Chrysomya putoria, a Putative Vector of Diarrheal Diseases

Background

Chrysomya spp are common blowflies in Africa, Asia and parts of South America and some species can reproduce in prodigious numbers in pit latrines. Because of their strong association with human feces and their synanthropic nature, we examined whether these flies are likely to be vectors of diarrheal pathogens.

Methodology/Principal Findings

Flies were sampled using exit traps placed over the drop holes of latrines in Gambian villages. Odor-baited fly traps were used to determine the relative attractiveness of different breeding and feeding media. The presence of bacteria on flies was confirmed by culture and bacterial DNA identified using PCR. A median of 7.00 flies/latrine/day (IQR = 0.0–25.25) was collected, of which 95% were Chrysomya spp, and of these nearly all were Chrysomya putoria (99%). More flies were collected from traps with feces from young children (median = 3.0, IQR = 1.75–10.75) and dogs (median = 1.50, IQR = 0.0–13.25) than from herbivores (median = 0.0, IQR = 0.0–0.0; goat, horse, cow and calf; p<0.001). Flies were strongly attracted to raw meat (median = 44.5, IQR = 26.25–143.00) compared with fish (median = 0.0, IQR = 0.0–19.75, ns), cooked and uncooked rice, and mangoes (median = 0.0, IQR = 0.0–0.0; p<0.001). Escherichia coli were cultured from the surface of 21% (15/72 agar plates) of Chrysomya spp and 10% of these were enterotoxigenic. Enteroaggregative E. coli were identified by PCR in 2% of homogenized Chrysomya spp, Shigella spp in 1.4% and Salmonella spp in 0.6% of samples.

Conclusions/Significance

The large numbers of C. putoria that can emerge from pit latrines, the presence of enteric pathogens on flies, and their strong attraction to raw meat and fish suggests these flies may be common vectors of diarrheal diseases in Africa.     

The association between N-acetyltransferase 2 gene polymorphisms and pancreatic cancer

Pancreatic cancer has been linked with exposure to environmental chemicals, which generally require metabolic activation to highly reactive toxic or carcinogenic intermediates. N-acetyltransferase 1 (NAT1) and N-acetyltransferase 2 (NAT2) are expressed primarily in extrahepatic and hepatic tissues, respectively. Both enzymes catalyze N- and O-acetylation of aromatic and heterocyclic amines. It is believed that these compounds are activated via O-acetylation and detoxified by N-acetylation. Several polymorphisms of these two genes have been associated with an increased risk of cancer. Twenty-seven cases of pancreatic cancer and 104 controls were included in this study. Blood was collected in EDTA-containing tubes, and genomic DNA was extracted from the white blood cells by using a high pure PCR template preparation kit. Genotyping of NAT2 polymorphisms was detected by a real time PCR instrument. There was a significant difference in the distribution of the NAT2*6A acetylators phenotype between cases and the controls. The odds ratio of pancreatic cancer for the NAT2*6A slow phenotype was 5.7 (95% CI = 1.27-25.55; p = 0.023) compared with the fast type. Our results suggest that slow acetylators have higher risk of developing pancreatic cancer than fast acetylators. NAT2 gene polymorphisms may be associated with genetic susceptibility to pancreatic cancer.     

Correlation of low molecular weight advanced glycation end products and nitric oxide metabolites with chronic complications in type 1 diabetic patients

Advanced glycation end products (AGEs) are involved in the occurrence of vascular complications in diabetes. The present study was undertaken to investigate the level of low-molecular weight products of AGEs (LMW-AGEs) in relation to microvascular complications in type 1 diabetes, and the possible relationship with nitric oxide (NO) as a marker of endothelial function. Patients with normal renal function (NRF) were classified into two groups: (1) without, and (2) with diabetic neuropathy; and patients with renal impairment also into two groups: (3) diabetic renal disease, and (4) end-stage renal disease. The fluorescence of LMW-AGEs and measurement of NO metabolites was assessed in 277 serum samples. In addition, multiple regression analysis was performed. In group 1, LMW-AGEs level (9.3±1.1 AF%) was higher than in the control group (2.4±0.3 AF%). A trend in the increase of LMW-AGEs with neuropathy (29.7±5.5 AF%, group 2), and further with renal impairment (47.0±8.0, group 3 and 137.8±25.5 AF%, group 4), was observed. In multivariate regression analysis LMW-AGEs were associated with NO metabolites in group 2. In NRF patients, diabetic neuropathy was significantly correlated with LMW-AGEs and NO metabolites, independently of serum creatinine and duration of diabetes. This relationship suggests that the NO and LMW-AGEs’ actions (possibly synergistic) in endothelial activation possess a role in the initiation and development of diabetic microvascular complications.     

