Activation of Matrix Metalloproteinase-3 is altered at the frog neuromuscular junction following changes in synaptic activity

The extracellular matrix surrounding the neuromuscular junction is a highly specialized and dynamic structure. Matrix Metalloproteinases are enzymes that sculpt the extracellular matrix. Since synaptic activity is critical to the structure and function of this synapse, we investigated whether changes in synaptic activity levels could alter the activity of Matrix Metalloproteinases at the neuromuscular junction. In particular, we focused on Matrix Metalloproteinase 3 (MMP3), since antibodies to MMP3 recognize molecules at the frog neuromuscular junction, and MMP3 cleaves a number of synaptic basal lamina molecules, including agrin. Here we show that the fluorogenic compound (M2300) can be used to perform in vivo proteolytic imaging of the frog neuromuscular junction to directly measure the activity state of MMP3. Application of this compound reveals that active MMP3 is concentrated at the normal frog neuromuscular junction, and is tightly associated with the terminal Schwann cell. Blocking presynaptic activity via denervation, or TTX nerve blockade, results in a decreased level of active MMP3 at the neuromuscular junction. The loss of active MMP3 at the neuromuscular junction in denervated muscles can result from decreased activation of pro-MMP3, or it could result from increased inhibition of MMP3. These results support the hypothesis that changes in synaptic activity can alter the level of active MMP3 at the neuromuscular junction.     

HaploBuild: an algorithm to construct non-contiguous associated haplotypes in family based genetic studies

We have created a program that searches densely genotyped regions for associated non-contiguous haplotypes using a standard family based haplotype association test. This program was designed to expand upon the 'sliding window' methodologies commonly used for haplotype construction by allowing the association of subsets of single nucleotide polymorphisms (SNPs) to drive the construction of the haplotype. This strategy permits HaploBuild to construct more biologically relevant haplotypes that are not constrained by arbitrary length and contiguous orientation. Availability: http://snp.bumc.bu.edu.     

Effect of short-term coyote removal on populations of coyote helminths

Coyote (Canis latrans) removal programs often are initiated despite the potential population regulatory mechanism of parasitism with increased coyote density. We investigated the effect of intensive, short-term coyote removal on population levels of helminths in juvenile and adult coyotes from western Texas. Coyotes were killed by aerial gunning every 3 mo for 2 yr on two 5,000 ha areas, which reduced the overall coyote density of these areas by about 50%. Two other 5,000 ha areas were used as comparison sites where a limited number of coyotes were killed each season. Densities on comparison sites remained stable throughout the study at a mean +/- 1 SE of 0.14 +/- 0.01 coyotes/km2. Twelve helminth species consisting of seven nematodes (Ancylostoma caninum, Physaloptera rara, Toxascaris leonina, Dirofilaria immitis, Spirocerca lupi, Oslerus osleri, and Capillaria aerophila), three cestodes (Taenia pisiformis, Taenia multiceps, and Mesocestoides sp.), one acanthocephalan (Oncicola canis), and one trematode (Alaria marcianae) were found in 252 coyotes. Of these, A. caninum, P. rara, T. multiceps, T. pisiformis, T. leonina, and S. lupi were common species. Rank-transformed values for the mean abundances of A. caninum and T. multiceps and A. caninum, T. multiceps, and S. lupi were reduced in juvenile and adult coyotes, respectively, from the removal sites compared to respective helminth abundances in similar age class coyotes from comparison sites. Because A. caninum has been suggested as a population regulator of coyotes, a coyote removal program that results in a reduced density of coyotes and at the same time causes a reduced abundance of A. caninum, may in fact negate the regulatory effect that A. caninum has on coyote populations.     

