Formalin fixing and paraffin embedding may lead to extra band development in PCR

The molecular biological analysis of infectious agents requires the availability of a reliable source of microorganisms to be used to recover DNA. Clinical samples can be obtained directly from infected patients or can be propagated using in vitro or in vivo systems. However, repeated sampling from patients is not always possible as the procedure may be invasive or unpleasant, or it is not possible to catch the same agent at the time of second sampling. Moreover, the techniques used may also produce false-positive and false-negative results. We therefore studied the impact of formalin-fixing and paraffin embedding on tissue sampling, and the methodologies such as DNA isolation and PCR amplification of DNAs from archival materials in the diagnosis of Mycobacterium tuberculosis. PCR analyses were done according to standard methods with some modifications. Demonstration of mycobacteria was successful both in tissue sections of the formalin-fixed lymph nodes and in stained fresh materials from patients. However, the results showed the presence of two extra bands in the gel. We accounted for extra band development due to the harshness of the methodology used to isolate nucleic acids from formalin-fixed and paraffin embedded tissue samples or the nature of the fixation procedure, or because of the time passed during storage in which alteration in the chromosomal DNA would take place. Thus, if disease- and tissue specific morphological features, such as sample size, type of fixation, and intralesional heterogeneity are ignored, errors because of sampling and methodologies used may lead to false-positive and false-negative results.     

Differential effects of nitrogen and sulfur deprivation on growth and biodiesel feedstock production of Chlamydomonas reinhardtii

Biodiesel production from microalgae is a promising approach for energy production; however, high cost of its process limits the use of microalgal biodiesel. Increasing the levels of triacylglycerol (TAG) levels, which is used as a biodiesel feedstock, in microalgae has been achieved mainly by nitrogen starvation. In this study, we compared effects of sulfur (S) and nitrogen (N) starvation on TAG accumulation and related parameters in wild-type Chlamydomonas reinhardtii CC-124 mt(-) and CC-125 mt(+) strains. Cell division was interrupted, protein and chlorophyll levels rapidly declined while cell volume, total neutral lipid, carotenoid, and carbohydrate content increased in response to nutrient starvation. Cytosolic lipid droplets in microalgae under nutrient starvation were monitored by three-dimensional confocal laser imaging of live cells. Infrared spectroscopy results showed that relative TAG, oligosaccharide and polysaccharide levels increased rapidly in response to nutrient starvation, especially S starvation. Both strains exhibited similar levels of regulation responses under mineral deficiency, however, the degree of their responses were significantly different, which emphasizes the importance of mating type on the physiological response of algae. Neutral lipid, TAG, and carbohydrate levels reached their peak values following 4 days of N or S starvation. Therefore, 4 days of N or S starvation provides an excellent way of increasing TAG content. Although increase in these parameters was followed by a subsequent decline in N-starved strains after 4 days, this decline was not observed in S-starved ones, which shows that S starvation is a better way of increasing TAG production of C. reinhardtii than N starvation.     

Soil fertility management effects on maize productivity and grain zinc content in smallholder farming systems of Zimbabwe

Biochar is produced as a by-product of the low temperature pyrolysis of biomass during bioenergy extraction and its incorporation into soil is of global interest as a potential carbon sequestration tool. Biochar influences soil nitrogen transformations and its capacity to take up ammonia is well recognized. Anthropogenic emissions of ammonia need to be mitigated due to negative environmental impacts and economic losses. Here we use an isotope of nitrogen to show that ammonia-N adsorbed by biochar is stable in ambient air, but readily bioavailable when placed in the soil. When biochars, containing adsorbed ¹⁵N labelled ammonia, were incorporated into soil the ¹⁵N recovery by roots averaged 6.8% but ranged from 26.1% to 10.9% in leaf tissue due to differing biochar properties with plant ¹⁵N recovery greater when acidic biochars were used to capture ammonia. Recovery of ¹⁵N as total soil nitrogen (organic+inorganic) ranged from 45% to 29% of ¹⁵N applied. We provide a proof of concept for a synergistic mitigation option where anthropogenic ammonia emissions could be captured using biochar, and made bioavailable in soils, thus leading to nitrogen capture by crops, while simultaneously sequestering carbon in soils.     

Biofortification of wheat with zinc through zinc fertilization in seven countries

Background and aims

Phosphorus (P) is a commonly limiting nutrient for plant growth in natural environments. Many legumes capable of N₂-fixation require more P than non-legumes do. Some legume crops can use sparingly soluble forms of P such as iron phosphate much better than other species, but reports on the ability of woody legumes to access iron phosphate are rare.

Methods

Plants of four Acacia species (Acacia stipuligera F. Muell., A. ancistrocarpa Maiden & Blakely, A. stellaticeps Kodela, Tindale & D. Keith and A. robeorum Maslin), native to the Great Sandy Desert in north-western Australia, were grown in a glasshouse in river sand with different levels of iron phosphate, between 0 and 16?μg P g^?1 sand. Plant growth, tissue P concentrations, and pH and carboxylates in the rhizosphere were measured.

Results

Growth of A. stipuligera and A. ancistrocarpa was not responsive to increased P supply; in contrast, A. stellaticeps and A. robeorum produced significantly more root and shoot dry mass at 8 and 16?μg P g^?1 sand than at 0?μg P g^?1 sand; differences in root mass ratio were significant between species but not between P treatments. A. robeorum was the only species colonised by mycorrhizal fungi, and the colonisation percentage decreased with increasing P supply. In all species, P-uptake rates and tissue P concentrations were significantly higher at greater P supply. Rhizosphere pH and the amount of carboxylates in the rhizosphere decreased with increasing P supply.

