A cationic cysteine-hydrazide as an enrichment tool for the mass spectrometric characterization of bacterial free oligosaccharides.

In Campylobacterales and related ε-proteobacteria with N-linked glycosylation (NLG) pathways, free oligosaccharides (fOS) are released into the periplasmic space from lipid-linked precursors by the bacterial oligosaccharyltransferase (PglB). This hydrolysis results in the same molecular structure as the oligosaccharide that is transferred to a protein to be glycosylated. This allowed for the general elucidation of the fOS-branched structures and monosaccharides from a number of species using standard enrichment and mass spectrometry methods. To aid characterization of fOS, hydrazide chemistry has often been used for chemical modification of the reducing part of oligosaccharides resulting in better selectivity and sensitivity in mass spectrometry; however, the removal of the unreacted reagents used for the modification often causes the loss of the sample. Here, we develop a more robust method for fOS purification and characterize glycostructures using complementary tandem mass spectrometry (MS/MS) analysis. A cationic cysteine hydrazide derivative was synthesized to selectively isolate fOS from periplasmic fractions of bacteria. The cysteine hydrazide nicotinamide (Cyhn) probe possesses both thiol and cationic moieties. The former enables reversible conjugation to a thiol-activated solid support, while the latter improves the ionization signal during MS analysis. This enrichment was validated on the well-studied Campylobacter jejuni by identifying fOS from the periplasmic extracts. Using complementary MS/MS analysis, we approximated data of a known structure of the fOS from Campylobacter concisus. This versatile enrichment technique allows for the exploration of a diversity of protein glycosylation pathways.

Effectiveness of osteopathic manipulative therapy for managing symptoms of irritable bowel syndrome: a systematic review.

CONTEXT: Irritable bowel syndrome (IBS) is a common and often lifelong functional gastrointestinal disorder. There is a scarcity of effective management options for IBS. OBJECTIVE: To assess the effectiveness of osteopathic manipulative therapy (OMTh) for managing the symptoms of IBS. DATA SOURCES: Articles without language or publication-date restriction were searched in PubMed, Embase, Cochrane Library, PEDro, OSTMED.DR, and Osteopathic Research Web. Search terms included irritable bowel syndrome, IBS, functional colonic disease, colon irritable, osteopath*, osteopathic manipulation, osteopathic medicine, clinical trial, and randomized clinical trial. Experts in the field of visceral osteopathy were also contacted to identify additional studies. STUDY SELECTION: The authors evaluated randomized controlled trials (RCTs) of OMTh for IBS in adults in whom IBS was diagnosed using Rome (I-III) criteria. If OMTh was not the sole intervention in the intervention group and if the same additional interventions were not applied to the control group, the study was excluded. DATA EXTRACTION: Citation identification, study selection, and data extraction were independently undertaken by 2 reviewers with a data extraction form from the Cochrane Collaboration. A consensus method was used to resolve disagreements concerning the assessment of the methodologic quality of the RCTs that were reviewed. RESULTS: The search identified 10 studies that examined OMTh for patients with IBS; 5 studies (204 patients) met the inclusion criteria. All studies were assessed as having low risk of bias according to the Cochrane Collaboration criteria, although there was heterogeneity in the outcome measures and control interventions. Three studies used visual analog scales for abdominal pain, whereas others used the IBS severity score and the Functional Bowel Disorder Severity Index. A variety of secondary outcomes were used. All studies reported more pronounced short-term improvements with OMTh compared with sham therapy or standard care only. These differences remained statistically significant after variable lengths of follow-up in 3 studies. CONCLUSION: The present systematic review provides preliminary evidence that OMTh may be beneficial in the treatment of patients with IBS. However, caution is required in the interpretation of these findings because of the limited number of studies available and the small sample sizes.

Calcium phosphate mineralization beneath a polycationic monolayer at the air-water interface.

The self-assembly of the amphiphilic block copolymer poly(n-butyl methacrylate)-block-poly[2-(dimethylamino)ethyl methacrylate] at the air-water interface has been investigated at different pH values. Similar to Rehfeldt et al. (J. Phys. Chem. B 2006, 110, 9171), the subphase pH strongly affects the monolayer properties. The formation of calcium phosphate beneath the monolayer can be tuned by the subphase pH and hence the monolayer charge. After 12 h of mineralization at pH 5, the polymer monolayers are still transparent, but transmission electron microscopy (TEM) shows that very thin calcium phosphate fibers form, which aggregate into cotton ball-like features with diameters of 20 to 50 nm. In contrast, after 12 h of mineralization at pH 8, the polymer film is very slightly turbid and TEM shows dense aggregates with sizes between 200 and 700 nm. The formation of calcium phosphate is further confirmed by Raman and energy dispersive X-ray spectroscopy. The calcium phosphate architectures can be assigned to the monolayer charge, which is high at low pH and low at high pH. The study demonstrates that the effects of polycations should not be ignored if attempting to understand the colloid chemistry of biomimetic mineralization. It also shows that basic block copolymers are useful complementary systems to the much more commonly studied acidic block copolymer templates.

