Proteomic characterisation of leech microglia extracellular vesicles (EVs): comparison between differential ultracentrifugation and Optiprep™ density gradient isolation.

In Mammals, microglial cells are considered as the resident immune cells in central nervous system (CNS). Many studies demonstrated that, after injury, these cells are activated and recruited at the lesion site. Leech microglia present a similar pattern of microglial activation and migration upon experimental lesion of CNS. This activation is associated with the release of a large amount of extracellular vesicles (EVs). We collected EVs released by microglia primary culture and compared two different protocols of isolation: one with differential ultracentrifugation (UC) and one using an additional Optiprep™ Density Gradient (ODG) ultracentrifugation. Nanoparticles tracking analysis (NTA) and transmission electron microscopy (TEM) were used to assess vesicles size and morphology. The protein content of isolated EVs was assessed by mass spectrometry approaches. Results showed the presence of EV-specific proteins in both procedures. The extensive proteomic analysis of each single ODG fractions confirmed the efficiency of this protocol in limiting the presence of co-isolated proteins aggregates and other membranous particles during vesicles isolation. The present study permitted for the first time the characterisation of microglial EV protein content in an annelid model. Interestingly, an important amount of proteins found in leech vesicles was previously described in EV-specific databases. Finally, purified EVs were assessed for neurotrophic activity and promote neurites outgrowth on primary cultured neurons.

Development of microsatellite markers for an outbreaking species of oak gall wasp, Zapatella davisae (Hymenoptera: Cynipidae), in the northeastern United States.

Populations of the recently described black oak gall wasp, Zapatella davisae Buffington (Hymenoptera: Cynipidae), have been identified as the cause of extensive tree damage and mortality to black oaks, Quercus velutina Lamarck (Fagales: Fagaceae), in the northeastern United States. Relatively little is known, however, about the distribution, phylogenetic placement, and lifecycle of this important tree pest. Therefore, we conducted next-generation sequencing using the Ion Torrent™ PGM (ThermoFisher Scientific, Inc.) platform to develop genomic resources for the study of Z. davisae and for other closely related species of oak gall wasps. Individual sequence reads were aligned, assembled into unique contigs, and the contigs were then utilized for the in silico isolation and development of microsatellite markers. In total, we screened 36 candidate microsatellite loci, of which 23 amplified consistently (five polymorphic and 18 monomorphic). We then examined whether the polymorphic loci could be used to infer whether populations of Z. davisae from Cape Cod and Nantucket are sexual or asexual by calculating several metrics of genetic diversity that might indicate the mode of reproduction. These included testing for statistical deviations from Hardy-Weinberg equilibrium (HWE) and for linkage disequilibrium (LD), observations for the presence of the Meselson effect, and by calculating the probability that clonal individuals are more prevalent than would be expected in a randomly mating population. While we found significant deviations from HWE and more clonal individuals than expected, our estimates of the Meselson effect were inconclusive due to limited sampling, and we found no evidence of LD. Therefore, the sexual/asexual status of Z. davisae populations remains uncertain.

Hm-MyD88 and Hm-SARM: two key regulators of the neuroimmune system and neural repair in the medicinal leech.

Unlike mammals, the CNS of the medicinal leech can regenerate damaged neurites, thus restoring neural functions after lesion. We previously demonstrated that the injured leech nerve cord is able to mount an immune response promoting the regenerative processes. Indeed neurons and microglia express sensing receptors like Hm-TLR1, a leech TLR ortholog, associated with chemokine release in response to a septic challenge or lesion. To gain insights into the TLR signaling pathways involved during these neuroimmune responses, members of the MyD88 family were investigated. In the present study, we report the characterization of Hm-MyD88 and Hm-SARM. The expression of their encoding gene was strongly regulated in leech CNS not only upon immune challenge but also during CNS repair, suggesting their involvement in both processes. This work also showed for the first time that differentiated neurons of the CNS could respond to LPS through a MyD88-dependent signalling pathway, while in mammals, studies describing the direct effect of LPS on neurons and the outcomes of such treatment are scarce and controversial. In the present study, we established that this PAMP induced the relocalization of Hm-MyD88 in isolated neurons.

Tag-mass: specific molecular imaging of transcriptome and proteome by mass spectrometry based on photocleavable tag.

MALDI tissue imaging of tissues has become a promising technique for tracking biomarkers while determining their location and structural characterization. We have now developed specific targeting probes (oligonucleotides, antibodies), named Tag-Mass. This approach is based on probes modified with a photocleavable linker coupled with a tag cleaved and detected using mass spectrometry. Tag-Mass development is the key for a rapid, sensitive, and accurate approach to correlate levels of expression of different mRNA or proteins in diseases.

Fatal yew (Taxus sp) poisoning in Willamette Valley, Oregon, horses.