Conclusions

Net P uptake increased with increasing P supply, showing that the present Acacia species can access P from iron phosphate. However, due to their inherently slow growth rate, enhanced P supply did not increase growth of two of the four studied species. The ability of the Acacia species to access P from iron phosphate is presumably related with carboxylate exudation and rhizosphere acidification.     

Biofortification of rice grain with zinc through zinc fertilization in different countries

Aims and background

The ability to suppress soil nitrification through the release of nitrification inhibitors from plant roots is termed ‘biological nitrification inhibition’ (BNI). Earlier, we reported that sorghum roots release higher BNI-activity when grown with NH ₄ ⁺ , but not with NO ₃ ^- as N source. Also for BNI release, rhizosphere pH of <5.0 is needed; beyond this, a negative effect on BNI release was observed with nearly 80% loss of BNI activity at pH >7.0. This study is aimed at understanding the inter-functional relationships associated with NH ₄ ⁺ uptake, rhizosphere-pH and plasma membrane H⁺-ATPase (PM H⁺-ATPase) activity in regulating the release of BNIs (biological nitrification inhibitors) from sorghum roots.

Methods

Sorghum was grown hydroponically and root exudates were collected from intact plants using a pH-stat system to separate the secondary acidification effects by NH ₄ ⁺ uptake on BNIs release. A recombinant luminescent Nitrosomonas europaea bioassay was used to determine BNI-activity. Root plasma membrane was isolated using a two-phase partitioning system. Hydrolytic H⁺-ATPase activity was determined. Split-root system setup was deployed to understand the localized responses to NH ₄ ⁺ , H⁺-ATPase-stimulator (fusicoccin) or H⁺-ATPase-inhibitor (vanadates) on BNI release by sorghum.

Results

Presence of NH ₄ ⁺ in the rhizosphere stimulated the expression of H⁺-ATPase activity and enhanced the release of BNIs from sorghum roots. Fusicoccin, which stimulates H⁺-ATPase activity, also stimulated BNIs release in the absence of NH ₄ ⁺ ; vanadate, which suppresses H⁺-ATPase activity, also suppressed the release of BNIs. NH ₄ ⁺ levels (in rhizosphere) positively influenced BNIs release and root H⁺-ATPase activity in the concentration range of 0-1.0 mM, indicating a close relationship between BNI release and root H⁺-ATPase activity with a possible involvement of carrier-mediated transport for the release of BNIs in sorghum.

Conclusion

Our results suggest that NH ₄ ⁺ uptake, PM H⁺-ATPase activity, and rhizosphere acidification are functionally inter-connected with BNI release in sorghum. Such knowledge is critical to gain insights into why BNI function is more effective in light-textured, mildly acidic soils compared to other soil types.     

Bioavailability of Trace Elements in Beans and Zinc-Biofortified Wheat in Pigs

The objectives of this experiment were to study bioavailability of trace elements in beans and wheat containing different levels of zinc and to study how the water solubility of trace elements was related to the bioavailability in pigs. Three wheat and two bean types were used: wheat of Danish origin as a control (CtrlW), two Turkish wheat types low (LZnW) and high (HZnW) in zinc, a common bean (Com), and a faba bean (Faba). Two diets were composed by combining 81?% CtrlW and 19?% Com or Faba beans. Solubility was measured as the trace element concentration in the supernatant of feedstuffs, and diets incubated in distilled water at pH?4 and 38°C for 3?h. The bioavailability of zinc and copper of the three wheat types and the two bean-containing diets were evaluated in the pigs by collection of urine and feces for 7?days. The solubility of zinc was 34?C63?%, copper 18?C42?%, and iron 3?C11?%. The zinc apparent digestibility in pigs was similar in the three wheat groups (11?C14?%), but was significantly higher in the CtrlW+Faba group (23?%) and negative in the CtrlW+Com group (?30?%). The apparent digestibility of copper was higher in the HZnW (27?%) and CtrlW+Faba (33?%) groups than in the CtrlW (17?%) and LZnW (18?%) groups. The apparent copper digestibility of the CtrlW+Com diet was negative (?7?%). The solubility and digestibility results did not reflect the concentration in feedstuffs. The in vitro results of water solubility showed no relationship to the results of trace mineral bioavailability in pigs.     

Blink test results in patients with central sleep apnea syndrome

Sleep and Biological Rhythms - The aim of the study was to evaluate whether there was a problem in the interneuronal junctions of patients with central sleep apnea using the blink reflex test....     

Inter‐ and intraspecific variation of spider mite susceptibility to fungal infections: Implications for the long‐term success of biological control

Spider mites are severe pests of several annual and perennial crops worldwide, often causing important economic damages. As rapid evolution of pesticide resistance in this group hampers the efficiency of chemical control, alternative control strategies, such as the use of entomopathogenic fungi, are being developed. However, while several studies have focused on the evaluation of the control potential of different fungal species and/or isolates as well as their compatibility with other control methods (e.g., predators or chemical pesticides), knowledge on the extent of inter‐ and intraspecific variation in spider mite susceptibility to fungal infection is as yet incipient. Here, we measured the mortality induced by two generalist fungi, Beauveria bassiana and Metarhizium brunneum, in 12 spider mite populations belonging to different Tetranychus species: T. evansi, T. ludeni, and T. urticae (green and red form), within a full factorial experiment. We found that spider mite species differed in their susceptibility to infection by both fungal species. Moreover, we also found important intraspecific variation for this trait. These results draw caution on the development of single strains as biocontrol agents. Indeed, the high level of intraspecific variation suggests that (a) the one‐size‐fits‐all strategy may fail to control spider mite populations and (b) hosts resistance to infection may evolve at a rapid pace. Finally, we propose future directions to better understand this system and improve the long‐term success of spider mite control strategies based on entomopathogenic fungi.