Subchronic toxicological evaluation of brea gum (Parkinsonia preacox) as a food additive in BALB/c mice.

Brea gum is a phloematic exudate from Parkinsonia praecox, an autoctonous tree that grows in the arid areas of Argentina. In this work, we propose its potential as a food additive. However, as no toxicological safety evaluation of brea gum has yet been reported, this preliminary study was conducted to evaluate its long-term toxicity over a 120-day period in BALB/c mice fed with brea gum at various levels in the diet. The results showed that animals on diets containing up to 5% brea gum were healthy, exhibiting growth curves similar to controls for both males (P = 0.9138) and females (P = 0.9459), thereby indicating that feed intake and utilization was not affected. A histopathological examination and weight recording of liver, kidneys, and intestine did not reveal any microscopic abnormalities or adverse toxicological effect (weights respect to control: P > 0.1). Moreover, hematological parameters and enzyme activities were within the normal values previously reported for mice. Our findings suggest that feeding brea gum at levels up to 5% to BALB/c mice do not exert any toxicological effects, supporting its potential use as a food additive for human consumption.

Desulfoglucosinolate sulfotransferases from Arabidopsis thaliana catalyze the final step in the biosynthesis of the glucosinolate core structure.

The phytotoxin coronatine is a structural analog of octadecanoid signaling molecules, which are well known mediators of plant defense reactions. To isolate novel coronatine-regulated genes from Arabidopsis thaliana, differential mRNA display was performed. Transcript levels of CORI-7 (coronatine induced-7) were rapidly and transiently increased in coronatine-treated plants, and the corresponding cDNA was found to encode the sulfotransferase AtST5a. Likewise, upon wounding, an immediate and transient increase in AtST5a mRNA levels could be observed in both locally wounded and unwounded (systemic) leaves. Furthermore, application of octadecanoids and ethylene as compounds involved in plant wound defense reactions resulted in AtST5a gene activation, whereas pathogen defense-related signals (yeast elicitor and salicylic acid) were inactive. AtST5a and its close homologs AtST5b and AtST5c were purified as His6-tagged proteins from Escherichia coli. The three enzymes were shown to catalyze the final step in the biosynthesis of the glucosinolate (GS) core structure, the sulfation of desulfoglucosinolates (dsGSs). They accept a broad range of dsGSs as substrates. However, in a competitive situation, AtST5a clearly prefers tryptophan- and phenylalanine-derived dsGSs, whereas long chain dsGSs derived from methionine are the preferred substrates of AtST5b and AtST5c. Treatment of Arabidopsis plants with low concentrations of coronatine resulted in an increase in the amounts of specific GSs, primarily glucobrassicin and neoglucobrassicin. Hence, it is suggested that AtST5a is the sulfotransferase responsible for the biosynthesis of tryptophan-derived GSs in vivo.

Technique and results of hyperthermic (41 degrees C) isolated lung perfusion with high-doses of cisplatin for the treatment of surgically relapsing or unresectable lung sarcoma metastasis.

OBJECTIVE: A technique of hyperthermic isolated lung perfusion (ILP) chemotherapy was developed. METHODS: Since April 1999, four patients with unilateral (n=2) or bilateral (n=2) sarcoma metastasis confined to a lobe (n=2) or entire lung (n=2) entered into a pilot study of hyperthermic (41 degrees C) ILP with high doses of cisplatin (70 mg/m(2)). Eligibility included drug resistant metastasis and at least four previous surgical metastectomies. The ILP of the lung segments was carried out following metastectomy, for 20-40 min at a rate of 0.3-0.5 l/min, a mean perfusion pressure lower than the own mean pulmonary artery pressure, and an inflow temperature of 41 degrees C or higher. Before and following ILP, the isolated lung segments were flushed with normothermic saline (1 l). Flow was continuously maintained by a centrifugal pump. RESULTS: All patients successfully completed 31.7+/-9 min perfusion time at 41.4+/-0.3 degrees C, and this time-point corresponded to the maximal platinum lung-uptake (93.8 ng/mg tissue). The total vascular isolation was confirmed by continuously low systemic cisplatin plasma levels. There was no systemic drug-related toxicity but all patients experienced transient pulmonary toxicity as non-cardiogenic edema of the treated lung segments. With a median follow-up of 12 months, three patients are alive and disease-free and one died from cerebral metastasis without autopsy evidence of local recurrence 13 months following ILP. CONCLUSION: Hyperthermic perfusion chemotherapy can be done safely and effectively. It represents a new treatment modality and deserves further investigations for patients with advanced, drug resistant or surgically refractory, lung sarcoma metastasis. However, further studies are needed to limit the ILP-induced pulmonary toxicity.