Fruit and vegetable consumption as a preventative strategy for non-communicable diseases.

A high intake of fruit and vegetables (FV) has consistently been associated with a reduced risk of a number of non-communicable diseases. This evidence base is largely from prospective cohort studies, with meta-analyses demonstrating an association between increased FV intake and reduced risk of both CHD and stroke, although the evidence is less certain for cancer and diabetes. Controlled intervention trials examining either clinical or intermediate risk factor endpoints are more scarce. Therefore, evidence that FV consumption reduces the risk of disease is so far largely confined to observational epidemiology, which is hampered by some methodological uncertainties. Although increased FV intake is promoted across all dietary guidelines, national surveys confirm that dietary intakes are suboptimal and are not increasing over time. A range of barriers to increasing FV intake exist, including economic, physical and behavioural barriers that must be considered when exploring potential opportunities to change this, considering the feasibility of different approaches to encourage increased FV consumption. Such interventions must include consideration of context, for example, challenges and uncertainties which exist with the whole food system.

Efficacy and safety of point-of-care ultrasound-guided intra-articular corticosteroid joint injections in patients with haemophilic arthropathy.

INTRODUCTION AND OBJECTIVES: Intra-articular corticosteroid injections are standard of care for managing joint pain secondary to osteoarthritis or rheumatoid arthritis but are rarely used in haemophilic arthropathy. We have introduced and evaluated the efficacy and safety of ultrasound-guided corticosteroid injections for pain relief in patients with haemophilic arthropathy. PATIENTS AND METHODS: Ultrasound-guided intra-articular injections performed on haemophilia patients at UCSD between March 2012 and January 2016 were analysed. Needle placement and injection (40 mg triamcinolone; 3-5 mL lidocaine) were performed with musculoskeletal ultrasound and Power Doppler. Analysis included patient demographics, joint-specific parameters such as tissue hypervascularity and effusions, pain relief, and procedure-associated complications. RESULTS: Forty-five injections (14 ankles, 13 elbows, 18 knees) were administered in 25 patients. Advanced arthropathy with hypervascularity and/or effusions was present in 91% and 61% of joints, respectively. Ninety-one per cent of injections resulted in pain relief which was significant in 84% (>30% reduction). Median pain score was reduced from 7 of 10 to 1 of 10 (P < 0.001), usually within 24 h. Median duration of pain relief was 8 weeks (range 1-16 weeks). Haemophilia B patients experienced longer periods of relief, and high Pettersson scores were associated with shorter duration of relief. There were no procedure-associated complications. Repeat ultrasound of eight joints within 4 weeks of injection demonstrated nearly complete resolution of hypervascularity. CONCLUSIONS: Point-of-care ultrasound enabled intra-articular corticosteroid injections that provided highly effective, safe, and relatively long-lasting pain relief in haemophilic arthropathy. This approach should be used to improve pain management in haemophilic arthropathy.

Reliability of self-reporting of antibiotic consumption in the community - Index of Reliability.

WHAT IS KNOWN AND OBJECTIVE: To date, there is no evidence to indicate the reliability of how patients self-report their own antibiotic usage in the community. Such data are fundamental in supporting antimicrobial stewardship practices, and so there is a need to determine its accuracy and reliability. COMMENT: Patients in the community (n = 476) were required to recollect their antibiotic usage in the past three months. Simultaneously, similar information was obtained by careful extraction from their respective medical notes, which was qualitatively compared with the patient's recollection. Overall, concordance was high (88·1%), but age (<20 and >80 years) and sex (female) were significant factors of reliability. WHAT IS NEW AND CONCLUSION: This study suggests that basic self-reporting of antibiotic usage amongst patients is relatively reliable, with increasing accuracy with years until 80 years. Where such information is critical, the current study can help decide who to interview and whose notes to interrogate, in the quest to obtain reliable and accurate information.

Mass spectrometry measurement of a therapeutic peptide for use in multiple sclerosis.

