Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30?kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.     

Predation drives stable coexistence ratios between red and green pea aphid morphs

We conducted field surveys and experiments to evaluate the hypothesis that predation is an important driving factor determining the degree of coexistence between red and green morphs of the pea aphid Acyrthosiphon pisum. Theory suggests that the different colour morphs are differentially susceptible to natural enemies and selection by predation which in turn leads to variable relative abundances of red and green morphs among host plants across landscapes. Our field surveys on pea and alfalfa revealed, however, that the colour morphs tended to coexist closely in a ratio of one red to three green aphids across fields with different host plant monocultures. Experimentation involving manipulation of the relative abundances of the two colour morphs on host plants pea and alfalfa with and without predator presence revealed that red morphs had higher or same fitness (per capita reproduction) than green morphs on both pea and alfalfa only when in the proportion of one red/three green proportion. Moreover, experimentation evaluating predator efficiency revealed that red morphs are safest from predation when in a 1 : 3 ratio with green morphs. These results suggest that in addition to predation selection effects, red morphs may behaviourally choose to associate with green morphs in a narrow 1 : 3 ratio to maximize their fitness. This evidence, along with existing published data on red and green morph anti‐predator behaviour indicates that a 1 : 3 red and green morph coexistence ratio is driven by a balance between predation pressure and behavioural assorting by red morphs across landscapes. In this way predators may have ecological‐evolutionary consequences for traits that affect the colour morphs' proportion and tolerances to selective pressure.     

Investigations on Aberrant Glycosylation of Glycosphingolipids in Colorectal Cancer Tissues Using Liquid Chromatography and Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF-MS)

Cancer is a leading cause of death and alterations of glycosylation are characteristic features of malignant cells. Colorectal cancer is one of the most common cancers and its exact causes and biology are not yet well understood. Here, we compared glycosylation profiles of colorectal tumor tissues and corresponding control tissues of 13 colorectal cancer patients to contribute to the understanding of this cancer. Using MALDI-TOF(/TOF)-MS and 2-dimensional LC-MS/MS we characterized enzymatically released and 2-aminobenzoic acid labeled glycans from glycosphingolipids. Multivariate data analysis revealed significant differences between tumor and corresponding control tissues. Main discriminators were obtained, which represent the overall alteration in glycosylation of glycosphingolipids during colorectal cancer progression, and these were found to be characterized by (1) increased fucosylation, (2) decreased acetylation, (3) decreased sulfation, (4) reduced expression of globo-type glycans, as well as (5) disialyl gangliosides. The findings of our current research confirm former reports, and in addition expand the knowledge of glycosphingolipid glycosylation in colorectal cancer by revealing new glycans with discriminative power and characteristic, cancer-associated glycosylation alterations. The obtained discriminating glycans can contribute to progress the discovery of biomarkers to improve diagnostics and patient treatment.Worldwide, cancer is a leading cause of death. With estimated 1.2 million diagnoses in 2008, colorectal cancer is the third most common cancer in the world and the fourth most common cause of death with an annual mortality of ∼600 000 (1). The exact causes of colorectal cancer are unknown, but different risk factors such as age, polyps, personal and family history, ulcerative colitis, or Crohn''s colitis have been proposed (2). Standard screening procedures include flexible sigmoidoscopy, colonoscopy, and immunological fecal occult blood testing. Each of them has its advantages and drawbacks such as invasiveness or low sensitivity and specificity (3). The method of choice for the treatment of colorectal cancer is surgery and therapeutic decisions are based on the tumor, lymph node, and metastasis staging-system as a prognostic factor (4). Current research has led to improved treatment strategies of colorectal cancer, however, the clinical outcome, the progression of the disease, and the response to the treatment remain variable among individuals. The heterogeneity of colorectal cancer at the molecular level—caused by accumulation of multiple genetic changes—may be one of the main reasons for this variability (5). Genetic factors such as instabilities, but also expression levels (6) can explain part of the cancer biology, but glycomics is gaining importance to complement the overall picture as aberrant glycosylation of proteins and lipids has been shown to be correlated with disease and malignancy (7, 8).Glycosylation is involved in many biological processes and especially its functional role in cellular interaction with respect to adhesion, cell growth, and signaling is prone to be affected in cancer progression, invasion, and metastasis (9). Several cancer-associated alterations in protein glycosylation have been reported: (1) increased branching of N-glycans, (2) higher density of O-glycans, and (3) incomplete synthesis of glycans. More particularly, an increased or induced expression of GlcNAc transferase V resulting in N-glycan structures with β1–6GlcNAc antennae (5, 10), and the expression of (sialyl) Tn-antigens (11) as aberrant O-glycosylation have been reported (10).Altered glycosphingolipid (GSL)¹ glycosylation of the cell surface membrane during malignancy can affect cell recognition, adhesion, and signal transduction (12) and is found to reflect: (1) incomplete synthesis with or without precursor accumulation, (2) neosynthesis (9), (3) increased sialylation, and (4) increased fucosylation (13). In many cancers, including colorectal cancer, an overexpression of the (sialyl) Lewis X antigen (10, 14) and the expression of (sialyl) Lewis A (15) are considered to be related to malignant transformation—reflecting incomplete synthesis of sialyl 6-sulfo Lewis X and disialyl Lewis A (16) as well as neosynthesis (17). Studies on gangliosides showed an overexpression of these sialylated GSLs in human malignant melanoma (18). Furthermore, the involvement of gangliosides in cell adhesion and motility was reported, which contributes to tumor metastasis (19). Specifically, the gangliosides GD3 (Hex2NeuAc2ceramide) and GM2 (Hex2HexNAc1NeuAc1ceramide) have been found to be associated with tumor-angiogenesis (19). The up-regulation of fucosyltransferases in cancer was shown to cause a higher degree of fucosylation in malignant tissues (20) and Moriwaki et al. proposed that the increase in the fucosylation for GSLs was an early event in cancer (21). Misonou et al. investigated glycans derived from GSLs in colorectal cancer tissues showing aberrant glycan structures based on linkage differences as well as increased sialylation and fucosylation compared with control tissue (22), which is in line with observed changes in GSL glycosylation with regard to cancer progression (9, 13).Recently, we investigated the N-glycosylation profiles of colorectal tumors and correlating control tissues for biomarker discovery. Statistical analyses revealed an increase of sulfated glycan structures as well as paucimannosidic glycans and glycans containing sialylated Lewis type epitopes in the tumor tissue, whereas structures with bisecting GlcNAc were found to be decreased in malignancy (23). To further progress the understanding of colorectal cancer biology and the improvement of diagnostic tools and patient treatment, we complemented this recent study on N-glycosylation by an investigation of the glycosphingolipid-derived glycans (named GSL-glycans in the following) from frozen tumor tissues and corresponding control tissues from the same 13 colorectal cancer patients. GSL-glycans were enzymatically released, labeled with 2-aminobenzoic acid (AA) and analyzed by hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection as well as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Employing multivariate statistical analysis, this approach revealed an intricate GSL-glycosylation pattern of tumor tissues and specific glycosylation differences in comparison to the corresponding control tissue.     