Breakfast in the nook and dinner in the dining room: time-of-day discrimination in rats

Four studies were conducted which demonstrate that most (63%) male Sprague-Dawley rats can attain criterion, nine correct choices over ten consecutive trials, on a time-of-day discrimination in an elevated T-maze, but that the task is relatively difficult. The discrimination required that the rats go to one goal arm during a morning session and the other in an afternoon session. The sessions always began at the same time and were at least 6 h apart. A larger proportion of rats attained criterion and required fewer trials when the discriminative cue was a maze insert providing visual and tactile stimulation (0.72), orientation and position of the maze in the room (0.88), or the rats were required to always make the same left or right turn (0.94). Also, once criterion was attained, rats trained on time-of-day discrimination only made about 70% correct choices with continued training. Housing the rats with continuous light, all laboratory noises masked with white noise, and a random feeding schedule did not prevent them from acquiring the time-place discrimination. Testing the rats with a random number of trials during morning and afternoon sessions and with added or omitted sessions revealed that the rats did not use response or session alternation strategies to perform the discrimination. Also, the particular experimenter administering the morning or afternoon sessions did not serve as a cue for the discrimination. The relative difficulty of the task suggests that time of day does not normally function as a discriminative stimulus for choices, but probably as a contextual stimulus. Further, performance of the task in the absence of time-of-day cues suggests that the discrimination is based on event memory combined with an internal timing mechanism.     

Differential effects of abscisic acid on desiccation tolerance and carbohydrates in three species of liverworts

Tissues of three species of in vitro grown liverworts, Riccia fluitans, Pallavicinia lyellii, and Marchantia polymorpha, were subjected to rapid drying with and without preculture for 1 week on medium containing 10 μM ABA. ABA preculture initiated total desiccation tolerance in R. fluitans, whereas control tissues were killed after 30 min of drying. Survival was also improved in P. lyellii, whereas ABA did not affect survival of M. polymorpha after rapid drying. ABA treatment did, however, reduce the rate of water loss in M. polymorpha. Total soluble carbohydrates were increased in ABA-treated R. fluitans and P. lyellii, but not in M. polymorpha, although there was no correlation between survival and changes in the percentage of these carbohydrates as reducing sugars. These differences in response to ABA and desiccation likely reflect different adaptations of these three species to conditions in situ.     

A Novel Strategy for Detection and Enumeration of Circulating Rare Cell Populations in Metastatic Cancer Patients Using Automated Microfluidic Filtration and Multiplex Immunoassay

Size selection via filtration offers an antigen-independent approach for the enrichment of rare cell populations in blood of cancer patients. We evaluated the performance of a novel approach for multiplex rare cell detection in blood samples from metastatic breast (n = 19) and lung cancer patients (n = 21), and healthy controls (n = 30) using an automated microfluidic filtration and multiplex immunoassay strategy. Captured cells were enumerated after sequential staining for specific markers to identify circulating tumor cells (CTCs), circulating mesenchymal cells (CMCs), putative circulating stem cells (CSCs), and circulating endothelial cells (CECs). Preclinical validation experiments using cancer cells spiked into healthy blood demonstrated high recovery rate (mean = 85%) and reproducibility of the assay. In clinical studies, CTCs and CMCs were detected in 35% and 58% of cancer patients, respectively, and were largely absent from healthy controls (3%, p = 0.001). Mean levels of CTCs were significantly higher in breast than in lung cancer patients (p = 0.03). Fifty-three percent (53%) of cancer patients harbored putative CSCs, while none were detectable in healthy controls (p<0.0001). In contrast, CECs were observed in both cancer and control groups. Direct comparison of CellSearch^® vs. our microfluidic filter method revealed moderate correlation (R² = 0.46, kappa = 0.47). Serial blood analysis in breast cancer patients demonstrated the feasibility of monitoring circulating rare cell populations over time. Simultaneous assessment of CTCs, CMCs, CSCs and CECs may provide new tools to study mechanisms of disease progression and treatment response/resistance.     

Evaluating costs for the <Emphasis Type="Italic">in vitro</Emphasis> propagation and preservation of endangered plants

