulness of the "craving" criterion. The criterion "quit/control" is the one that presents the best centrality indices and expected influence, showing strong relationships with the criteria of "craving," "tolerance," "neglect roles" and "activities given up."Conclusions: Network analysis appears to be a useful and complementary technique to factor analysis and IRT for understanding CUD. The "quit/control" criterion emerges as a central criterion to understand CUD.

Comparison of two coprological methods for the diagnosis of Eurytrema ssp. in cattle and sheep.

Eurytrematosis is a disease caused by flukes of the genus Eurytrema. These parasites infect the pancreatic ducts of a wide variety of species, including cattle, sheep and humans. Diagnosing eurytrematosis through the analysis of faecal samples can be difficult because most of the available techniques are considered of low sensitivity. In this context, a modification of the Dennis, Stone and Swanson technique (Belem Sedimentation Technique, BST) was previously developed to increase the probability of detecting infected animals; nevertheless, the values of eggs per gram obtained using the modified technique are generally low. We proposed a modification of the this technique (MBST), to increase the sensitivity and detection rate of infected animals. The objective of this work was to describe MBST and compare it with BST. Faecal samples of 212 clinically healthy animals (174 from cattle and 38 from sheep) from 20 farms were taken by the intra-rectal route and stored at 4°C. The samples were processed using BST and MBST. Positive samples amounted to 55 (25.9%) using BST and 121 (57.1%) using MBST. In the simples from cattle, 52 (29.8%) and 107 (61.4%) were positive in BST and MBST, respectively. In sheep, three (7.8%) and 14 (36.8%) positive samples were obtained in BST and MBST, respectively.The results obtained using the two methods were significantly different, indicating a lack of agreement between their findings. The results suggest that MBST is a more sensitive method to detect Eurytrema spp. eggs in faeces than BST.

Stingray spear injury to the pediatric spinal cord: case report and review of the literature.

Stingray injuries are rare, mostly causing injuries to the lower extremities but occasionally fatal if there is direct puncture of the thorax, abdomen, or neck. Direct combined stingray injury to the central nervous system has not been reported in the literature. Herein we present the case of a 12-year-old boy who, while wading at the seashore of the Costa Rica's Pacific Ocean, sustained a combined oblique penetrating injury to the C6 vertebra caused by a Stingray. He initially presented to the hospital with a complete asymmetric right C6/left T1 ASIA A examination, priapism, and loss of anal sphincter tone. Imaging revealed fracture of the posterior elements of C6 with an oblique trajectory into the left radicular foramen. T2W images did not reveal anatomical section but rather edema and minor bleeding in the epidural space. The patient underwent medical management and serial imaging. During the next 3 months, there was recovery of sensation on the right hemi body, bilateral paresthesias and asymmetric progressive improvement in strength on both legs. Acute care management and midterm term follow up are provided, along with a review of the literature for salient management considerations when evaluating and treating combined penetrating and envenomation injuries caused by stingrays. To our knowledge, this is the first report of such injury to the spine.

Correction to: Stingray spear injury to the pediatric spinal cord: case report and review of the literature.

The original version of this article unfortunately contained an error in the spelling of the last name of one of the co-authors. The corresponding author did not notice that the last name of one of the co-authors, Nathan Shlobin, was misspelled as "Schlobin". The correct spelling of his last name is "Shlobin". Given in this article is the corrected author name.

Mutations during the Adaptation of H9N2 Avian Influenza Virus to the Respiratory Epithelium of Pigs Enhance Sialic Acid Binding Activity and Virulence in Mice.

