Genome Wide Analysis of Fertility and Production Traits in Italian Holstein Cattle

A genome wide scan was performed on a total of 2093 Italian Holstein proven bulls genotyped with 50K single nucleotide polymorphisms (SNPs), with the objective of identifying loci associated with fertility related traits and to test their effects on milk production traits. The analysis was carried out using estimated breeding values for the aggregate fertility index and for each trait contributing to the index: angularity, calving interval, non-return rate at 56 days, days to first service, and 305 day first parity lactation. In addition, two production traits not included in the aggregate fertility index were analysed: fat yield and protein yield. Analyses were carried out using all SNPs treated separately, further the most significant marker on BTA14 associated to milk quality located in the DGAT1 region was treated as fixed effect. Genome wide association analysis identified 61 significant SNPs and 75 significant marker-trait associations. Eight additional SNP associations were detected when SNP located near DGAT1 was included as a fixed effect. As there were no obvious common SNPs between the traits analyzed independently in this study, a network analysis was carried out to identify unforeseen relationships that may link production and fertility traits.     

Functional and Spectroscopic Characterization of Chlamydomonas reinhardtii Truncated Hemoglobins

The single-cell green alga Chlamydomonas reinhardtii harbors twelve truncated hemoglobins (Cr-TrHbs). Cr-TrHb1-1 and Cr-TrHb1-8 have been postulated to be parts of the nitrogen assimilation pathway, and of a NO-dependent signaling pathway, respectively. Here, spectroscopic and reactivity properties of Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4, all belonging to clsss 1 (previously known as group N or group I), are reported. The ferric form of Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 displays a stable 6cLS heme-Fe atom, whereas the hexa-coordination of the ferrous derivative appears less strongly stabilized. Accordingly, kinetics of azide binding to ferric Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are independent of the ligand concentration. Conversely, kinetics of CO or NO₂⁻ binding to ferrous Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are ligand-dependent at low CO or NO₂⁻ concentrations, tending to level off at high ligand concentrations, suggesting the presence of a rate-limiting step. In agreement with the different heme-Fe environments, the pH-dependent kinetics for CO and NO₂−binding to ferrous Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are characterized by different ligand-linked protonation events. This raises the question of whether the simultaneous presence in C. reinhardtii of multiple TrHb1s may be related to different regulatory roles.     

Climate warming could increase recruitment success in glacier foreland plants

Background and Aims Glacier foreland plants are highly threatened by global warming. Regeneration from seeds on deglaciated terrain will be crucial for successful migration and survival of these species, and hence a better understanding of the impacts of climate change on seedling recruitment is urgently needed to predict future plant persistence in these environments. This study presents the first field evidence of the impact of climate change on recruitment success of glacier foreland plants.Methods Seeds of eight foreland species were sown on a foreland site at 2500 m a.s.l., and at a site 400 m lower in altitude to simulate a 2·7 °C increase in mean annual temperature. Soil from the site of origin was used to reproduce the natural germination substrate. Recruitment success, temperature and water potential were monitored for 2 years. The response of seed germination to warming was further investigated in the laboratory.Key Results At the glacier foreland site, seedling emergence was low (0 to approx. 40 %) and occurred in summer in all species after seeds had experienced autumn and winter seasons. However, at the warmer site there was a shift from summer to autumn emergence in two species and a significant increase of summer emergence (13–35 % higher) in all species except two. Survival and establishment was possible for 60–75 % of autumn-emerged seedlings and was generally greater under warmer conditions. Early snowmelt in spring caused the main ecological factors enhancing the recruitment success.Conclusions The results suggest that warming will influence the recruitment of glacier foreland species primarily via the extension of the snow-free period in spring, which increases seedling establishment and results in a greater resistance to summer drought and winter extremes. The changes in recruitment success observed here imply that range shifts or changes in abundance are possible in a future warmer climate, but overall success may be dependent on interactions with shifts in other components of the plant community.     