The FSHD2 Gene SMCHD1 Is a Modifier of Disease Severity in Families Affected by FSHD1

Facioscapulohumeral muscular dystrophy type 1 (FSHD1) is caused by contraction of the D4Z4 repeat array on chromosome 4 to a size of 1–10 units. The residual number of D4Z4 units inversely correlates with clinical severity, but significant clinical variability exists. Each unit contains a copy of the DUX4 retrogene. Repeat contractions are associated with changes in D4Z4 chromatin structure that increase the likelihood of DUX4 expression in skeletal muscle, but only when the repeat resides in a genetic background that contains a DUX4 polyadenylation signal. Mutations in the structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) gene, encoding a chromatin modifier of D4Z4, also result in the increased likelihood of DUX4 expression in individuals with a rare form of FSHD (FSHD2). Because SMCHD1 directly binds to D4Z4 and suppresses somatic expression of DUX4, we hypothesized that SMCHD1 may act as a genetic modifier in FSHD1. We describe three unrelated individuals with FSHD1 presenting an unusual high clinical severity based on their upper-sized FSHD1 repeat array of nine units. Each of these individuals also carries a mutation in the SMCHD1 gene. Familial carriers of the FSHD1 allele without the SMCHD1 mutation were only mildly affected, suggesting a modifier effect of the SMCHD1 mutation. Knocking down SMCHD1 in FSHD1 myotubes increased DUX4 expression, lending molecular support to a modifier role for SMCHD1 in FSHD1. We conclude that FSHD1 and FSHD2 share a common pathophysiological pathway in which the FSHD2 gene can act as modifier for disease severity in families affected by FSHD1.     