The natural reservoir for influenza viruses is waterfowl, and from there they succeeded in crossing the barrier to different mammalian species. We analyzed the adaptation of avian influenza viruses to a mammalian host by passaging an H9N2 strain three times in differentiated swine airway epithelial cells. Using precision-cut slices from the porcine lung to passage the parental virus, isolates from each of the three passages (P1 to P3) were characterized by assessing growth curves and ciliostatic effects. The only difference noted was an increased growth kinetics of the P3 virus. Sequence analysis revealed four mutations: one each in the PB2 and NS1 proteins and two in the HA protein. The HA mutations, A190V and T212I, were characterized by generating recombinant viruses containing either one or both amino acid exchanges. Whereas the parental virus recognized α2,3-linked sialic acids preferentially, the HA190 mutant bound to a broad spectrum of glycans with α2,6/8/9-linked sialic acids. The HA212 mutant alone differed only slightly from the parental virus; however, the combination of both mutations (HA190+HA212) increased the binding affinity to those glycans recognized by the HA190 mutant. Remarkably, only the HA double mutant showed a significantly increased pathogenicity in mice. In contrast, none of those mutations affected the ciliary activity of the epithelial cells which is characteristic for virulent swine influenza viruses. Taken together, our results indicate that shifts in the HA receptor affinity are just an early adaptation step of avian H9N2 strains; further mutational changes may be required to become virulent for pigs.IMPORTANCE Swine play an important role in the interspecies transmission of influenza viruses. Avian influenza A viruses (IAV) of the H9N2 subtype have successfully infected hosts from different species but have not established a stable lineage. We have analyzed the adaptation of IAV-H9N2 virus to target cells of a new host by passaging the virus three times in differentiated porcine respiratory epithelial cells. Among the four mutations detected, the two HA mutations were analyzed by generating recombinant viruses. Depending on the infection system used, the mutations differed in their phenotypic expression, e.g., sialic acid binding activity, replication kinetics, plaque size, and pathogenicity in inbred mice. However, none of the mutations affected the ciliary activity which serves as a virulence marker. Thus, early adaptive mutation enhances the replication kinetics, but more mutations are required for IAV of the H9N2 subtype to become virulent.

Enolase from Trypanosoma cruzi is inhibited by its interaction with metallocarboxypeptidase-1 and a putative acireductone dioxygenase.

Purification of enolase (ENO) from the cytosol of Trypanosoma cruzi indicated that it may interact with at least five other proteins. Two of them were identified as metallocarboxypeptidase-1 (TcMCP-1) and a putative acireductone dioxygenase (ARDp). Subcellular localization studies confirmed the presence of ARDp in the cytosol, as is the case for ENO and TcMCP-1. Analysis of the ARDp sequence showed that this protein has two domains, an N-terminal ARD and a C-terminal TRP14 (thioredoxin-related protein) domain. The interactions between ENO, TcMCP-1 and ARDp were confirmed for the natural proteins from the trypanosome (using size-exclusion chromatography and co-immunoprecipitation from a cytosolic fraction) and recombinant forms (using ELISA ligand-binding assay and ENO activity assays). The ELISA ligand-binding assays permitted to verify the optimal physicochemical conditions for the interactions (representative for the physiological conditions) and to determine the affinity constants (Kd): ENO/ARDp: 9.54 ±â€¯0.82 nM, ARDp/ENO 10.05 ±â€¯1.11 nM, and ENO/TcMCP-1: 5.66 ±â€¯0.61 nM. The data also show that the interaction between TcMCP-1 and ARDp is mediated by ENO acting as a "bridge". Furthermore, considerable inhibition of the ENO activity, up to 85%, is observed when the enzyme interacts with TcMCP-1 and ARDp simultaneously. All these data confirm that the interaction between ENO, TcMCP-1 and ARDp, occurring in T. cruzi's cytosol, modulates the ENO activity and suggest a possible physiological mechanism for regulation of the ENO activity by the protein-protein interaction.

Molecular and biochemical characterization of natural and recombinant phosphoglycerate kinase B from Trypanosoma rangeli.

T. rangeli epimastigotes contain only a single detectable phosphoglycerate kinase (PGK) enzyme in their cytosol. Analysis of this parasite's recently sequenced genome showed a gene predicted to code for a PGK with the same molecular mass as the natural enzyme, and with a cytosolic localization as well. In this work, we have partially purified the natural PGK from T. rangeli epimastigotes. Furthermore, we cloned the predicted PGK gene and expressed it as a recombinant active enzyme. Both purified enzymes were kinetically characterized and displayed similar substrate affinities, with KmATP values of 0.13 mM and 0.5 mM, and Km3PGA values of 0.28 mM and 0.71 mM, for the natural and recombinant enzyme, respectively. The optimal pH for activity of both enzymes was in the range of 8-10. Like other PGKs, TrPGK is monomeric with a molecular mass of approximately 44 kDa. The enzyme's kinetic characteristics are comparable with those of cytosolic PGK isoforms from related trypanosomatid species, indicating that, most likely, this enzyme is equivalent with the PGKB that is responsible for generating ATP in the cytosol of other trypanosomatids. This is the first report of a glycolytic enzyme characterization from T. rangeli.