Despite the well-founded reputation of plants of the genus Taxus as being amongst the most toxic plants for domestic livestock in the US, there are surprisingly few published case reports of yew poisoning in horses. This report documents 2 acute fatalities in horses in the central Willamette Valley, OR associated with the consumption of Taxus sp. The predominant features of the intoxication were peracute death, with no signs of struggling or convulsions, in otherwise fit and well managed adult horses. The most significant gross necropsy findings were limited to pulmonary congestion and hemorrhage, suggestive of acute circulatory disturbance. A diagnosis of Taxus sp poisoning was confirmed on the basis of a history of potential exposure, by the identification of yew leaves in the gastric contents of the horses, and by the subsequent identification of yew clippings in the pasture. The literature relevant to Taxus sp poisoning in horses is reviewed.

The ontogeny of face recognition: eye contact and sweet taste induce face preference in 9- and 12-week-old human infants.

Nine and 12-week-old infants (N = 140) who were either calm or crying sat facing a researcher for 3.5 min. The researcher gazed into the infant's eyes with a smiling face or looked above the infant's forehead. She delivered a 12% sucrose solution via a syringe or a pacifier, or she did not deliver anything. After the exposure period, the mother held her infant over her shoulder. Infant gaze direction was recorded while the infant faced the same researcher and a stranger. The confluence of sweet taste and eye contact was necessary and sufficient for calm 9- and 12-week-olds to form a preference for the researcher. Crying infants never did so, even though eye contact and sweet taste arrested crying. Different visual-gustatory combinations induced unanticipated affective states and are discussed within the contexts of cognitive mechanisms that mediate face learning and preference, the proximate mechanisms involved, and the evolutionary significance of face recognition.

Identifying possible contingencies during descriptive analyses of severe behavior disorders.

Contingencies of reinforcement involve, in part, relations between behavior and subsequent environmental events. In this study we observed 11 individuals with developmental disabilities and severe behavior problems while they interacted with their primary care providers in simulated environments (hospital therapy rooms). We compared the probability of obtaining attention, escape from instructional demands, or access to materials following instances of problem behavior with the background probability of those events. However, the focus of our analysis was the evaluation of comparative probabilities ("contingency values") in the context of relevant establishing operations such as diverted attention, instructional demands, and restricted access to materials. Results showed that the method was useful in identifying relations between behavior and subsequent environmental events. Implications for linking descriptive and functional analyses are discussed, and difficulties in identifying naturally occurring contingencies are considered.

One thousand video-urodynamic studies in children with non-neurogenic bladder sphincter dysfunction.

OBJECTIVE: To ascertain the aetiology and epidemiology of non-neurogenic bladder sphincter dysfunction (NNBSD) by assessing the results of prospective video-urodynamic studies (VUD) in 1000 children. PATIENTS AND METHODS: During a 4-year study period (January 1995 to December 1998) 1000 children prospectively underwent VUD to further define their NNBSD. After a noninvasive screening assessment consisting of a history, voiding diary, clinical examination, urine analysis, ultrasonography and uroflowmetry, those children who would benefit from further VUD were selected. The selection criteria included a history of urinary tract infection (UTI), a small bladder capacity not responding to training, dysfunctional uroflow, ultrasonographic abnormalities and resistance to therapy. During the study period 3500 children were screened for incontinence problems, including monosymptomatic nocturnal enuresis; 1000 of these were selected for VUD (524 boys and 476 girls). RESULTS: The urodynamic diagnosis was of normal bladder-sphincter function in 62 (6.2%, male : female 44 : 56), urge syndrome in 582 (58%, 58 : 42), dysfunctional voiding in 316 (32%, 49 : 51) and 'lazy bladder' in 40 (4%, 20 : 80). Boys diagnosed with a 'lazy bladder' were younger than those with urge syndrome and dysfunctional voiding. Girls with dysfunctional voiding were younger than those with urge syndrome. The incidence of UTI was significantly higher in girls than in boys; boys with NNBSD had no greater risk for UTI and in girls the general risk was 34%. Only in girls with a lazy bladder was there a significantly higher incidence of UTI (53%). Reflux occurred equally in all groups, with an overall incidence of 15%. The incidence of obstipation was significantly higher in girls with a lazy bladder, and overall was 17%. CONCLUSION: These results from a large series provide a new insight into the epidemiology and pathophysiology of NNBSD. The age distribution provides evidence against a dysfunctional voiding sequence. The risk of developing UTI in NNBSD is greater only in girls. In children with a lazy bladder the risk is also significantly higher, indicating that residual urine is a greater risk factor than detrusor instability. Urge syndrome and dysfunctional voiding in girls carry the same risk for developing UTI, indicating that bladder instability is a higher risk factor for UTI than detrusor sphincter discoordination. All dysfunctions carry an equal risk for developing secondary reflux. Children with NNBSD have different primary diseases but all have a common risk of incontinence, UTIs, reflux and obstipation.

A method for analyzing changes in response efficiency.

Although experimental effects typically are evaluated by summarizing levels of responding across time (e.g., calculating the mean levels of problem behavior during 10-min sessions), these data summaries may obscure important mechanisms that may be responsible for changes in responding. A case study is reported to illustrate alternative methods of data analysis when decreasing trends in responding may be due to increases in response efficiency.