Atomistic details of effect of disulfide bond reduction on active site of Phytase B from Aspergillus niger: A MD Study

The molecular integrity of the active site of phytases from fungi is critical for maintaining phytase function as efficient catalytic machines. In this study, the molecular dynamics (MD) of two monomers of phytase B from Aspergillus niger, the disulfide intact monomer (NAP) and a monomer with broken disulfide bonds (RAP), were simulated to explore the conformational basis of the loss of catalytic activity when disulfide bonds are broken. The simulations indicated that the overall secondary and tertiary structures of the two monomers were nearly identical but differed in some crucial secondary–structural elements in the vicinity of the disulfide bonds and catalytic site. Disulfide bonds stabilize the β-sheet that contains residue Arg66 of the active site and destabilize the α-helix that contains the catalytic residue Asp319. This stabilization and destabilization lead to changes in the shape of the active–site pocket. Functionally important hydrogen bonds and atomic fluctuations in the catalytic pocket change during the RAP simulation. None of the disulfide bonds are in or near the catalytic pocket but are most likely essential for maintaining the native conformation of the catalytic site.

Abbreviations

PhyB - 2.5 pH acid phophatese from Aspergillus niger, NAP - disulphide intact monomer of Phytase B, RAP - disulphide reduced monomer of Phytase B, Rg - radius of gyration, RMSD - root mean square deviation, MD - molecular dynamics.     

Predation Determines Different Selective Pressure on Pea Aphid Host Races in a Complex Agricultural Mosaic

Field assessments were conducted to examine the interplay between host plant and predation in complex agricultural mosaic on pea aphid clover and alfalfa races. In one experiment, we examined the relative fitness on clover race (CR) and alfalfa race (AR) pea aphids on broad bean, red clover and alfalfa alone. But because clover is typically grown in a more complex agricultural mosaic with alfalfa and broad bean, a second experiment was conducted to assess the fitness consequences under predation in a more complex agricultural field setting that also included potential apparent competition with AR pea aphids. In a third experiment we tested for the effect of differential host race density on the fitness of the other host race mediated by a predator effect. CR pea aphids always had fitness losses when on broad bean (had lower fitness on broad bean relative to red clover) and fitness benefits when on red clover (higher fitness on red clover relative to broad bean), whether or not in apparent competition with alfalfa race aphids on bean and alfalfa. AR suffered fitness loss on both alfalfa and bean in apparent competition with CR on clover. Therefore we can conclude that the predation rate between host races was highly asymmetrical. The complexity of the agricultural mosaic thus can influence prey selection by predators on different host plants. These may have evolutionary consequences through context dependent fitness benefits on particular host plants.     

Effect of weight loss and exercise on angiogenic factors in the circulation and in adipose tissue in obese subjects

Background:

Vascular growth is a prerequisite for adipose tissue (AT) development and expansion. Some AT cytokines and hormones have effects on vascular development, like vascular endothelial growth factor (VEGF‐A), angiopoietin (ANG‐1), ANG‐2 and angiopoietin‐like protein‐4 (ANGPTL‐4).

Methods:

In this study, the independent and combined effects of diet‐induced weight loss and exercise on AT gene expression and proteins levels of those angiogenic factors were investigated. Seventy‐nine obese males and females were randomized to: 1. Exercise‐only (EXO; 12‐weeks exercise without diet‐restriction), 2. Hypocaloric diet (DIO; 8‐weeks very low energy diet (VLED) + 4‐weeks weight maintenance diet) and 3. Hypocaloric diet and exercise (DEX; 8‐weeks VLED + 4‐weeks weight maintenance diet combined with exercise throughout the 12 weeks). Blood samples and fat biopsies were taken before and after the intervention.

Results:

Weight loss was 3.5 kg in the EXO group and 12.3 kg in the DIO and DEX groups. VEGF‐A protein was non‐significantly reduced in the weight loss groups. ANG‐1 protein levels were significantly reduced 22‐25% after all three interventions (P < 0.01). The ANG‐1/ANG‐2 ratio was also decreased in all three groups (P < 0.05) by 27‐38%. ANGPTL‐4 was increased in the EXO group (15%, P < 0.05) and 9% (P < 0.05) in the DIO group. VEGF‐A, ANG‐1, and ANGPTL‐4 were all expressed in human AT, but only ANGPTL‐4 was influenced by the interventions.

Conclusions:

Our data show that serum VEGF‐A, ANG‐1, ANG‐2, and ANGPTL‐4 levels are influenced by weight changes, indicating the involvement of these factors in the obese state. Moreover, it was found that weight loss generally was associated with a reduced angiogenic activity in the circulation.