Shifts in the ecological niche of Lutzomyia peruensis under climate change scenarios in Peru.

The Peruvian Andes presents a climate suitable for many species of sandfly that are known vectors of leishmaniasis or bartonellosis, including Lutzomyia peruensis (Diptera: Psychodidae), among others. In the present study, occurrences data for Lu. peruensis were compiled from several items in the scientific literature from Peru published between 1927 and 2015. Based on these data, ecological niche models were constructed to predict spatial distributions using three algorithms [Support vector machine (SVM), the Genetic Algorithm for Rule-set Prediction (GARP) and Maximum Entropy (MaxEnt)]. In addition, the environmental requirements of Lu. peruensis and three niche characteristics were modelled in the context of future climate change scenarios: (a) potential changes in niche breadth; (b) shifts in the direction and magnitude of niche centroids, and (c) shifts in elevation range. The model identified areas that included environments suitable for Lu. peruensis in most regions of Peru (45.77%) and an average altitude of 3289 m a.s.l. Under climate change scenarios, a decrease in the distribution areas of Lu. peruensis was observed for all representative concentration pathways. However, the centroid of the species' ecological niche showed a northwest direction in all climate change scenarios. The information generated in this study may help health authorities responsible for the supervision of strategies to control leishmaniasis to coordinate, plan and implement appropriate strategies for each area of risk, taking into account the geographic distribution and potential dispersal of Lu. peruensis.

Fleas and Flea-Associated Bartonella Species in Dogs and Cats from Peru.

In the present study, we investigated 238 fleas collected from cats and dogs in three regions of Peru (Ancash, Cajamarca, and Lima) for the presence of Bartonella DNA. Bartonella spp. were detected by amplification of the citrate synthase gene (16.4%) and the 16S-23S intergenic spacer region (20.6%). Bartonella rochalimae was the most common species detected followed by Bartonella clarridgeiae and Bartonella henselae. Our results demonstrate that dogs and cats in Peru are infested with fleas harboring zoonotic Bartonella spp. and these infected fleas could pose a disease risk for humans.

Seroprevalence of trypanosomosis and associated risk factors in cattle from coast and amazonian provinces of Ecuador.

Trypanosomosis is a tropical disease caused by various protozoan haemoparasites, which affects wild and domestic animals, the latter ones related to worldwide livestock production systems. Species such as Trypanosoma vivax and Trypanosoma evansi have been described using serological and molecular tools in several countries from South and Central America. However, Ecuador presents a relevant knowledge gap in the associated general epidemiology and risk factors of the disease. Therefore, the objective of this study was to determine the seroprevalence of trypanosomosis in cattle from different regions of Ecuador. 745 serum samples from 7 Coastal and 3 Amazon provinces were screened for IgG anti-Trypanosoma spp. antibodies, using an in-house indirect ELISA. The seropositivity was explored and associated with several variables such as sex, age, breed, region, management, and province, using statistical tools. The general seroprevalence of trypanosomosis was 19.1% (95% CI: 16.30-22.1%). The Amazonian provinces of Sucumbíos and Napo and the Coastal province of Esmeraldas presented the highest seroprevalence values of 36.7% (95% CI: 27.67-46.47%), 23.64% (95% CI: 16.06-32.68%) and 25% (95% CI: 15.99-35.94%), respectively. Statistical significance was found for the region, province, and management variables, indicating as relevant risk factors the extensive management and Amazon location of the cattle analyzed. Specific actions should be taken to identify the exact species on reservoirs and susceptible hosts, evaluate the implication of farm management and cattle movement as risk factors, and implement surveillance and treatment plans for affected herds.

Consensus recommendations on sleeping problems in Phelan-McDermid syndrome.