Multiple sclerosis is an autoimmune disease of the central nervous system believed to be mediated by pathogenic T lymphocytes. We have developed a next-generation therapy in which cells secrete specific therapeutic molecules to silence these aberrant T cells. We have shown that fibroblasts, transduced to secrete a myelin basic protein-derived peptide, abrogate disease in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis, which we hypothesized using a low-zone tolerance mechanism. To determine the efficacy (or not) of this therapy in humans, we must ensure that patients receive comparable doses of therapeutic peptide. To this end, we have used liquid chromatography coupled to tandem mass spectrometry to detect a tryptic peptide, derived from the secreted therapeutic product, at nanomolar concentrations. Success depended on growing the transduced fibroblasts in defined PC-1 medium in the presence of a cocktail of protease inhibitors.

The role of neural activity in the migration and differentiation of enteric neuron precursors.

BACKGROUND: As they migrate through the developing gut, a sub-population of enteric neural crest-derived cells (ENCCs) begins to differentiate into neurons. The early appearance of neurons raises the possibility that electrical activity and neurotransmitter release could influence the migration or differentiation of ENNCs. METHODS: The appearance of neuronal sub-types in the gut of embryonic mice was examined using immunohistochemistry. The effects of blocking various forms of neural activity on ENCC migration and neuronal differentiation were examined using explants of cultured embryonic gut. KEY RESULTS: Nerve fibers were present in close apposition to many ENCCs. Commencing at E11.5, neuronal nitric oxide synthase (nNOS), calbindin and IK(Ca) channel immunoreactivities were shown by sub-populations of enteric neurons. In cultured explants of embryonic gut, tetrodotoxin (TTX, an inhibitor of action potential generation), nitro-L-arginine (NOLA, an inhibitor of nitric oxide synthesis) and clotrimazole (an IK(Ca) channel blocker) did not affect the rate of ENCC migration, but tetanus toxin (an inhibitor of SNARE-mediated vesicle fusion) significantly impaired ENCC migration as previously reported. In explants of E11.5 and E12.5 hindgut grown in the presence of TTX or tetanus toxin there was a decrease in the number nNOS+ neurons close to the migratory wavefront, but no significant difference in the proportion of all ENCC that expressed the pan-neuronal marker, Hu. CONCLUSIONS & INFERENCES: (i) Some enteric neuron sub-types are present very early during the development of the enteric nervous system. (ii) The rate of differentiation of some sub-types of enteric neurons appears to be influenced by TTX- and tetanus toxin-sensitive mechanisms.

Minimally invasive incisional herniorrhaphy: a review of 208 cases.

BACKGROUND: Minimally invasive incisional herniorrhaphy has become an accepted approach for incisional hernia. However, the ideal technique for this procedure is not known. The authors present their technique and personal experience with minimally invasive incisional herniorrhaphy. METHODS: A retrospective review investigated 208 consecutive patients who underwent minimally invasive incisional hernia repair under the supervision of a single surgeon between 1995 and 2002. RESULTS: An intraperitoneal mesh repair was performed in all cases. There were no conversions. The mean operative time was 2.1 h (range, 0.8-4.5 h). The mean length of hospital stay was 2.5 days (range, 0-13 days). There were six complications, including two bowel perforations, and zero mortality. There were three recurrences during the follow-up period, which ranged from 6 to 72 months (median, 24 months). CONCLUSIONS: Minimally invasive incisional herniorrhaphy yielded an acceptable morbidity and recurrence rate during the follow-up period. The outcome compares favorably with that for open incisional hernia repair. Although long-term follow-up evaluation is desirable, the data support the contention that the minimally invasive approach is an appropriate option for incisional hernia.

Biochemical and genetic analyses of the U5, U6, and U4/U6 x U5 small nuclear ribonucleoproteins from Saccharomyces cerevisiae.

We have purified the yeast U5 and U6 pre-mRNA splicing small nuclear ribonucleoproteins (snRNPs) by affinity chromatography and analyzed the associated polypeptides by mass spectrometry. The yeast U5 snRNP is composed of the two variants of U5 snRNA, six U5-specific proteins and the 7 proteins of the canonical Sm core. The U6 snRNP is composed of the U6 snRNA, Prp24, and the 7 Sm-Like (LSM) proteins. Surprisingly, the yeast DEAD-box helicase-like protein Prp28 is stably associated with the U5 snRNP, yet is absent from the purified U4/U6 x U5 snRNP. A novel yeast U5 and four novel yeast U4/U6 x U5 snRNP polypeptides were characterized by genetic and biochemical means to demonstrate their involvement in the pre-mRNA splicing reaction. We also show that, unlike the human tri-snRNP, the yeast tri-snRNP dissociated upon addition of ATP or dATP.