The peculiar heme pocket of the 2/2 hemoglobin of cold-adapted Pseudoalteromonas haloplanktis TAC125

The genome of the cold-adapted bacterium Pseudoalteromonas haloplanktis TAC125 contains multiple genes encoding three distinct monomeric hemoglobins exhibiting a 2/2 ??-helical fold. In the present work, one of these hemoglobins is studied by resonance Raman, electronic absorption and electronic paramagnetic resonance spectroscopies, kinetic measurements, and different bioinformatic approaches. It is the first cold-adapted bacterial hemoglobin to be characterized. The results indicate that this protein belongs to the 2/2 hemoglobin family, Group II, characterized by the presence of a tryptophanyl residue on the bottom of the heme distal pocket in position G8 and two tyrosyl residues (TyrCD1 and TyrB10). However, unlike other bacterial hemoglobins, the ferric state, in addition to the aquo hexacoordinated high-spin form, shows multiple hexacoordinated low-spin forms, where either TyrCD1 or TyrB10 can likely coordinate the iron. This is the first example in which both TyrCD1 and TyrB10 are proposed to be the residues that are alternatively involved in heme hexacoordination by endogenous ligands.     

Oxygen-Linked S-Nitrosation in Fish Myoglobins: A Cysteine-Specific Tertiary Allosteric Effect

The discovery that cysteine (Cys) S-nitrosation of trout myoglobin (Mb) increases heme O₂ affinity has revealed a novel allosteric effect that may promote hypoxia-induced nitric oxide (NO) delivery in the trout heart and improve myocardial efficiency. To better understand this allosteric effect, we investigated the functional effects and structural origin of S-nitrosation in selected fish Mbs differing by content and position of reactive cysteine (Cys) residues. The Mbs from the Atlantic salmon and the yellowfin tuna, containing two and one reactive Cys, respectively, were S-nitrosated in vitro by reaction with Cys-NO to generate Mb-SNO to a similar yield (∼0.50 SH/heme), suggesting reaction at a specific Cys residue. As found for trout, salmon Mb showed a low O₂ affinity (P ₅₀ = 2.7 torr) that was increased by S-nitrosation (P ₅₀ = 1.7 torr), whereas in tuna Mb, O₂ affinity (P ₅₀ = 0.9 torr) was independent of S-nitrosation. O₂ dissociation rates (k _off) of trout and salmon Mbs were not altered when Cys were in the SNO or N-ethylmaleimide (NEM) forms, suggesting that S-nitrosation should affect O₂ affinity by raising the O₂ association rate (k _on). Taken together, these results indicate that O₂-linked S-nitrosation may occur specifically at Cys107, present in salmon and trout Mb but not in tuna Mb, and that it may relieve protein constraints that limit O₂ entry to the heme pocket of the unmodified Mb by a yet unknown mechanism. UV-Vis and resonance Raman spectra of the NEM-derivative of trout Mb (functionally equivalent to Mb-SNO and not photolabile) were identical to those of the unmodified Mb, indicating that S-nitrosation does not affect the extent or nature of heme-ligand stabilization of the fully ligated protein. The importance of S-nitrosation of Mb in vivo is confirmed by the observation that Mb-SNO is present in trout hearts and that its level can be significantly reduced by anoxic conditions.     

Human Circulating Antibody-Producing B Cell as a Predictive Measure of Mucosal Immunity to Poliovirus

Background

The “gold standard” for assessing mucosal immunity after vaccination with poliovirus vaccines consists in measuring virus excretion in stool after challenge with oral poliovirus vaccine (OPV). This testing is time and resource intensive, and development of alternative methods is a priority for accelerating polio eradication. We therefore evaluated circulating antibody-secreting cells (ASCs) as a potential means to evaluate mucosal immunity to poliovirus vaccine.

