Identification of a single genome by electron paramagnetic resonance (EPR) with nitroxide-labeled oligonucleotide probes

Nitroxide-labeled nucleic acids are used as a molecular size sensor to identify as few as one genome under polymerase chain reaction (PCR) conditions by electron paramagnetic resonance (EPR) spectroscopy. DNA identification is based on differences in the EPR spectra of mono-nitroxide-labeled nucleic acids. The experimental data imply that rapid DNA identification can be achieved in many systems by EPR at the molecular level.     

Direct immobilization of gangliosides onto gold-carboxymethyldextran sensor surfaces by hydrophobic interaction: applications to antibody characterization

We describe a novel immobilization technique to investigate interactions between immobilized gangliosides (GD3, GM1, and GM2) and their respective antibodies, antibody fragments, or binding partners using an optical biosensor. Immobilization was performed by direct injection onto a carboxymethyldextran sensor chip and did not require derivatization of the sensor surface or the ganglioside. The ganglioside appeared to bind to the sensor surface by hydrophobic interaction, leaving the carbohydrate epitope available for antibody or, in the case of GM1, cholera toxin binding. The carboxyl group of the dextran chains on the sensor surface did not appear to be involved in the immobilization as evidenced by equivalent levels of immobilization following conversion of the carboxyl groups into acyl amino esters, but rather the dextran layer provided a hydrophilic coverage of the sensor chip which was essential to prevent nonspecific binding. This technique gave better reactivity and specificity for anti- ganglioside monoclonal antibodies (anti-GD3: KM871, KM641, R24; and anti-GM2: KM966) than immobilization by hydrophobic interaction onto a gold sensor surface or photoactivated cross-linking onto carboxymethydextran. This rapid immobilization procedure has facilitated detailed kinetic analysis of ganglioside/antibody interactions, with the surface remaining viable for a large number of cycles (>125). Kinetic constants were determined from the biosensor data using linear regression, nonlinear least squares and equilibrium analysis. The values of kd, ka, and KAobtained by nonlinear analysis (KAKM871 = 1.05, KM641 = 1.66, R24 = 0.14, and KM966 = 0.65 x 10(7) M- 1) were essentially independent of concentration and showed good agreement with data obtained by other analytical methods.      

Nucleic binding affinity of bacteriophage T4 gene 32 protein in the cooperative binding mode

This study reports on various parameters which affect the binding stoichiometry for complexes of bacteriophage T4 gene 32 protein (P32) and single stranded polynucleotides (determined by UV absorbance and fluorescence quenching) and presents results of a quantitative electron spin resonance assay to determine physiologically effective binding affinity differences of nucleic acid binding proteins. The assay employs macromolecular spin probes (spin-labeled nucleic acids) which are used to determine the fraction of saturation in competition experiments with unlabeled nucleic acids. It was found that the fraction of complexed spin-labeled polynucleotides can be directly monitored by ESR with a two-component analysis approach when ligands such as poly(L-lysine), gene 5 protein (P5) of filamentous bacteriophage fd, and gene 32 protein (P32) of bacteriophage T4 are used. The ESR data unequivocally show that: 1) the binding stoichiometry for poly(L-lysine), P5 and P32 is nucleotide/lysine, 4 nucleotides/P5 monomer, and 10 nucleotides/P32 monomer, respectively; and 2) under physiologically relevant buffer conditions the relative affinity of P32 in the cooperative binding mode for polythymidylic acid is about 4 times greater than for polydeoxyinosinic acid and about 12 times greater than for polyinosinic acid, and the relative affinity of P32 for polydeoxyinosinic acid is about 3 times greater than for polyinosinic acid.     

Influence of a natural and a synthetic inhibitor of factor XIIIa on fibrin clot rheology

We investigated the origins of greater clot rigidity associated with FXIIIa-dependent cross-linking. Fibrin clots were examined in which cross-linking was controlled through the use of two inhibitors: a highly specific active-center-directed synthetic inhibitor of FXIIIa, 1,3-dimethyl-4,5-diphenyl-2[2(oxopropyl)thio]imidazolium trifluoromethylsulfonate, and a patient-derived immunoglobulin directed mainly against the thrombin-activated catalytic A subunits of thrombin-activated FXIII. Cross-linked fibrin chains were identified and quantified by one- and two-dimensional gel electrophoresis and immunostaining with antibodies specific for the alpha- and gamma-chains of fibrin. Gamma-dimers, gamma-multimers, alpha(n)-polymers, and alpha(p)gamma(q)-hybrids were detected. The synthetic inhibitor was highly effective in preventing the production of all cross-linked species. In contrast, the autoimmune antibody of the patient caused primarily an inhibition of alpha-chain cross-linking. Clot rigidities (storage moduli, G') were measured with a cone and plate rheometer and correlated with the distributions of the various cross-linked species found in the clots. Our findings indicate that the FXIIIa-induced dimeric cross-linking of gamma-chains by itself is not sufficient to stiffen the fibrin networks. Instead, the augmentation of clot rigidity was more strongly correlated with the formation of gamma-multimers, alpha(n)-polymers, and alpha(p)gamma(q)-hybrid cross-links. A mechanism is proposed to explain how these cross-linked species may enhance clot rigidity.     