In vitro bioassays for anticancer drug screening: effects of cell concentration and other assay parameters on growth inhibitory activity.

In vitro growth inhibition assays were performed using human cancer cell lines at various concentrations with experimental anticancer drugs such as the cryptophycins and other cytotoxins. The effect of variations in assay parameters on the observed growth inhibition of these anticancer therapeutic agents was determined. The results demonstrated that the observed inhibitory activity of these compounds varied inversely with the cell concentrations used. The observed differences in activity between different cytotoxins were not necessarily proportionate. Thus, the relative activities of two toxins also varied with cell concentration. Furthermore, the sensitivity of these cell lines to the cytostatic purine analog, 6-mercaptopurine (used as a control), varied with cell concentration as well. The activity of this compound was dependent on the medium used for cell growth, yielding good activity in Eagle's minimum essential medium, but not in Ham's F-12 (Kaigin) medium. Moreover, growth inhibition by cryptophycin as well as 6-mercaptopurine was also dependent on the serum concentration in the medium. Finally, the sensitivity of the cancer cell lines to various organic solvents commonly used as drug vehicles for in vitro testing, such as ethanol, dimethylformamide, and dimethylsulfoxide, was likewise found to vary inversely with cell concentration.

Isolation of dolastatin 10 from the marine cyanobacterium Symploca species VP642 and total stereochemistry and biological evaluation of its analogue symplostatin 1.

The potent antitumor agent dolastatin 10 (1) was originally isolated from the sea hare Dolabella auricularia, and we now report its isolation from the marine cyanobacterium Symploca sp. VP642 from Palau. The chemically related analogue symplostatin 1 (2) has been reisolated from Guamanian and Hawaiian varieties of S. hydnoides and its total stereochemistry completed by determining the N,N-dimethylisoleucine unit to be L. Symplostatin 1 (2), like dolastatin 10 (1), is a potent microtubule inhibitor. The antitumor activity of 2 was assessed in vivo against several murine tumors. Symplostatin 1 (2) was effective against a drug-insensitive mammary tumor and a drug-insensitive colon tumor; however, it was only slightly effective against two MDR tumors.

Total structure determination of apratoxin A, a potent novel cytotoxin from the marine cyanobacterium Lyngbya majuscula.

Apratoxin A (1), a potent cytotoxin with a novel skeleton, has been isolated from the marine cyanobacterium Lyngbya majuscula Harvey ex Gomont. This cyclodepsipeptide of mixed peptide-polyketide biogenesis bears a thiazoline ring flanked by polyketide portions, one of which possesses an unusual methylation pattern. Its gross structure has been elucidated by spectral analysis, including various 2D NMR techniques. The absolute configurations of the amino acid-derived units were determined by chiral HPLC analysis of hydrolysis products. The relative stereochemistry of the new dihydroxylated fatty acid unit, 3,7-dihydroxy-2,5,8,8-tetramethylnonanoic acid, was elucidated by successful application of the J-based configuration analysis originally developed for acyclic organic compounds using carbon-proton spin-coupling constants ((2,3)J(C,H)) and proton-proton spin-coupling constants ((3)J(H,H)); its absolute stereochemistry was established by Mosher analysis. The conformation of 1 in solution was mimicked by molecular modeling, employing a combination of distance geometry and restrained molecular dynamics. Apratoxin A (1) possesses IC(50) values for in vitro cytotoxicity against human tumor cell lines ranging from 0.36 to 0.52 nM; however, it was only marginally active in vivo against a colon tumor and ineffective against a mammary tumor.

Pitipeptolides A and B, new cyclodepsipeptides from the marine cyanobacterium Lyngbya majuscula.