Design, structural, and immuno-analytical properties of antigenic bioconjugates comprising a beta-amyloid-plaque specific epitope

Immunotherapeutic approaches are investigated for treatment of neurodegenerative diseases of the Alzheimer's dementia (AD) type. The identification of a beta-amyloid-plaque specific epitope, Abeta(4-10) (4FRHDSGY10), recognized by therapeutically active antibodies from transgenic AD mice could provide the basis for the development of AD vaccines. Here we report on the synthesis, structural and immuno-analytical characterization of bioconjugates comprising the beta-amyloid(4-10) epitope as new vaccine lead structures against Alzheimer's disease. To produce antigenic bioconjugates, potential immunogens, the epitope peptide elongated by a cysteine residue or a cysteinyl-pentaglycine hexapeptide unit either at the N- or C-terminus was attached via a thioether bond to synthetic oligopeptide carriers, such as oligotuftsin derivatives, sequential oligopeptide carrier, or lysine dendrimer. The antigenic properties of these constructs were determined by enzyme-linked immunosorbent assay (ELISA) using an anti-Abeta(1-17) monoclonal antibody. Our results indicate that the major factors which influence the antibody binding of the Abeta(4-10) epitope are (i) the epitope topology and (ii) the presence of a spacer moiety between the carrier and the epitope peptide. Interestingly, the carrier type had no marked effect on the binding of the antibody to the epitope-conjugates. The conformational preferences of the conjugates were examined by circular dichroism spectroscopy in water and in trifluoroethanol. In water, the conjugates adopt random coil conformation independently on their primary structure. However, differences related to the attachment site of the epitope to the carriers were determined in TFE, conjugates in which the epitope was attached to the carrier through the N-terminus exhibiting more ordered secondary structure.     

Synthesis and structural characterization of bioactive peptide conjugates using thioether linkage approaches.

Applications of cysteine-insertion and thioether linkage approaches to the preparation of a number of bioactive peptide conjugates are reported. Peptides containing epitopes from (i) herpes simplex virus type 1 glycoprotein D, (ii) a specific N-terminal beta-amyloid epitope recognized by therapeutically active antibodies, and (iii) a GnRH-III peptide from sea lamprey with antitumour activity, were elongated with Cys residues and attached to a chloroacetylated tetratuftsin derivative carrier via a thioether linkage either directly, or by insertion of a spacer. The structures and molecular homogeneity of all the peptide conjugates were ascertained by HPLC, MALDI and electrospray mass spectrometry. The use of a spacer such as an oligoglycine or GFLG-tetrapeptide gave an increased yield in the conjugation reaction and enhanced reaction rates. In the formation of cysteinyl-thioether linkages, it was found that the position of flanking Cys residues markedly influenced the conjugation reaction and the formation of intermolecular epitope disulfide-dimers. C-terminal Cys residues gave thioether conjugates with significantly diminished epitope-dimerization, while Cys at the N-terminal caused rapid disulfide-dimerization, thereby preventing efficient conjugation.     

A GABAergic inhibitory microcircuit controlling cholinergic outflow to the airways.