Further analysis of idiosyncratic antecedent influences during the assessment and treatment of problem behavior.

Recent research findings suggest that idiosyncratic variables can influence the outcomes of functional analyses (E. G. Carr, Yarbrough, & Langdon, 1997). In the present study, we examined idiosyncratic environment-behavior relations more precisely after identifying stimuli (i.e., a particular toy and social interaction) associated with increased levels of problem behavior. Two children, an 8-year-old boy with moderate mental retardation and a 5-year-old boy with no developmental delays, participated. Results of functional analyses for both children indicated that idiosyncratic antecedent stimuli set the occasion for occurrences of problem behavior (hand biting or hand flapping) and that problem behavior persisted in the absence of social contingencies. Further analyses were conducted to identify specific components of the stimuli that occasioned problem behavior. Treatments based on results of the analyses successfully reduced self-injury and hand flapping.

Montessori-based activities for long-term care residents with advanced dementia: effects on engagement and affect.

Sixteen residents in long-term care with advanced dementia (14 women; average age = 88) showed significantly more constructive engagement (defined as motor or verbal behaviors in response to an activity), less passive engagement (defined as passively observing an activity), and more pleasure while participating in Montessori-based programming than in regularly scheduled activities programming. Principles of Montessori-based programming, along with examples of such programming, are presented. Implications of the study and methods for expanding the use of Montessori-based dementia programming are discussed.

Variable-time reinforcement schedules in the treatment of socially maintained problem behavior.

Noncontingent reinforcement (NCR) consists of delivering a reinforcer on a time-based schedule, independent of responding. Studies evaluating the effectiveness of NCR as treatment for problem behavior have used fixed-time (FT) schedules of reinforcement. In this study, the efficacy of NCR with variable-time (VT) schedules was evaluated by comparing the effects of VT and FT reinforcement schedules with 2 individuals who engaged in problem behavior maintained by positive reinforcement. Both FT and VT schedules were effective in reducing problem behavior. These findings suggest that VT schedules can be used to treat problem behavior maintained by social consequences.

Effects of reinforcement magnitude on spontaneous recovery.

Extinction of operant behavior has been associated with a number of undesirable effects. One such effect is the temporary reappearance of behavior after responding appears to be completely extinguished, known as spontaneous recovery. In this report, the occurrence of spontaneous recovery and its attenuation with large amounts of reinforcement were examined during the treatment of disruption.

Within-session patterns of responding during functional analyses: the role of establishing operations in clarifying behavioral function.

Functional analysis procedures have been demonstrated to be effective for identifying the operant mechanisms underlying problem behavior. However, functional analyses sometimes yield results that are undifferentiated (i.e., show similar levels of responding across test conditions). Within-session (i.e., minute-by-minute) analyses of response patterns during undifferentiated functional analyses have proven useful in clarifying behavioral function. This study extends previous research by examining within-session changes in responding associated with variations in relevant establishing operations. Levels of problem behavior during the presentation and removal of reinforcement were compared when responding occurred in test conditions associated with sources of social reinforcement (i.e., access to attention, materials, escape). Results showed that changes in responding associated with changes in relevant establishing operations could be examined to clarify behavioral function.

Fixed-time schedules attenuate extinction-induced phenomena in the treatment of severe aberrant behavior.

We compared the effects of extinction (EXT) and fixed-time (FT) schedules as treatment for severe problem behavior displayed by 3 individuals with developmental disabilities. First, functional analyses identified the reinforcers maintaining aberrant behavior for all 3 individuals. Next, EXT and FT schedules were compared using a multielement design. During EXT, the reinforcer maintaining problem behavior was withheld. During FT, the reinforcers were presented response independently at preset intervals. Results showed that FT schedules were generally more effective than EXT schedules in reducing aberrant behavior. FT schedules may be used in situations when extinction-induced phenomena are problematic.

An intergenerational program for persons with dementia using Montessori methods.

An intergenerational program bringing together older adults with dementia and preschool children in one-on-one interactions is described. Montessori activities, which have strong ties to physical and occupational therapy, as well as to theories of developmental and cognitive psychology, are used as the context for these interactions. Our experience indicates that older adults with dementia can still serve as effective mentors and teachers to children in an appropriately structured setting.

Vertebral osteomyelitis: nontypeable beta-lactamase-negative Haemophilus influenzae in an adult: case report.

Adult pyogenic vertebral osteomyelitis due to Haemophilus influenzae is exceedingly rare. After a search of the literature, we deemed our case to be the seventh case of H. influenzae pyogenic osteomyelitis. Vertebral osteomyelitis in itself is a rarity. The most common organisms associated with vertebral osteomyelitis are Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Of the six previously reported cases of adult pyogenic vertebral osteomyelitis due to H. influenzae, four of the six cases were caused by Type B H. influenzae, one case was attributed to Type C, and the other strain was not typed.