Paternal levels of DNA damage in spermatozoa and maternal parity influence offspring mortality in an endangered ungulate

Understanding which factors influence offspring mortality rates is a major challenge since it influences population dynamics and may constrain the chances of recovery among endangered species. Most studies have focused on the effects of maternal and environmental factors, but little is known about paternal factors. Among most polygynous mammals, males only contribute the haploid genome to their offspring, but the possibility that sperm DNA integrity may influence offspring survival has not been explored. We examined several maternal, paternal and individual factors that may influence offspring survival in an endangered species (Gazella cuvieri). Levels of sperm DNA damage had the largest impact upon offspring mortality rates, followed by maternal parity. In addition, there was a significant interaction between these two variables, so that offspring born to primiparous mothers were more likely to die if their father had high levels of sperm DNA damage, but this was not the case among multiparous mothers. Thus, multiparous mothers seem to protect their offspring from the deleterious effects of sperm DNA damage. Since levels of sperm DNA damage seem to be higher among endangered species, more attention should be paid to the impact of this largely ignored factor among the viability of endangered species.     

Apoptotic cells activate NKT cells through T cell Ig-like mucin-like-1 resulting in airway hyperreactivity

T cell Ig-like mucin-like-1 (TIM-1) is an important asthma susceptibility gene, but the immunological mechanisms by which TIM-1 functions remain uncertain. TIM-1 is also a receptor for phosphatidylserine (PtdSer), an important marker of cells undergoing programmed cell death, or apoptosis. We now demonstrate that NKT cells constitutively express TIM-1 and become activated by apoptotic cells expressing PtdSer. TIM-1 recognition of PtdSer induced NKT cell activation, proliferation, and cytokine production. Moreover, the induction of apoptosis in airway epithelial cells activated pulmonary NKT cells and unexpectedly resulted in airway hyperreactivity, a cardinal feature of asthma, in an NKT cell-dependent and TIM-1-dependent fashion. These results suggest that TIM-1 serves as a pattern recognition receptor on NKT cells that senses PtdSer on apoptotic cells as a damage-associated molecular pattern. Furthermore, these results provide evidence for a novel innate pathway that results in airway hyperreactivity and may help to explain how TIM-1 and NKT cells regulate asthma.     

Phylogenetic analysis of nuclear and mitochondrial DNA reveals a complex of cryptic species in Crassicutis cichlasomae (Digenea: Apocreadiidae), a parasite of Middle-American cichlids

We obtained nuclear ITS-1 and mitochondrial cox1 sequences from 225 Crassicutis cichlasomae adults collected in 12 species of cichlids from 32 localities to prospect for the presence of cryptic species. This trematode is commonly found in species of cichlids over a wide geographic range in Middle-America. Population-level phylogenetic analyses of ITS-1 and cox1, assessments of genetic and haplotype diversity, and morphological observations revealed that C. cichlasomae represents a complex of seven cryptic species for which no morphological diagnostic characters have been discovered thus far. Bayesian and Maximum Likelihood analyses of concatenated datasets (906 bp) recovered eight lineages of C. cichlasomae, all with high posterior probabilities and bootstrap branch support. Values of genetic divergence between clades ranged from 1.0% to 5.2% for ITS-1, and from 7.2% to 30.0% for cox1. Morphological study of more than 300 individuals did not reveal structural diagnostic traits for the species defined using molecular evidence. These observations indicate that some traditional morphological characters (e.g., testes position) have substantial intra-specific variation, and should be used with caution when classifying C. cichlasomae and their sister taxa. Additionally, phylogenetic analyses did not reveal a strict correlation between these cryptic species and their host species or geographic distribution, however it appears that genetic distinctiveness of these cryptic species was influenced by the diversification and biogeographical history of Middle-American cichlids.     

Site Mapping and Characterization of O-Glycan Structures on α-Dystroglycan Isolated from Rabbit Skeletal Muscle

The main extracellular matrix binding component of the dystrophin-glycoprotein complex, α-dystroglycan (α-DG), which was originally isolated from rabbit skeletal muscle, is an extensively O-glycosylated protein. Previous studies have shown α-DG to be modified by both O-GalNAc- and O-mannose-initiated glycan structures. O-Mannosylation, which accounts for up to 30% of the reported O-linked structures in certain tissues, has been rarely observed on mammalian proteins. Mutations in multiple genes encoding defined or putative glycosyltransferases involved in O-mannosylation are causal for various forms of congenital muscular dystrophy. Here, we explore the glycosylation of purified rabbit skeletal muscle α-DG in detail. Using tandem mass spectrometry approaches, we identify 4 O-mannose-initiated and 17 O-GalNAc-initiated structures on α-DG isolated from rabbit skeletal muscle. Additionally, we demonstrate the use of tandem mass spectrometry-based workflows to directly analyze glycopeptides generated from the purified protein. By combining glycomics and tandem mass spectrometry analysis of 91 glycopeptides from α-DG, we were able to assign 21 different residues as being modified by O-glycosylation with differing degrees of microheterogeneity; 9 sites of O-mannosylation and 14 sites of O-GalNAcylation were observed with only two sites definitively exhibiting occupancy by either type of glycan. The distribution of identified sites of O-mannosylation suggests a limited role for local primary sequence in dictating sites of attachment.     

