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A field experiment was carried out to quantify the effect of a native bacterial inoculant on the growth, yield, and quality of the wheat crop, under different nitrogen (N) fertilizer rates in two agricultural seasons. Wheat was sown under field conditions at the Experimental Technology Transfer Center (CETT-910), as a representative wheat crop area from the Yaqui Valley, Sonora México. The experiment was conducted using different doses of nitrogen (0, 130, and 250 kg N ha−1) and a bacterial consortium (BC) (Bacillus subtilis TSO9, B. cabrialesii subsp. tritici TSO2T, B. subtilis TSO22, B. paralicheniformis TRQ65, and Priestia megaterium TRQ8). Results showed that the agricultural season affected chlorophyll content, spike size, grains per spike, protein content, and whole meal yellowness. The highest chlorophyll and Normalized Difference Vegetation Index (NDVI) values, as well as lower canopy temperature values, were observed in treatments under the application of 130 and 250 kg N ha−1 (the conventional Nitrogen dose). Wheat quality parameters such as yellow berry, protein content, Sodium dodecyl sulfate (SDS)-Sedimentation, and whole meal yellowness were affected by the N dose. Moreover, the application of the native bacterial consortium, under 130 kg N ha−1, resulted in a higher spike length and grain number per spike, which led to a higher yield (+1.0 ton ha−1 vs. un-inoculated treatment), without compromising the quality of grains. In conclusion, the use of this bacterial consortium has the potential to significantly enhance wheat growth, yield, and quality while reducing the nitrogen fertilizer application, thereby offering a promising agro-biotechnological alternative for improving wheat production.

In order to prepare protein extracts of Plasmodium falciparum blood stages for western blot analysis, infected red blood cells (iRBC) need to be separated from uninfected red blood cells (uRBC) which make up the bulk of the parasite culture. Depending on the localisation of the parasite protein of interest, different methods are available to achieve this. If the protein is present within the parasite or is attached to a cellular structure of the iRBC cell, saponin can be used. This reagent lyses the membranes of infected and uninfected erythrocytes, the Maurer´s clefts (vesicular structures in the iRBC) and the parasitophorous vacuole membrane containing the parasite but leaves the parasite plasma membrane intact, providing a convenient procedure to isolate intact parasites without uRBCs. However, this method has the disadvantage that the host cell cytosol and the parasitophorous vacuole (PV) content of iRBCs are lost. If this has to be avoided, it is possible to use a Percoll gradient to separate intact iRBCs from uRBCs. Sequential treatment with Tetanolysin and saponin can then be used to selectively release the iRBC cytosol and the PV content from the parasite. These selective lysis methods are also suitable to determine the subcellular localisation of a protein of interest.

This protocol was used to prepare pre-embedding samples of Plasmodium falciparum blood stage parasites that overexpressed the parasite protein PF13_0191 tagged with GFP. Using GFP-specific antibodies and Protein A-Gold the localisation of the overexpressed protein in the infected host cell was determined using standard transmission electron microscopy (EM). Pre-embedding EM is a common method where the antibodies are introduced before embedding and sectioning. This method avoids the problem that antigens are often difficult to detect on EM-sections after embedding. In the method described here antigens in the parasite-infected host cell are detected. Entry of the antibody is made possible through permeabilisation of the host cell with tetanolysin. In principle this method could also be used to detect antigens within the parasite if the sample is appropriately fixed and permeabilised before addition of the relevant antibody. While access of the antibody will avoid the detection problems often seen with post-embedding methods, this procedure will produce comparably poorer morphology.

The study of above- and below-ground organ plant coordination is crucial for understanding the biophysical constraints and trade-offs involved in species’ performance under different environmental conditions. Environmental stress is expected to increase constraints on species trait combinations, resulting in stronger coordination among the organs involved in the acquisition and processing of the most limiting resource. To test this hypothesis, we compared the coordination of trait combinations in 94 tree seedling species from two tropical forest systems in Mexico: dry and moist. In general, we expected that the water limitation experienced by dry forest species would result in stronger leaf-stem-root coordination than light limitation experienced by moist forest species. Using multiple correlations analyses and tools derived from network theory, we found similar functional trait coordination between forests. However, the most important traits differed between the forest types. While in the dry forest the most central traits were all related to water storage (leaf and stem water content and root thickness), in the moist forest they were related to the capacity to store water in leaves (leaf water content), root efficiency to capture resources (specific root length), and stem toughness (wood density). Our findings indicate that there is a shift in the relative importance of mechanisms to face the most limiting resource in contrasting tropical forests.

Adenylate energy charge (AEC) has been used as a practical index of the physiological status and health in several disciplines, such as ecotoxicology and aquaculture. This study standardizes several procedures for AEC determination in penaeid shrimp that are very sensitive to sampling. We concluded that shrimp can be frozen in liquid nitrogen and then stored at -76°C for up to two years for further analysis, or freshly dissected and immediately homogenized in acid. Other cooling procedures, such as immersion in cold water or placing shrimp on ice for 15 min resulted in 50% and 73% decreases in ATP levels, and 9-fold and 10-fold increases in IMP levels, respectively. Optimal values of AEC (0.9) were obtained in shrimp recently transferred from ponds to indoor conditions, but decreased to 0.77 after one month in indoor tanks when stocked at high densities; the AEC re-established to 0.85 when the shrimps were transferred to optimal conditions (lower density and dark tanks). While the levels of arginine phosphate followed the same pattern, its levels did not fully re-establish. Comparison of different devices for sample homogenization indicated that a cryogenic ball mill mixer is the more suitable procedure.

Malaria blood stage parasites export a large number of proteins into their host erythrocyte to change it from a container of predominantly hemoglobin optimized for the transport of oxygen into a niche for parasite propagation. To understand this process, it is crucial to know which parasite proteins are exported into the host cell. This has been aided by the PEXEL/HT sequence, a five-residue motif found in many exported proteins, leading to the prediction of the exportome. However, several PEXEL/HT negative exported proteins (PNEPs) indicate that this exportome is incomplete and it remains unknown if and how many further PNEPs exist. Here we report the identification of new PNEPs in the most virulent malaria parasite Plasmodium falciparum. This includes proteins with a domain structure deviating from previously known PNEPs and indicates that PNEPs are not a rare exception. Unexpectedly, this included members of the MSP-7 related protein (MSRP) family, suggesting unanticipated functions of MSRPs. Analyzing regions mediating export of selected new PNEPs, we show that the first 20 amino acids of PNEPs without a classical N-terminal signal peptide are sufficient to promote export of a reporter, confirming the concept that this is a shared property of all PNEPs of this type. Moreover, we took advantage of newly found soluble PNEPs to show that this type of exported protein requires unfolding to move from the parasitophorous vacuole (PV) into the host cell. This indicates that soluble PNEPs, like PEXEL/HT proteins, are exported by translocation across the PV membrane (PVM), highlighting protein translocation in the parasite periphery as a general means in protein export of malaria parasites.