Early onset sleep problems and disorders are very common in individuals with Phelan-McDermid Syndrome (PMS) with rates of up to 90%. These sleep problems and disorders cannot be taken lightly. Not only do they have a major impact on the health, behaviour, functioning and learning opportunities of affected individuals, they can also have detrimental effects on the well-being and resilience of parents and caregivers, ultimately affecting the physical health, mental health and well-being of the whole social system. In this review we aim to understand the types and frequencies of sleeping problems in PMS as the basis for recommendations on their management and treatment and to provide general guidelines for clinicians and practitioners. We conducted an in-depth literature search, summarised findings, and participated in a series of consensus meetings with other consortium members - experts on PMS and stakeholders - to agree on guidelines and recommendations. In parallel, a world-wide survey was created and distributed amongst parents to include their perspective. Our literature search found only four articles specifically focused on sleeping problems in PMS, although some other articles mentioned prevalence and associated factors. Country-specific prevalence rates ranged between 24% and 46%, whereas our parental survey reported 59%. The main problems reported involved difficulty falling asleep and numerous night awakenings, with being restless in sleep, night-time incontinence, and tooth grinding also commonly reported. Only a small number of individuals had undergone a sleep study monitored by a specialist. Bedtime resistance normally decreases with age, but sleep-onset delay, sleep anxiety, parasomnias, problems falling and remaining asleep remain throughout lifespan, with total sleep time improving during adulthood. However, this improvement was also accompanied by a substantial increase in parasomnias. Ultimately, an increase in sleep disorders in children correlates with increased sleep disorders and daytime sleepiness in parents/caregivers. No study to date has focused on the underlying causes of sleeping problems in PMS, but comorbid mental health conditions, somatic causes, or (poly)pharmacy have been proposed as triggers for sleeping disturbances. Currently there is no PMS-specific treatment for sleeping problems, and current recommendations are mostly based on individuals with intellectual disability and/or neurodevelopmental conditions.

Assessing Internalizing Symptoms and Their Relation with Levels of Impairment: Evidence-Based Cutoffs for Interpreting Inventory of Depression and Anxiety Symptoms (IDAS-II) Scores.

Tests and scales measuring psychological disorders should provide information about how scores relate to other constructs such as quality of life or functional impairment. Such information is necessary to allow that their scores contribute to clinical decision making. The current study analyzes the clinical utility of the Spanish version of the Inventory for Depression and Anxiety Symptoms (IDAS-II) to discriminate between different levels of functional impairment and identify the IDAS-II scales that contribute most to explaining impairment. The total sample (N = 1390) consists of two subsamples: a community sample of the general population (n = 1072) selected by random sampling; and a sample of patients (n = 318) from public and private mental health services. The Spanish IDAS-II for measuring internalizing symptoms and WHODAS 2.0 for measuring impairment were administered to all participants. All scales show statistically significant higher scores in the patient sample, with Cohen's d effect sizes values greater than 0.30, except for well-being (d = 0.19). The cutoff values and their confidence intervals do not overlap with the means of either the community or patient sample. AUC values for most of the scales are above .70, except for appetite gain, ordering, euphoria, cleaning, and well-being. Multiple linear regression model using IDAS-II scales explain 57.1% of the variance of the WHODAS 2.0 (F 12.1377 = 155.305; p < .001). Cutoff values provided allow us to reliably differentiate between the patients and community samples. Spanish IDAS-II scores show greater sensitivity and specificity in detecting those with greater impairment. General Depression, Lassitude, Panic and Claustrophobia contribute to impairment in a greater extent. Knowledge of which symptoms are most related with impairment, allows healthcare providers to improve treatment planning based on empirical evidence.

Clinical validation of the CANFOR scale (Camberwell Assessment of Need-Forensic version) for the needs assessment of people with mental health problems in the forensic services.

INTRODUCTION: One of seven people admitted in prison has a diagnosis of a severe mental disorder and it is necessary to assess their needs. CANFOR was developed in 2004 to assess the specific needs of the forensic mental health services users. Adaptation and validation of CANFOR into Spanish was done and the psychometric properties were tested. METHOD: After translation and back translation, we administered the CANFOR to a sample of 90 users of the penitentiary mental health services in Catalonia. RESULTS: Inter-rater and test-retest reliability coefficients for each of the 25 domains were high: moderate to almost perfect (0,44 to 1). Regarding concurrent validity a negative correlation was found between GAF (p<0,01); LSP in all its subscales(p<0,01-p<0,05 ) and needs as assessed with CANFOR. CONCLUSIONS: the Spanish version of CANFOR retains the psychometric properties described in the original version. It has been proved that the Spanish version is valid and reliable, so it can be considered an optimal instrument for the assessment of needs of people with a severe mental disorder admitted in prison.