Food environment and diabetes mellitus in South Asia: A geospatial analysis of health outcome data

BackgroundThe global epidemic of type 2 diabetes mellitus (T2DM) renders its prevention a major public health priority. A key risk factor of diabetes is obesity and poor diets. Food environments have been found to influence people’s diets and obesity, positing they may play a role in the prevalence of diabetes. Yet, there is scant evidence on the role they may play in the context of low- and middle-income countries (LMICs). We examined the associations of food environments on T2DM among adults and its heterogeneity by income and sex.Methods and findingsWe linked individual health outcome data of 12,167 individuals from a network of health surveillance sites (the South Asia Biobank) to the density and proximity of food outlets geolocated around their homes from environment mapping survey data collected between 2018 and 2020 in Bangladesh and Sri Lanka. Density was defined as share of food outlets within 300 m from study participant’s home, and proximity was defined as having at least 1 outlet within 100 m from home. The outcome variables include fasting blood glucose level, high blood glucose, and self-reported diagnosed diabetes. Control variables included demographics, socioeconomic status (SES), health status, healthcare utilization, and physical activities. Data were analyzed in ArcMap 10.3 and STATA 15.1. A higher share of fast-food restaurants (FFR) was associated with a 9.21 mg/dl blood glucose increase (95% CI: 0.17, 18.24; p < 0.05). Having at least 1 FFR in the proximity was associated with 2.14 mg/dl blood glucose increase (CI: 0.55, 3.72; p < 0.01). A 1% increase in the share of FFR near an individual’s home was associated with 8% increase in the probability of being clinically diagnosed as a diabetic (average marginal effects (AMEs): 0.08; CI: 0.02, 0.14; p < 0.05). Having at least 1 FFR near home was associated with 16% (odds ratio [OR]: 1.16; CI: 1.01, 1.33; p < 0.05) and 19% (OR: 1.19; CI: 1.03, 1.38; p < 0.05) increases in the odds of higher blood glucose levels and diagnosed diabetes, respectively. The positive association between FFR density and blood glucose level was stronger among women than men, but the association between FFR proximity and blood glucose level was stronger among men as well as among those with higher incomes. One of the study’s key limitations is that we measured exposure to food environments around residency geolocation; however, participants may source their meals elsewhere.ConclusionsOur results suggest that the exposure to fast-food outlets may have a detrimental impact on the risk of T2DM, especially among females and higher-income earners. Policies should target changes in the food environments to promote better diets and prevent T2DM.

Dian Kusuma and colleagues investigate the associations between exposure to the density and proximity of healthy and unhealthy food outlets and diabetes in Bangladesh and Sri Lanka.     

Marsupials and monotremes sort genome treasures from junk

A recent landmark paper demonstrates the unique contribution of marsupials and monotremes to comparative genome analysis, filling an evolutionary gap between the eutherian mammals (including humans) and more distant vertebrate species.     

Spectroscopic and functional properties of novel 2[4Fe-4S] cluster-containing ferredoxins from the green sulfur bacterium Chlorobium tepidum