Assessment of oxidative damage induced by acute doses of morphine sulfate in postnatal and adult rat brain

The aim of the present study is to evaluate the oxidative damage in rats of different ages. Weaned rats of 25 g and adults of 300 g were used in groups of 6, a single i.p. dose of morphine sulfate of 3, 6 or 12 mg/kg was administered. All animals were sacrificed to measure GSH and 5-HT levels in brain by liquid chromatography, as well as Na⁺, K⁺-ATPase and total ATPase enzymatic activity. 5-HT levels decreased significantly (p<0.05) in adult animals that received 3 and 6 mg morphine. Na⁺, K⁺-ATPase activity increased significantly (p<0.05) in all groups of weaned animals. In adult animals, Na⁺, K⁺-ATPase and total ATPase partially diminished. GSH levels diminished significantly (p<0.05) both in weaned and in adult groups. The results indicate age-induced changes in cellular regulation and biochemical responses to oxidative stress induced by morphine.     

Immunologic memory response induced by a meningococcal serogroup C conjugate vaccine using the P64k recombinant protein as carrier

In this study, we used an adoptive lymphocyte transfer experiment to evaluate the ability of the P64k recombinant protein to recruit T-helper activity and induce immunologic memory response to the polysaccharide moiety in a meningococcal serogroup C conjugate vaccine. Adoptive transfer of splenocytes from mice immunized with the glycoconjugate conferred antipolysaccharide immunologic memory to naive recipient mice. The observed anamnestic immune response was characterized by more rapid kinetics, isotype switching from IgM to IgG and higher antipolysaccharide antibody titers compared with those reached in groups transferred with splenocytes from plain polysaccharide or phosphate-immunized mice. The memory response generated was also long lasting. Sera from mice transferred with cells from conjugate-immunized mice were the only protective in the infant rat passive protection assay, and also showed higher bactericidal titers. We demonstrated that priming the mice immune system with the glycoconjugate using the P64k protein as carrier induced a memory response to the polysaccharide, promoting a switch of the T-cell-independent response to a T-cell dependent one.     

Comparison of three ELISA protocols to measure antibody responses elicited against serogroup C meningococcal polysaccharide in mouse, monkey and human sera

In this study we compared the following ELISA protocols to measure antibody levels against serogroup C meningococcal polysaccharide: a traditional protocol using poly-L-Lysine mixed with the polysaccharide as coating antigen, a second protocol coating with a mixture of methylated human serum albumin with the C polysaccharide, a modified protocol coating with derivatized polysaccharide and a modification to the last one, specifically without adding ammonium thiocyanate to the sample buffer. Serum bactericidal activity of mouse, monkey and human sera were measured and correlation coefficients were calculated. For all serum types the modified ELISA protocol showed the highest correlation coefficients while the traditional protocol showed the lower ones. We demonstrated that the traditional protocol measures non-specific antibodies to the C polysaccharide, because no differences were detected between pre-immune and post-immune human sera (P>0.05), while the modified protocol detected the highest difference (P<0.01).     

Bioconversion of agrowastes by Lentinula edodes: the high potential of viticulture residues

The production of four strains of edible mushroom Lentinula edodes was evaluated through solid-state fermentation (SSF) of vineyard pruning (VP), barley straw (BS), and wheat straw (WS). Biological efficiency, proximal composition, and energy value of the fruiting bodies, as well as substrate chemical changes after harvest, were determined. The shortest primordium formation time (28 days), highest biological efficiency (93.25%), highest yield (37.46%), and shortest production cycle (6 days) were observed in VP. The fruiting bodies obtained from VP had high energy value (379.09 to 392.95 kcal) and contents of protein (12.37 to 17.19%), but low contents of fat (1.82 to 2.15%). After SSF, phenol concentration decreased on VP (1.2 mmol/L) and BS (0.31 mmol/L), but on WS remained practically the same. Hemicellulose decreased in all substrates; cellulose increased on WS and decreased in the rest of the treatments. Lignin decreased on WS and BS, but its concentration increased on VP. The variability observed in the degradation capacity of lignocellulosic components was influenced by the substrate's nature, environmental factors, and genetic factors among strains. VP has great potential for shiitake production due to its low cost, short production cycles, and high biological efficiency.Research was conducted at Instituto de Ecología, AC, and Centro de Investigación en Alimentación y Desarrollo, AC     

BTA, a novel reagent for DNA attachment on glass and efficient generation of solid-phase amplified DNA colonies

The tricarboxylate reagent benzene-1,3,5-triacetic acid (BTA) was used to attach 5′-aminated DNA primers and templates on an aminosilanized glass surface for subsequent generation of DNA colonies by in situ solid-phase amplification. We have characterized the derivatized surfaces for the chemical attachment of oligonucleotides and evaluate the properties relevant for the amplification process: surface density, thermal stability towards thermocycling, functionalization reproducibility and storage stability. The derivatization process, first developed for glass slides, was then adapted to microfabricated glass channels containing integrated fluidic connections. This implementation resulted in an important reduction of reaction times, consumption of reagents and process automation. Innovative analytical methods for the characterization of attached DNA were developed for assessing the surface immobilized DNA content after amplification. The results obtained showed that the BTA chemistry is compatible and suitable for forming highly dense arrays of DNA colonies with optimal surface coverage of about 10 million colonies/cm² from the amplification of initial single-template DNA molecules immobilized. We also demonstrate that the dsDNA colonies generated can be quantitatively processed in situ by restriction enzymes digestion. DNA colonies generated using the BTA reagent can be used for further sequence analysis in an unprecedented parallel fashion for low-cost genomic studies.