In vitro methods provide opportunities for propagating and preserving endangered plant species when seed-based methods are not adequate. Such species include those that produce few or no seeds, as well as species with recalcitrant seeds. Tissue culture propagation methods can be used to produce such plants for reintroduction, research, education, display, and commerce. They can also be the basis for tissue banking as a way to preserve genetic diversity when seeds cannot be banked. With some recalcitrant species, embryo banking, a method which also utilizes in vitro culture for recovery germination, is possible. The number of endangered species that will require in vitro methods is estimated to be at least 5,000 worldwide. Further information is needed to identify these species, and the ongoing collection of information into databases on endangered species and recalcitrant species will help provide this. The costs of these methods are higher than for traditional propagation and preservation, but they may be necessary for species under higher threat. The multiplication rate of a culture, as well as the rates of rooting and acclimatization, has a major effect on the number of transfers needed for producing plants or tissue for banking, and improvements that will increase the efficiency of these steps can help lower costs. Further research into factors affecting the growth of tissues in vitro, as well as coordination of efforts among institutions with infrastructure for in vitro work, should facilitate the application of in vitro methods to the endangered species that cannot be propagated or preserved using seeds.     

Performance of LED-based fluorescence microscopy to diagnose tuberculosis in a peripheral health centre in Nairobi

Background

Sputum microscopy is the only tuberculosis (TB) diagnostic available at peripheral levels of care in resource limited countries. Its sensitivity is low, particularly in high HIV prevalence settings. Fluorescence microscopy (FM) can improve performance of microscopy and with the new light emitting diode (LED) technologies could be appropriate for peripheral settings. The study aimed to compare the performance of LED-FM versus Ziehl-Neelsen (ZN) microscopy and to assess feasibility of LED-FM at a low level of care in a high HIV prevalence country.

Methods

A prospective study was conducted in an urban health clinic in Nairobi, Kenya. Three sputum specimens were collected over 2 days from suspected TB patients. Each sample was processed with Auramine O and ZN methods and a 4^th specimen was collected for TB culture reference standard. Auramine smears were read using the same microscope, equipped with the FluoLED™ fluorescence illuminator. Inter-reader agreement, reading time and technicians'' acceptability assessed feasibility.

Results

497 patients were included and 1394 specimens were collected. The detection yields of LED-FM and ZN microscopy were 20.3% and 20.6% (p = 0.64), respectively. Sensitivity was 73.2% for LED-FM and 72% for ZN microscopy, p = 0.32. It was 96.7% and 95.9% for specificity, p = 0.53. Inter-reader agreement was high (kappa = 0.9). Mean reading time was three times faster than ZN microscopy with very good acceptance by technicians.

Conclusions

Although it did not increase sensitivity, the faster reading time combined with very good acceptance and ease of use supports the introduction of LED-FM at the peripheral laboratory level of high TB and HIV burden countries.     

Three-Year Change in the Wellbeing of Orphaned and Separated Children in Institutional and Family-Based Care Settings in Five Low- and Middle-Income Countries

Background

With more than 2 million children living in group homes, or “institutions”, worldwide, the extent to which institution-based caregiving negatively affects development and wellbeing is a central question for international policymakers.

Methods

A two-stage random sampling methodology identified community representative samples of 1,357 institution-dwelling orphaned and separated children (OSC) and 1,480 family-dwelling OSC aged 6–12 from 5 low and middle income countries. Data were collected from children and their primary caregivers. Survey-analytic techniques and linear mixed effects models describe child wellbeing collected at baseline and at 36 months, including physical and emotional health, growth, cognitive development and memory, and the variation in outcomes between children, care settings, and study sites.

Findings

At 36-month follow-up, institution-dwelling OSC had statistically significantly higher height-for-age Z-scores and better caregiver-reported physical health; family-dwelling OSC had fewer caregiver-reported emotional difficulties. There were no statistically significant differences between the two groups on other measures. At both baseline and follow-up, the magnitude of the differences between the institution- and family-dwelling groups was small. Relatively little variation in outcomes was attributable to differences between sites (11–27% of total variation) or care settings within sites (8–14%), with most variation attributable to differences between children within settings (60–75%). The percent of variation in outcomes attributable to the care setting type, institution- versus family-based care, ranged from 0–4% at baseline, 0–3% at 36-month follow-up, and 0–4% for changes between baseline and 36 months.

Interpretation

These findings contradict the hypothesis that group home placement universally adversely affects child wellbeing. Without substantial improvements in and support for family settings, the removal of institutions, broadly defined, would not significantly improve child wellbeing and could worsen outcomes of children who are moved from a setting where they are doing relatively well to a more deprived setting.