Two distinct ferredoxins, Fd I and Fd II, were isolated and purified to homogeneity from photoautotrophically grown Chlorobium tepidum, a moderately thermophilic green sulfur bacterium that assimilates carbon dioxide by the reductive tricarboxylic acid cycle. Both ferredoxins serve a crucial role as electron donors for reductive carboxylation, catalyzed by a key enzyme of this pathway, pyruvate synthase/pyruvate ferredoxin oxidoreductase. The reduction potentials of Fd I and Fd II were determined by cyclic voltammetry to be -514 and -584 mV, respectively, which are more electronegative than any previously studied Fds in which two [4Fe-4S] clusters display a single transition. Further spectroscopic studies indicated that the CD spectrum of oxidized Fd I closely resembled that of Fd II; however, both spectra appeared to be unique relative to ferredoxins studied previously. Double integration of the EPR signal of the two Fds yielded approximately approximately 2.0 spins per molecule, compatible with the idea that C. tepidum Fd I and Fd II accept 2 electrons upon reduction. These results suggest that the C. tepidum Fd I and Fd II polypeptides each contain two bound [4Fe-4S] clusters. C. tepidum Fd I and Fd II are novel 2[4Fe-4S] Fds, which were shown previously to function as biological electron donors or acceptors for C. tepidum pyruvate synthase/pyruvate ferredoxin oxidoreductase (Yoon, K.-S., Hille, R., Hemann, C. F., and Tabita, F. R. (1999) J. Biol. Chem. 274, 29772-29778). Kinetic measurements indicated that Fd I had approximately 2.3-fold higher affinity than Fd II. The results of amino acid sequence alignments, molecular modeling, oxidation-reduction potentials, and spectral properties strongly indicate that the C. tepidum Fds are chimeras of both clostridial-type and chromatium-type Fds, suggesting that the two Fds are likely intermediates in the evolutional development of 2[4Fe-4S] clusters compared with the well described clostridial and chromatium types.     

Mapping the Naked Neck (NA) and Polydactyly (PO) mutants of the chicken with microsatellite molecular markers

The bulked segregant analysis methodology has been used to map, with microsatellite markers, two morphological mutations in the chicken: polydactyly (PO) and naked neck (NA). These autosomal mutations show partial dominance for NA, and dominance with incomplete penetrance for PO. They were mapped previously to different linkage groups of the classical map, PO to the linkage group IV and NA being linked to the erythrocyte antigen CPPP. An informative family of 70 offspring was produced by mating a sire, heterozygous for each of the mutations, to 7 dams homozygous recessive for each locus. Three DNA pools were prepared, pool PO included 20 chicks exhibiting at least one extra-toe, pool NA included 20 non-polydactyly chicks showing the typical phenotype associated with heterozygosity for the naked neck mutation, and pool NP included 20 chicks exhibiting neither of the mutant phenotypes. Typings were done on an ABI-373 automatic sequencer with 147 microsatellite markers covering most of the genome. An unbalanced distribution of sire marker alleles were detected between pool PO, and pools NA and NP, for two markers of chromosome 2p, MCW0082 and MCW0247. A linkage analysis taking into account the incomplete penetrance of polydactyly (80%) was performed with additional markers of this region and showed that the closest marker to the PO locus was MCW0071 (5 cM, lod score = 9). MCW0071 lies within the engrailed gene EN2 in the chicken. In the mouse, the homologous gene maps on chromosome 5, close to the hemimelic extra-toes mutation Hx. In the case of the NA locus, markers of chromosome 3 were selected because CPPP was mapped on this chromosome. Analysis of individual typings showed a linkage of 5.7 cM (lod score = 13) between the NA locus and ADL0237 in the distal region of chromosome 3q. These results contribute to connecting the former classical map to the molecular genetic map of the chicken, and open the way to the identification of the molecular nature of two developmental mutations of the chicken that are known to occur in many breeds of chickens.     

Genome sequencing reveals fine scale diversification and reticulation history during speciation in Sus

Background

Elucidating the process of speciation requires an in-depth understanding of the evolutionary history of the species in question. Studies that rely upon a limited number of genetic loci do not always reveal actual evolutionary history, and often confuse inferences related to phylogeny and speciation. Whole-genome data, however, can overcome this issue by providing a nearly unbiased window into the patterns and processes of speciation. In order to reveal the complexity of the speciation process, we sequenced and analyzed the genomes of 10 wild pigs, representing morphologically or geographically well-defined species and subspecies of the genus Sus from insular and mainland Southeast Asia, and one African common warthog.

Results

Our data highlight the importance of past cyclical climatic fluctuations in facilitating the dispersal and isolation of populations, thus leading to the diversification of suids in one of the most species-rich regions of the world. Moreover, admixture analyses revealed extensive, intra- and inter-specific gene-flow that explains previous conflicting results obtained from a limited number of loci. We show that these multiple episodes of gene-flow resulted from both natural and human-mediated dispersal.

Conclusions

Our results demonstrate the importance of past climatic fluctuations and human mediated translocations in driving and complicating the process of speciation in island Southeast Asia. This case study demonstrates that genomics is a powerful tool to decipher the evolutionary history of a genus, and reveals the complexity of the process of speciation.