Two new cyclodepsipeptides have been isolated from a population of the marine cyanobacterium Lyngbya majuscula collected at Piti Bomb Holes, Guam. They appear to be unique to this particular Guamanian collection and have been named pitipeptolides A (1) and B (2). Their structures have been elucidated by spectroscopic techniques and by characterization of degradation products. Distinctive features include the presence of a 2,2-dimethyl-3-hydroxy-7-octynoic acid residue in 1 and a 2,2-dimethyl-3-hydroxy-7-octenoic acid residue in 2, previously shown to be biosynthetic signatures of cyanobacterial metabolites. Pitipeptolides A (1) and B (2) exhibit weak cytotoxicity against LoVo cancer cells, but possess moderate antimycobacterial activity and stimulate elastase activity.

Osteoclasts secrete the chemotactic cytokine mim-1.

Osteoclasts are terminally differentiated, multinucleated cells of monocytic origin. In this study, we report that osteoclasts secrete a 35 kD protein and that phorbol myristate acetate treatment stimulates secretion dramatically. Peptide digests of the protein were analyzed by mass spectroscopy. The protein was identified as myb induced myeloid protein-1 precursor (mim-1 protein). Mim-1 is expressed specifically by hematopoietic cells and has no known function. It is homologous with the neutrophil chemokine, chondromodulin II, which stimulates proliferation of osteoblasts and chondrocytes. Western analysis showed that osteoclasts secrete mim-1 into culture media. Immunofluorescence studies demonstrated a cytoplasmic and perinuclear distribution of mim-1 in both avian osteoclasts and human osteoclast-like cells. Expression and secretion of a chemokine-like protein by osteoclasts suggests a novel signaling pathway in the bone microenvironment that may be involved in coordinating bone remodeling.

Factors associated with use of mental health services for depression by children and adolescents.

OBJECTIVE: This study examined patterns of mental health service use among depressed children and adolescents and factors associated with help seeking and treatment modalities. METHODS: The sample consisted of 206 children and adolescents aged 9 to 17 years who were assessed as part of a larger survey of mental health service use in five service systems and in the community and who met DSM-III-R criteria for depressive disorders (major depression or dysthymia). RESULTS: Among the 206 children, 75 (36 percent) never received professional help for depressive symptoms. Among the 131 children who received professional help for depression, antidepressants were prescribed for 40 (31 percent) in the year before the interview. The findings indicate possible undertreatment of depression among children and adolescents, especially among African Americans. Socioeconomic factors, such as the mother's education and the child's health insurance, were not associated with receiving professional help for depressive symptoms but were associated with receiving antidepressants. Parental perception of a child's mental health service need was associated with receiving professional help but not with receiving antidepressants. Also, depressed children were more likely to receive antidepressants when they had life-threatening or severe symptoms, such as a suicide attempt or drug abuse. CONCLUSIONS: Whether a depressed child receives mental health services and the types of treatment received are influenced by different individual and family factors and by the type of symptoms exhibited. Better understanding of these factors will help in meeting the service needs of depressed children and adolescents.

Isolation and structure determination of nostocyclopeptides A1 and A2 from the terrestrial cyanobacterium Nostoc sp. ATCC53789.

The isolation and total structure determination of nostocyclopeptides A1 (1) and A2 (2) are described. These cyclic heptapeptides, which possess a unique imino linkage in the macrocyclic ring, are characteristic constituents of the cryptophycin-producing cyanobacterium Nostoc sp. ATCC53789. 1D TOCSY experiments proved to be very useful in identifying the seven amino acid residues in each compound, and HMBC and NOESY correlations made it possible to sequence the seven units into a total gross structure. The absolute stereochemistry was determined by directly comparing the amino acids in the acid hydrolyzate of each natural product and its peroxide oxidation and borohydride reduction products with authentic standards. Studies were carried out on the biosynthesis and initiated on the biological activity of these cyclic peptides.

The role of the FRE family of plasma membrane reductases in the uptake of siderophore-iron in Saccharomyces cerevisiae.