Methods

199 subjects, aged 10 years, and previously immunized repeatedly with OPV, were selected. Subjects were assigned to receive either a booster dose of inactivated poliovirus vaccine (IPV), bivalent OPV (bOPV), or no vaccine. Using a micro-modified whole blood-based ELISPOT assay designed for field setting, circulating poliovirus type-specific IgA- and IgG-ASCs, including gut homing α4β7+ ASCs, were enumerated on days 0 and 7 after booster immunization. In addition, serum samples collected on days 0, 28 and 56 were tested for neutralizing antibody titers against poliovirus types 1, 2, and 3. Stool specimens were collected on day 28 (day of bOPV challenge), and on days 31, 35 and 42 and processed for poliovirus isolation.

Results

An IPV dose elicited blood IgA- and IgG-ASC responses in 84.8 to 94.9% of subjects, respectively. In comparison, a bOPV dose evoked corresponding blood ASC responses in 20.0 to 48.6% of subjects. A significant association was found between IgA- and IgG-ASC responses and serum neutralizing antibody titers for poliovirus type 1, 2, 3 (p<0.001). In the IPV group, α4β7⁺ ASCs accounted for a substantial proportion of IgA-ASCs and the proportion of subjects with a positive α4β7⁺ IgA-ASC response to poliovirus types 1, 2 and 3 was 62.7%, 89.8% and 45.8%, respectively. A significant association was observed between virus excretion and α4β7⁺ IgA^- and/or IgG-ASC responses to poliovirus type 3 among immunized children; however, only a weak association was found for type 1 poliovirus.

Discussion

Our results suggest that virus-specific blood ASCs, especially for type 3 poliovirus, can serve as surrogate of mucosal immunity after vaccination. Further studies are needed to evaluate the duration of such memory responses and to assess the programmatic utility of this whole blood-based mucosal ASC testing for the polio eradication program.     

The role of CyaY in iron sulfur cluster assembly on the E. coli IscU scaffold protein

Progress in understanding the mechanism underlying the enzymatic formation of iron-sulfur clusters is difficult since it involves a complex reaction and a multi-component system. By exploiting different spectroscopies, we characterize the effect on the enzymatic kinetics of cluster formation of CyaY, the bacterial ortholog of frataxin, on cluster formation on the scaffold protein IscU. Frataxin/CyaY is a highly conserved protein implicated in an incurable ataxia in humans. Previous studies had suggested a role of CyaY as an inhibitor of iron sulfur cluster formation. Similar studies on the eukaryotic proteins have however suggested for frataxin a role as an activator. Our studies independently confirm that CyaY slows down the reaction and shed new light onto the mechanism by which CyaY works. We observe that the presence of CyaY does not alter the relative ratio between [2Fe2S](2+) and [4Fe4S](2+) but directly affects enzymatic activity.     

Electroolfactogram responses from organotypic cultures of the olfactory epithelium from postnatal mice

Organotypic cultures of the mouse olfactory epithelium connected to the olfactory bulb were obtained with the roller tube technique from postnatal mice aged between 13 and 66 days. To test the functionality of the cultures, we measured electroolfactograms (EOGs) at different days in vitro (DIV), up to 7 DIV, and we compared them with EOGs from identical acute preparations (0 DIV). Average amplitudes of EOG responses to 2 mixtures of various odorants at concentrations of 1 mM or 100 microM decreased in cultures between 2 and 5 DIV compared with 0 DIV. The percentage of responsive cultures was 57%. We also used the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) to trigger the olfactory transduction cascade bypassing odorant receptor activation. Average amplitudes of EOG responses to 500 microM IBMX were not significantly different in cultures up to 6 DIV or 0 DIV, and the average percentage of responsive cultures between 2 and 5 DIV was 72%. The dose-response curve to IBMX measured in cultures up to 7 DIV was similar to that at 0 DIV. Moreover, the percentage of EOG response to IBMX blocked by niflumic acid, a blocker of Ca-activated Cl channels, was not significantly different in cultured or acute preparations.