Detection of recombinant and endogenous mouse melatonin receptors by monoclonal antibodies targeting the C‐terminal domain

Melatonin receptors play important roles in the regulation of circadian and seasonal rhythms, sleep, retinal functions, the immune system, depression, and type 2 diabetes development. Melatonin receptors are approved drug targets for insomnia, non‐24‐hour sleep‐wake disorders, and major depressive disorders. In mammals, two melatonin receptors (MTRs) exist, MT₁ and MT₂, belonging to the G protein‐coupled receptor (GPCR) superfamily. Similar to most other GPCRs, reliable antibodies recognizing melatonin receptors proved to be difficult to obtain. Here, we describe the development of the first monoclonal antibodies (mABs) for mouse MT₁ and MT₂. Purified antibodies were extensively characterized for specific reactivity with mouse, rat, and human MT₁ and MT₂ by Western blot, immunoprecipitation, immunofluorescence, and proximity ligation assay. Several mABs were specific for either mouse MT₁ or MT₂. None of the mABs cross‐reacted with rat MTRs, and some were able to react with human MTRs. The specificity of the selected mABs was validated by immunofluorescence microscopy in three established locations (retina, suprachiasmatic nuclei, pituitary gland) for MTR expression in mice using MTR‐KO mice as control. MT₂ expression was not detected in mouse insulinoma MIN6 cells or pancreatic beta‐cells. Collectively, we report the first monoclonal antibodies recognizing recombinant and native mouse melatonin receptors that will be valuable tools for future studies.     

Antimicrobial activities of novel bipyridine compounds produced by a new strain of Saccharothrix isolated from Saharan soil

The actinobacterium strain ABH26 closely related to Saccharothrix xinjiangensis, isolated from an Algerian Saharan soil sample, exhibited highly antagonist activity against Gram-positive bacteria, yeasts and filamentous fungi. Its ability to produce antimicrobial compounds was investigated using several solid culture media. The highest antimicrobial activity was obtained on Bennett medium. The antibiotics secreted by strain ABH26 on Bennett medium were extracted by methanol and purified by reverse-phase HPLC using a C18 column. The chemical structures of the compounds were determined after spectroscopic (¹H NMR, ¹³C NMR, ¹H-¹H COSY and ¹H-¹³C HMBC spectra), and spectrometric (mass spectrum) analyses. Two new cyanogriside antibiotics named cyanogriside I (1) and cyanogriside J (2), were characterized along with three known caerulomycins, caerulomycin A (3), caerulomycin F (4) and caerulomycinonitrile (5). This is the first report of cyanogrisides and caerulomycins production by a member of the Saccharothrix genus. The minimum inhibitory concentrations (MIC) of these antibiotics were determined against pathogenic microorganisms.     

Hydration and Cognitive Function in Children

Adequate fluid intake is critical for survival. While adults are at liberty to drink fluids as wanted, children and infants are dependent upon caregivers for food and fluid. Children are at greater risk for dehydration than adults due to their higher surface-to-mass ratio. Additionally, children have different thirst sensitivities and body cooling mechanisms than adults. Children differ from adults in total body water content, and boys and girls differ in body water content with maturation. Research in young adults shows that mild dehydration corresponding to only 1% to 2% of body weight loss can lead to significant impairment in cognitive function. Dehydration in infants is associated with confusion, irritability, and lethargy; in children, it may produce decrements in cognitive performance.     

Aging pathways for organophosphate-inhibited human butyrylcholinesterase, including novel pathways for isomalathion, resolved by mass spectrometry.

Some organophosphorus compounds are toxic because they inhibit acetylcholinesterase (AChE) by phosphylation of the active site serine, forming a stable conjugate: Ser-O-P(O)-(Y)-(XR) (where X can be O, N, or S and Y can be methyl, OR, or SR). The inhibited enzyme can undergo an aging process, during which the X-R moiety is dealkylated by breaking either the P-X or the X-R bond depending on the specific compound, leading to a nonreactivatable enzyme. Aging mechanisms have been studied primarily using AChE. However, some recent studies have indicated that organophosphate-inhibited butyrylcholinesterase (BChE) may age through an alternative pathway. Our work utilized matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry to study the aging mechanism of human BChE inhibited by dichlorvos, echothiophate, diisopropylfluorophosphate (DFP), isomalathion, soman, sarin, cyclohexyl sarin, VX, and VR. Inhibited BChE was aged in the presence of H2O18 to allow incorporation of (18)O, if cleavage was at the P-X bond. Tryptic-peptide organophosphate conjugates were identified through peptide mass mapping. Our results showed no aging of VX- and VR-treated BChE at 25 degrees C, pH 7.0. However, BChE inhibited by dichlorvos, echothiophate, DFP, soman, sarin, and cyclohexyl sarin aged exclusively through O-C bond cleavage, i.e., the classical X-R scission pathway. In contrast, isomalathion aged through both X-R and P-X pathways; the main aged product resulted from P-S bond cleavage and a minor product resulted from O-C and/or S-C bond cleavage.     

Illuminating Negative Results in Evaluation of Smoking Prevention Programs

Evaluation of program implementation can help illuminate negative results of school-based smoking prevention programs. In three conventional quasiexperimental evaluations, no statistically significant impacts of smoking prevention programs on children's knowledge, attitudes, intentions, or behavior were detected. Complementary evaluations of program implementation along several dimensions using naturalistic methods suggested reasons for null effects were different at each site. These data were used to form hypotheses and recommendations for future interventions.     

Lumbar Puncture Ordering and Results in the Pediatric Population: A Promising Data Source for Surveillance Systems

Background: The Centers for Disease Control and Prevention is incorporating laboratory data into real-time surveillance systems. When normal patterns of laboratory test orders and results are modeled, aberrations can be detected. Because many test orders are available electronically well before results, atypical patterns of test ordering may signal outbreaks.
Objectives: The authors sought to characterize baseline patterns in the ordering and early results of lumbar punctures, motivated by the possibility of using these data for real-time surveillance for early detection of meningitis or encephalitis outbreaks.
Methods: Retrospective cohorts of pediatric emergency department patients at a single hospital (1993–2003) and from the National Hospital and Ambulatory Medical Care Survey (1992–2000) were used for analysis.
Results: Test ordering exhibits seasonal patterns, with monthly peaks in January and August (p < 0.0001). For the hospital cohort, the rate of cerebrospinal fluid pleocytosis exhibits seasonal patterns (p < 0.0001), with a peak from August to October. This is strongly associated with the rate and pattern of clinical neurologic disease (p < 0.0001). A long-term secular decline in daily test ordering is evident, dropping from 5.3 to 2.9 in the hospital sample, and from 371.8 to 185.3 in the national sample (p < 0.001). The long-term rate of pleocytosis has declined (p < 0.0001), though the yield of testing for pleocytosis has improved (p = 0.0104).
Conclusions: Laboratory test patterns correspond with those of clinical disease and are a promising source of surveillance data. Using such data for real-time monitoring requires specific adjustments for patient age, periodicities, and secular trends.