GABA is the main inhibitory neurotransmitter that participates in the regulation of cholinergic outflow to the airways. We have tested the hypothesis that a monosynaptic GABAergic circuit modulates the output of airway-related vagal preganglionic neurons (AVPNs) in the rostral nucleus ambiguus by using a dual-labeling electron microscopic method combining immunocytochemistry for glutamic acid decarboxylase (GAD) with retrograde tracing from the trachea. We also determined the effects of blockade of GABAA receptors on airway smooth muscle tone. The results showed that retrogradely labeled AVPNs received a significant GAD-immunoreactive (GAD-IR) terminal input. Out of a pooled total of 3,161 synaptic contacts with retrogradely labeled somatic and dendritic profiles, 20.2% were GAD-IR. GAD-IR terminals formed significantly more axosomatic synapses than axodendritic synapses (P < 0.02). A dense population of GABAergic synaptic contacts on AVPNs provides a morphological basis for potent physiological effects of GABA on the excitability of AVPNs. GAD-IR terminals formed exclusively symmetric synaptic specializations. GAD-IR terminals were significantly larger (P < 0.05) in both length and width than unlabeled terminals synapsing on AVPNs. Therefore, the structural characteristics of certain nerve terminals may be closely correlated with their function. Pharmacological blockade of GABAA receptors within the rostral nucleus ambiguus increased activity of putative AVPNs and airway smooth muscle tone. We conclude that a tonically active monosynaptic GABAergic circuit utilizing symmetric synapses regulates the discharge of AVPNs.     

Ionic blockade of the rat connexin40 gap junction channel by large tetraalkylammonium ions.

The rat connexin40 gap junction channel is permeable to monovalent cations including tetramethylammonium and tetraethylammonium ions. Larger tetraalkyammonium (TAA(+)) ions beginning with tetrabutylammonium (TBA(+)) reduced KCl junctional currents disproportionately. Ionic blockade by tetrapentylammonium (TPeA(+)) and tetrahexylammonium (THxA(+)) ions were concentration- and voltage-dependent and occurred only when TAA(+) ions were on the same side as net K(+) efflux across the junction, indicative of block of the ionic permeation pathway. The voltage-dependent dissociation constants (K(m)(V(j))) were lower for THxA(+) than TPeA(+), consistent with steric effects within the pore. The K(m)-V(j) relationships for TPeA(+) and THxA(+) were fit with different reaction rate models for a symmetrical (homotypic) connexin gap junction channel and were described by either a one- or two-site model that assumed each ion traversed the entire V(j) field. Bilateral addition of TPeA(+) ions confirmed a common site of interaction within the pore that possessed identical K(m)(V(j)) values for cis-trans concentrations of TPeA(+) ions as indicated by the modeled I-V relations and rapid channel block that precluded unitary current measurements. The TAA(+) block of K(+) currents and bilateral TPeA(+) interactions did not alter V(j)-gating of Cx40 gap junctions. N-octyl-tributylammonium and -triethylammonium also blocked rCx40 channels with higher affinity and faster kinetics than TBA(+) or TPeA(+), indicative of a hydrophobic site within the pore near the site of block.     

Activation of the midbrain periaqueductal gray induces airway smooth muscle relaxation.

In this study, we examined effects of chemical stimulation of the ventrolateral region of the midbrain periaqueductal gray (vl PAG) on airway smooth muscle tone. We observed that in anesthetized, paralyzed, and artificially ventilated ferrets, vl PAG stimulation elicited airway smooth muscle relaxation. To clarify the mechanisms underlying this observation, we examined the GABA-GABA(A) receptor signaling pathway by 1) examining the expression of GABA(A) receptors on airway-related vagal preganglionic neurons (AVPNs) located in the rostral nucleus ambiguus region (rNA), by use of receptor immunochemistry and confocal microscopy; 2) measuring GABA release within the rNA by using microdialysis; and 3) performing physiological experiments to determine the effects of selective blockade of GABA(A) receptors expressed by AVPNs in the rNA region on vl PAG-induced airway relaxation, thereby defining the role of the GABA(A) receptor subtype in this process. We observed that AVPNs located in the rNA region do express the GABA(A) receptor beta-subtype. In addition, we demonstrated that activation of vl PAG induced GABA release within the rNA region, and this release was associated with airway smooth muscle relaxation. Blockade of the GABA(A) receptor subtype expressed by AVPNs in the rNA by bicuculline diminished the inhibitory effects of vl PAG stimulation on airway smooth muscle tone. These data indicate, for the first time, that activation of vl PAG dilates the airways by a release of GABA and activation of GABA(A) receptors expressed by AVPNs.     

The effect of follicular fluid hormones on oocyte recovery after ovarian stimulation: FSH level predicts oocyte recovery

Background  

Ovarian stimulation for assisted reproductive technology (ART) overcomes the physiologic process to develop a single dominant follicle. However, following stimulation, egg recovery rates are not 100%. The objective of this study is to determine if the follicular fluid hormonal environment is associated with oocyte recovery.