First record of clinical coccidiosis (Eimeria ovinoidalis) in adult sheep from northwestern Argentina.

Coccidiosis of sheep is an intestinal infection caused by protozoa of the genus Eimeria. An outbreak of the disease in adult sheep from Salta province, northwestern Argentina, was studied to establish its clinical, epidemiological, pathological and etiological aspects. The affected animals were part of a flock of 20 sheep brought from Formosa province about 10 days before. Most sheep (80% incidence) showed hemorrhagic diarrhea, dehydration and loss of body condition; six of them died and two that became permanently recumbent were euthanized. Three necropsied sheep showed mild mesenteric lymphadenomegaly, diffuse proliferative enteritis in the small and large intestines, and mucosal thickening. Histopathological studies exhibited diffuse proliferative enteritis and presence of structures compatible with intracellular coccidia at different stages of development. Parasitological studies (n = 12) resulted in an average of 16,636.6 (± 15,266.8) Eimeria oocysts per gram of feces (range 1680-46,400). Taxonomy of Eimeria species based on analysis of sporulated oocysts derived from 4 fecal samples (n = 100 oocyst per sample) showed, on average, a high prevalence of E. ovinoidalis (61.5%), followed by E. parva (27.2%), and lower proportions of E. crandallis (5.3%), E. ahsata (3.2%) and E. intricata (2.8%). Clinical and pathological findings confirmed the diagnosis of coccidiosis in the affected sheep; parasitological results showed that E. ovinoidalis was the main species responsible for the clinical signs. Clinical coccidiosis is considered unusual in adult sheep, but the present case shows that under favorable environmental and/or management conditions, this infection may be highly deleterious for adult sheep.

Spanish Adaptation of the Inventory of Depression and Anxiety Symptoms (IDAS-II) and a study of its psychometric properties.

BACKGROUND: The Inventory of Depression and Anxiety Symptoms (IDAS-II) is composed of 99 items organized into 18 specific scales that provides dimensional assessment of depression, anxiety and bipolar symptoms. To date, IDAS-II is only available in the English and Turkish population. The main purpose of this study is to adapt the IDAS-II to the Spanish population and to assess the psychometric properties. METHODS: Participants included community adults (n = 620) and college students (n = 378). All participants completed the Beck Depression Inventory-II, Beck Anxiety Inventory, Hypomania Check List-32, Post-traumatic Stress Disorder Checklist-Civilian Version and Obsessive-Compulsive Inventory-Revised, in addition to the Spanish version of the IDAS-II. RESULTS: The results indicate good internal consistency and high temporal stability of the Spanish version of the IDAS-II. Confirmatory factor analyses show for the first time that the three-factor structure of the IDAS-II (Distress, Obsessions/Fear, and Positive Mood) loads on a second order factor, labeled "Internalizing" according to the Hierarchical Taxonomy Of Psychopathology (HiTOP). LIMITATIONS: Study was conducted exclusively on student and community samples and some of the measures used as gold-standard have presented limitations CONCLUSIONS: According to previous studies, the results supported the convergent and discriminant validity of the majority of IDAS-II scales. IDAS-II is useful in assessing the severity of depression, anxiety and bipolar symptoms in research contexts in a Spanish population according to the HiTOP model. However, more evidence is required to prove the adequate functioning of the IDAS-II in clinical samples.

Suppression of KIF2 in PC12 cells alters the distribution of a growth cone nonsynaptic membrane receptor and inhibits neurite extension.

