In vitro and in vivo evaluation of Alexa Fluor 680-bombesin[7-14]NH2 peptide conjugate, a high-affinity fluorescent probe with high selectivity for the gastrin-releasing peptide receptor

Gastrin-releasing peptide (GRP) receptors are overexpressed on several types of human cancer cells, including breast, prostate, small cell lung, and pancreatic cancers. Bombesin (BBN), a 14-amino acid peptide that is an analogue of human GRP, binds to GRP receptors with very high affinity and specificity. The aim of this study was to develop a new fluorescent probe based on BBN having high tumor uptake and optimal pharmacokinetics for specific targeting and optical imaging of human breast cancer tissue. In this study, solid-phase peptide synthesis was used to produce H(2)N-glycylglycylglycine-BBN[7-14]NH(2) peptide with the following general sequence: H(2)N-G-G-G-Q-W-A-V-G-H-L-M-(NH(2)). This conjugate was purified by reversed-phase high-performance liquid chromatography and characterized by electrospray-ionization mass spectra. The fluorescent probe Alexa Fluor 680-G-G-G-BBN[7-14]NH(2) conjugate was prepared by reaction of Alexa Fluor 680 succinimidyl ester to H(2)N-G-G-G-BBN[7-14]NH(2) in dimethylformamide (DMF). In vitro competitive binding assays, using (125)I-Tyr(4)-BBN as the radiolabeling gold standard, demonstrated an inhibitory concentration 50% value of 7.7 +/- 1.4 nM in human T-47D breast cancer cells. Confocal fluorescence microscopy images of Alexa Fluor 680-G-G-G-BBN[7-14]NH(2) in human T-47D breast cancer cells indicated specific uptake, internalization, and receptor blocking of the fluorescent bioprobe in vitro. In vivo investigations in SCID mice bearing xenografted T-47D breast cancer lesions demonstrated the ability of this new conjugate to specifically target tumor tissue with high selectivity and affinity.     

Convenient conversion of wheat hemicelluloses pentoses (D-xylose and L-arabinose) into a common intermediate

The transformation of D-xylose and L-arabinose, the two major components of wheat straw and bran, into a unique multifunctional, optically pure, five-carbon synthon has been achieved. The synthetic sequence requires three steps: suitable protection of the hydroxyl groups of the pentoses, introduction of an iodide at the C-5 position and zinc-mediated opening of the furanose ring leading to the formation of a common substituted pent-4-enal.     

Brain-derived neurotrophic factor in the hypothalamic paraventricular nucleus reduces energy intake

Recent studies show that brain-derived neurotrophic factor (BDNF) decreases feeding and body weight after peripheral and ventricular administration. BDNF mRNA and protein, and its receptor tyrosine kinase B (TrkB) are widely distributed in the hypothalamus and other brain regions. However, there are few reports on specific brain sites of actions for BDNF. We evaluated the effect of BDNF in the hypothalamic paraventricular nucleus (PVN) on feeding. BDNF injected unilaterally or bilaterally into the PVN of food-deprived and nondeprived rats significantly decreased feeding and body weight gain within the 0- to 24-h and 24- to 48-h postinjection intervals. Effective doses producing inhibition of feeding behavior did not establish a conditioned taste aversion. PVN BDNF significantly decreased PVN neuropeptide Y (NPY)-induced feeding at 1, 2, and 4 h following injection. BDNF administration in the PVN abolished food-restriction-induced NPY gene expression in the hypothalamic arcuate nucleus. In conclusion, BDNF in the PVN significantly decreases food intake and body weight gain, suggesting that the PVN is an important site of action for BDNF in its effects on energy metabolism. Furthermore, BDNF appears to interact with NPY in its anorectic actions, although a direct effect on NPY remains to be established.     

Prediction of Severe Disease in Children with Diarrhea in a Resource-Limited Setting

Objective

To investigate the accuracy of three clinical scales for predicting severe disease (severe dehydration or death) in children with diarrhea in a resource-limited setting.

Methods

Participants included 178 children admitted to three Rwandan hospitals with diarrhea. A local physician or nurse assessed each child on arrival using the World Health Organization (WHO) severe dehydration scale and the Centers for Disease Control (CDC) scale. Children were weighed on arrival and daily until they achieved a stable weight, with a 10% increase between admission weight and stable weight considered severe dehydration. The Clinical Dehydration Scale was then constructed post-hoc using the data collected for the other two scales. Receiver Operator Characteristic (ROC) curves were constructed for each scale compared to the composite outcome of severe dehydration or death.

Results

The WHO severe dehydration scale, CDC scale, and Clinical Dehydration Scale had areas under the ROC curves (AUCs) of 0.72 (95% CI 0.60, 0.85), 0.73 (95% CI 0.62, 0.84), and 0.80 (95% CI 0.71, 0.89), respectively, in the full cohort. Only the Clinical Dehydration Scale was a significant predictor of severe disease when used in infants, with an AUC of 0.77 (95% CI 0.61, 0.93), and when used by nurses, with an AUC of 0.78 (95% CI 0.63, 0.93).

Conclusions

While all three scales were moderate predictors of severe disease in children with diarrhea, scale accuracy varied based on provider training and age of the child. Future research should focus on developing or validating clinical tools that can be used accurately by nurses and other less-skilled providers to assess all children with diarrhea in resource-limited settings.