Role of mass spectrometry in the purification of peptides and proteins

Experiments were carried out to evaluate the fractionation of proteins and peptides according to mass. Model mixtures were separated by either reversed-phase or ion-exchange chromatography with mass spectrometry-compatible mobile phase additives. Fraction collection was triggered by the mass/charge ratio of each one of the components of the mixture. Chromatography was additionally monitored with a UV-Vis detector in order to compare the new technique with generally accepted in separations. The results indicated that adequate purification is achieved by this new technique. Fraction collection triggered by changes in the mass/charge ratio reduces sample handling and analysis time. This study demonstrates the utility of mass-directed fractionation of peptides and proteins when mass spectrometry-compatible mobile phase additives are used.     

Adipokines and Obesity Are Associated with Colorectal Polyps in Adult Males: A Cross-Sectional Study

Background

Obesity increases the risk of colon cancer. It is also known that most colorectal cancers develop from adenomatous polyps. However, the effects of obesity and adipokines on colonic polyp formation are unknown.

Methods

To determine if BMI, waist circumference or adipokines are associated with colon polyps in males, 126 asymptomatic men (48–65 yr) were recruited at time of colonoscopy, and anthropometric measures as well as blood were collected. Odds ratios were determined using polytomous logistic regression for polyp number (0 or ≥3) and polyp type (no polyp, hyperplastic polyp, tubular adenoma).

Results

41% of the men in our study were obese (BMI ≥30). The odds of an obese individual having ≥3 polyps was 6.5 (CI: 1.3–33.0) times greater than those of a lean (BMI<25) individual. Additionally, relative to lean individuals, obese individuals were 7.8 (CI: 2.0–30.8) times more likely to have a tubular adenoma than no polyp. As BMI category increased, participants were 2.9 (CI: 1.5–5.4) times more likely to have a tubular adenoma than no polyps. Serum leptin, IP-10 and TNF-α were significantly associated with tubular adenoma presence. Serum leptin and IP-10 were significantly associated with increased likelihood of ≥3 polyps, and TNF-α showed a trend (p = 0.09).

Conclusions

Obese men are more likely to have at least three polyps and adenomas. This cross-sectional study provides evidence that colonoscopy should be recommended for obese, white males.     

Signalling between the hindbrain and paraxial tissues dictates neural crest migration pathways.

Cranial neural crest cells are a pluripotent population of cells derived from the neural tube that migrate into the branchial arches to generate the distinctive bone, connective tissue and peripheral nervous system components characteristic of the vertebrate head. The highly conserved segmental organisation of the vertebrate hindbrain plays an important role in patterning the pathways of neural crest cell migration and in generating the distinct or separate streams of crest cells that form unique structures in each arch. We have used focal injections of DiI into the developing mouse hindbrain in combination with in vitro whole embryo culture to map the patterns of cranial neural crest cell migration into the developing branchial arches. Our results show that mouse hindbrain-derived neural crest cells migrate in three segregated streams adjacent to the even-numbered rhombomeres into the branchial arches, and each stream contains contributions of cells from three rhombomeres in a pattern very similar to that observed in the chick embryo. There are clear neural crest-free zones adjacent to r3 and r5. Furthermore, using grafting and lineage-tracing techniques in cultured mouse embryos to investigate the differential ability of odd and even-numbered segments to generate neural crest cells, we find that odd and even segments have an intrinsic ability to produce equivalent numbers of neural crest cells. This implies that inter-rhombomeric signalling is less important than combinatorial interactions between the hindbrain and the adjacent arch environment in specific regions, in the process of restricting the generation and migration of neural crest cells. This creates crest-free territories and suggests that tissue interactions established during development and patterning of the branchial arches may set up signals that the neural plate is primed to interpret during the progressive events leading to the delamination and migration of neural crest cells. Using interspecies grafting experiments between mouse and chick embryos, we have shown that this process forms part of a conserved mechanism for generating neural crest-free zones and contributing to the separation of migrating crest populations with distinct Hox expression during vertebrate head development.     

Sporulation and Enterotoxin Production by Mutants of Clostridium perfringens

The ability of Clostridium perfringens type A to produce an enterotoxin active in human food poisoning has been shown to be directly related to the ability of the organism to sporulate. Enterotoxin was produced only in a sporulation medium and not in a growth medium in which sporulation was repressed. Mutants with an altered ability to sporulate were isolated from an sp(+) ent(+) strain either as spontaneous mutants or after mutagenesis with acridine orange or nitrosoguanidine. All sp(0) (-) mutants were ent(-). Except for one isolate, these mutants were not disturbed in other toxic functions characteristic of the wild type and unrelated to sporulation. A total of four of seven osp(0) mutants retained the ability to produce detectable levels of enterotoxin. None of the ent(-) mutants produced gene products serologically homologous to enterotoxin. A total of three sp(-) mutants, blocked at intermediate stages of sporulation, produced enterotoxin. Of these mutants, one was blocked at stage III, one probably at late stage IV, and one probably at stage V. A total of three sp(+) revertants isolated from an sp(-) ent(-) mutant regained not only the ability to sporulate but also the ability to produce enterotoxin. The enterotoxin appears to be a sporulation-specific gene product; however, the function of the enterotoxin in sporulation is unknown.     