In the present study, we present evidence about the cellular functions of KIF2, a kinesin-like superfamily member having a unique structure in that its motor domain is localized at the center of the molecule (Noda Y., Y. Sato-Yoshitake, S. Kondo, M. Nangaku, and N. Hirokawa. 1995. J. Cell Biol. 129:157-167.). Using subcellular fractionation techniques, isopicnic sucrose density centrifugation of microsomal fractions from developing rat cerebral cortex, and immunoisolation with KIF2 antibodies, we have now identified a type of nonsynaptic vesicle that associates with KIF2. This type of organelle lacks synaptic vesicle markers (synapsin, synaptophysin), amyloid precursor protein, GAP-43, or N-cadherin. On the other hand, it contains betagc, which is a novel variant of the beta subunit of the IGF-1 receptor, which is highly enriched in growth cone membranes. Both betagc and KIF2 are upregulated by NGF in PC12 cells and highly concentrated in growth cones of developing neurons. We have also analyzed the consequences of KIF2 suppression by antisense oligonucleotide treatment on nerve cell morphogenesis and the distribution of synaptic and nonsynaptic vesicle markers. KIF2 suppression results in a dramatic accumulation of betagc within the cell body and in its complete disappearance from growth cones; no alterations in the distribution of synapsin, synaptophysin, GAP-43, or amyloid percursor protein are detected in KIF2-suppressed neurons. Instead, all of them remained highly enriched at nerve terminals. KIF2 suppression also produces a dramatic inhibition of neurite outgrowth; this phenomenon occurs after betagc has disappeared from growth cones. Taken collectively, our results suggest an important role for KIF2 in neurite extension, a phenomenon that may be related with the anterograde transport of a type of nonsynaptic vesicle that contains as one of its components a growth cone membrane receptor for IGF-1, a growth factor implicated in nerve cell development.

Evidence for the involvement of KIF4 in the anterograde transport of L1-containing vesicles.

In this study we present evidence about the cellular functions of KIF4. Using subcellular fractionation techniques and immunoisolation, we have now identified a type of vesicle that associates with KIF4, an NH(2)-terminal globular motor domain kinesin-like protein. This vesicle is highly concentrated in growth cones and contains L1, a cell adhesion molecule implicated in axonal elongation. It lacks synaptic vesicle markers, receptors for neurotrophins, and membrane proteins involved in growth cone guidance. In cultured neurons, KIF4 and L1 predominantly localize to the axonal shaft and its growth cone. Suppression of KIF4 with antisense oligonucleotides results in the accumulation of L1 within the cell body and in its complete disappearance from axonal tips. In addition, KIF4 suppression prevents L1-enhanced axonal elongation. Taken collectively, our results suggest an important role for KIF4 during neuronal development, a phenomenon which may be related to the anterograde transport of L1-containing vesicles.

Kinesin-mediated organelle translocation revealed by specific cellular manipulations.

The distribution of membrane-bound organelles was studied in cultured hippocampal neurons after antisense oligonucleotide suppression of the kinesin-heavy chain (KHC). We observed reduced 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) fluorescent staining in neurites and growth cones. In astrocytes, KHC suppression results in the disappearance of the DiOC6(3)-positive reticular network from the cell periphery, and a parallel accumulation of label within the cell center. On the other hand, mitochondria microtubules and microfilaments display a distribution that closely resembles that observed in control cells. KHC suppression of neurons and astrocytes completely inhibited the Brefeldin A-induced spreading and tubulation of the Golgi-associated structure enriched in mannose-6-phosphate receptors. In addition, KHC suppression prevents the low pH-induced anterograde redistribution of late endocytic structures. Taken collectively, these observations suggest that in living neurons, kinesin mediates the anterograde transport of tubulovesicular structures originated in the central vacuolar system (e.g., the endoplasmic reticulum) and that the regulation of kinesin-membrane interactions may be of key importance for determining the intracellular distribution of selected organelles.

Suppression of radixin and moesin alters growth cone morphology, motility, and process formation in primary cultured neurons.

In this study we have examined the cellular functions of ERM proteins in developing neurons. The results obtained indicate that there is a high degree of spatial and temporal correlation between the expression and subcellular localization of radixin and moesin with the morphological development of neuritic growth cones. More importantly, we show that double suppression of radixin and moesin, but not of ezrin-radixin or ezrin-moesin, results in reduction of growth cone size, disappearance of radial striations, retraction of the growth cone lamellipodial veil, and disorganization of actin filaments that invade the central region of growth cones where they colocalize with microtubules. Neuritic tips from radixin-moesin suppressed neurons displayed high filopodial protrusive activity; however, its rate of advance is 8-10 times slower than the one of growth cones from control neurons. Radixin-moesin suppressed neurons have short neurites and failed to develop an axon-like neurite, a phenomenon that appears to be directly linked with the alterations in growth cone structure and motility. Taken collectively, our data suggest that by regulating key aspects of growth cone development and maintenance, radixin and moesin modulate neurite formation and the development of neuronal polarity.