Saccharomyces cerevisiae takes up siderophore-bound iron through two distinct systems, one that requires siderophore transporters of the ARN family and one that requires the high affinity ferrous iron transporter on the plasma membrane. Uptake through the plasma membrane ferrous iron transporter requires that the iron first must dissociate from the siderophore and undergo reduction to the ferrous form. FRE1 and FRE2 encode cell surface metalloreductases that are required for reduction and uptake of free ferric iron. The yeast genome contains five additional FRE1 and FRE2 homologues, four of which are regulated by iron and the major iron-dependent transcription factor, Aft1p, but whose function remains unknown. Fre3p was required for the reduction and uptake of ferrioxamine B-iron and for growth on ferrioxamine B, ferrichrome, triacetylfusarinine C, and rhodotorulic acid in the absence of Fre1p and Fre2p. By indirect immunofluorescence, Fre3p was expressed on the plasma membrane in a pattern similar to that of Fet3p, a component of the high affinity ferrous transporter. Enterobactin, a catecholate siderophore, was not a substrate for Fre3p, and reductive uptake required either Fre1p or Fre2p. Fre4p could facilitate utilization of rhodotorulic acid-iron when the siderophore was present in higher concentrations. We propose that Fre3p and Fre4p are siderophore-iron reductases and that the apparent redundancy of the FRE genes confers the capacity to utilize iron from a variety of siderophore sources.

Protein identification using a quadrupole ion trap mass spectrometer and SEQUEST database matching.

A procedure is described for rapid, sensitive protein identification utilizing liquid chromatography--tandem mass spectrometry. The analysis is performed on mixtures of peptides obtained by enzyme digestion. The SEQUEST computer program is used to match the sequence information in the spectra to a database of known protein sequences.

Introducing samples directly into electrospray ionization mass spectrometers using microscale capillary liquid chromatography.

This unit describes the design and operation of a microscale electrospray (ES) interface suitable for the on-line liquid chromatography (LC) separation and mass spectrometry (MS) analysis of mixtures of peptides and proteins. The interface utilizes an ES needle packed with reversed-phase support. Such a design has the advantage of minimizing any void volume between the end of the column and point of electrospray ionization, thus maintaining the integrity of the LC separation and maximizing sensitivity. Here, protocols are presented for construction of an integrated LC column ES needle in-house, packing the ES needle, and mounting and using the microscale ES LC/MS interface assembly. Various options for low-flow solvent delivery systems are also discussed.

Introducing samples directly into electrospray ionization mass spectrometers using a nanospray interface.

Electrospray (ES) is a concentration-dependent phenomenon, and operation at high flow rates results in a corresponding high rate of sample consumption. Sensitivity increases are most readily achieved by decreasing the flow rate of sample solution into the electrospray source. In recent years, a number of methodologies have been developed to achieve ES analyses at sub-ml/min flow rates. There are a large number of microscale ES devices currently in use, but this unit describes in detail a micro-scale interface for direct liquid introduction without separation.

Isolation and structure of the cytotoxin lyngbyabellin B and absolute configuration of lyngbyapeptin A from the marine cyanobacterium Lyngbya majuscula.

An analogue of the potent microfilament-disrupter lyngbyabellin A (1) has been isolated as a minor metabolite from the marine cyanobacterium Lyngbya majuscula collected at Apra Harbor, Guam. It possesses slightly weaker cytotoxicity than 1 and has been named lyngbyabellin B (2). Primarily NMR spectroscopy was used to determine its structure. The absolute configuration of 2 has been ascertained by chiral HPLC analysis of degradation products and by comparison with lyngbyabellin A (1). The known modified tetrapeptide lyngbyapeptin A (3) has also been found in the same extract, and its absolute stereochemistry could be determined for the first time.

Apramides A-G, novel lipopeptides from the marine cyanobacterium Lyngbya majuscula.

Six new metabolites have been isolated from a lyngbyastatin 2-producing strain of the marine cyanobacterium Lyngbya majuscula collected at Apra Harbor, Guam, and their structures elucidated. These linear lipopeptides have been assigned the trivial names apramides A-F (1-6). From a more recent collection of this cyanobacterium, a structurally related compound, apramide G (7), has been found instead of apramides A-F (1-6). Structure elucidation of the lipopeptides 1-7 is based on spectroscopic techniques and chiral chromatography of hydrolysis products. The apramides appear as NMR-spectroscopically distinguishable conformers in solution, and this has been ascribed to the presence of a thiazole-containing modified amino acid unit.