Isolation of Shigellae: V. Comparison of Enrichment Broths with Stools

Many enteric media are more efficient for the detection of salmonellae than of shigellae. Comparisons of three enrichment broths and three plating media were made during analysis of 1,405 stool specimens to choose a combination of media which would enhance detection of shigellae as well. Gram-Negative (GN), Selenite, and Silliker's Broths were streaked to E M B, Salmonella-Shigella (SS), and xylose lysine deoxycholate (XLD) Agars. The enrichment broths produced a twofold increase in isolations of both salmonellae and shigellae over direct streaking. All three broths performed equally well for Salmonella detection, but GN and Silliker's produced twice as many Shigella isolates as did Selenite. Comparison of the plating media showed that XLD was markedly more efficient than either E M B or SS Agar for the recovery of both genera. SS Agar was superior to E M B for isolation of salmonellae after enrichment, whereas E M B was better for isolation of shigellae by direct streaking. Both E M B and SS were more effective when used after GN and Silliker's than after Selenite. GN Broth and XLD Agar were the most efficient combination of media. During these analyses, 158 salmonellae and 49 shigellae isolates were obtained.     

The impact of interventions in the global land and agri‐food sectors on Nature’s Contributions to People and the UN Sustainable Development Goals

Interlocked challenges of climate change, biodiversity loss, and land degradation require transformative interventions in the land management and food production sectors to reduce carbon emissions, strengthen adaptive capacity, and increase food security. However, deciding which interventions to pursue and understanding their relative co‐benefits with and trade‐offs against different social and environmental goals have been difficult without comparisons across a range of possible actions. This study examined 40 different options, implemented through land management, value chains, or risk management, for their relative impacts across 18 Nature's Contributions to People (NCPs) and the 17 Sustainable Development Goals (SDGs). We find that a relatively small number of interventions show positive synergies with both SDGs and NCPs with no significant adverse trade‐offs; these include improved cropland management, improved grazing land management, improved livestock management, agroforestry, integrated water management, increased soil organic carbon content, reduced soil erosion, salinization, and compaction, fire management, reduced landslides and hazards, reduced pollution, reduced post‐harvest losses, improved energy use in food systems, and disaster risk management. Several interventions show potentially significant negative impacts on both SDGs and NCPs; these include bioenergy and bioenergy with carbon capture and storage, afforestation, and some risk sharing measures, like commercial crop insurance. Our results demonstrate that a better understanding of co‐benefits and trade‐offs of different policy approaches can help decision‐makers choose the more effective, or at the very minimum, more benign interventions for implementation.     

RADIOAUTOGRAPHIC LOCALIZATION OF THE INCREASED SYNTHESIS OF PHOSPHATIDYLINOSITOL IN RESPONSE TO PANCREOZYMIN OR ACETYLCHOLINE IN GUINEA PIG PANCREAS SLICES

A technique is described for measuring the incorporation of myo-inositol-2-³H into the lipid of various regions of the guinea pig pancreatic acinar cell by radioautography. Stimulation of enzyme secretion with either pancreozymin or acetylcholine was associated with increased graining in both the basophilic cytoplasm and the nonbasophilic cytoplasm. Kinetic studies suggested that the incorporation of myo-inositol-2-³H was stimulated independently in the two regions. Most of the increment in graining due to stimulation with pancreozymin or acetylcholine plus eserine was abolished if the tissue was extracted with 2:1 chloroform-methanol before radioautography. On chromatography of lipid extracts of pancreas, the only lipid showing a detectable increment in radioactivity on stimulation with pancreozymin was phosphatidylinositol. Thus, essentially all of the increment in graining is likely to be due to increased incorporation of tritium into phosphatidylinositol. These studies, coupled with earlier studies employing differential centrifugation, indicate that on stimulation of enzyme secretion there is increased synthesis of phosphatidylinositol in the rough-surfaced endoplasmic reticulum and in the smooth-surfaced Golgi membranes. The significance of these observations is discussed in connection with membrane circulation presumed to occur in the pancreatic acinar cell on stimulation of protein secretion. It is suggested that the increased synthesis of phosphatidylinositol may be concerned with the formation of new endoplasmic reticulum and possibly Golgi membrane to replace that which is presumably converted to membrane of the zymogen granules during intracellular protein transport.     

Endophytic and Biological Control Potential of Bacillus mojavensis and Related Species

The identity of a patented endophytic bacterium was established by 16S rRNA sequence analysis as a strain of Bacillus mojavensis, a recently erected species within one of the B. subtilis subgroups. This strain of B. mojavensis is antagonistic to the fungus Fusarium moniliforme, an endophytic mycotoxin-producing pathogen of maize and other plants. There are five other species within this subgroup: Bacillus amyloliquefaciens, B. atrophaeus, B. licheniformis, Brevibacterium halotolerans, Paenibacillus lentimorbus, and P. popilliae. The objectives of this research were to screen other isolates of B. mojavensis, B. subtilis, and the other closely related Bacillus species for endophytic colonizing capacity and to determine the in vitro antagonism to F. moniliforme in an effort to survey the distribution of these traits, which are desirable biological control qualities within the Bacillaceae. Antagonism was determined on nutrient agar, and endophytic colonization was established with maize plants following recovery of rifampin-resistant mutants generated from all strains used in the study. The study established that all 13 strains of B. mojavensis, isolated from major deserts of the world, endophytically colonized maize and were antagonists to F. moniliforme. The endophytic colonization of maize by B. subtilis and other species within this subgroup of the Bacillaceae varied, as did antagonism, to F. moniliforme. Thus, this study suggests that endophytic colonization is another characteristic of the species B. mojavensis. The endophytic habit and demonstrated antagonism to the test fungus indicate that isolates of this species might prove to be important biological control organisms where the endophytic habit is desired.