Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

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sperm cryopreservation glycerol mithun

Not AvailableThe effect of concentration and addition method of glycerol on the quality of cryopreserved mithun (Bos frontalis) spermatozoa was investigated. Semen samples were collected from five healthy mithun bulls through rectal massage method and cryopreserved in liquid nitrogen. The samples were diluted in Tris–egg yolk–glycerol extender, equilibrated for 4 h at 4 °C and loaded into 0.50‐ml straws. The straws were then frozen in liquid nitrogen vapour for 10 min and finally plunged into liquid nitrogen for storage. The required amount of glycerol was added into the diluted samples either in a single dose (3%, 4%, 5%, 6% or 7%; added at 37 °C immediately before equilibration) or in split doses (5%, 6% or 7%; the total amount was divided into four equal parts, and a part was added at 37 °C immediately before equilibration, and the remaining parts were added subsequently at 1, 2 and 3 h of equilibration at 4 °C). In the single‐dose addition method, following freeze‐thawing, greater (p < 0.05) motility (%) and proportion of live spermatozoa with intact acrosome (LSIA, %) in 5% glycerol (40.6 ± 1.7 and 43.4 ± 1.8 respectively) and lesser (p < 0.05) total morphological abnormalities (%) in 5% (14.1 ± 0.8) and 6% (13.7 ± 1.0) glycerol were observed compared to the other glycerol concentrations. In the split‐dose addition method, following freeze‐thawing, greater (p < 0.05) motility (%) and LSIA proportion (%) were found in 5% (50.2 ± 1.9 and 53.3 ± 1.8 respectively) compared to 6% or 7% glycerol, but the total morphological abnormalities were not different among the glycerol concentrations. In addition, in all the glycerol concentrations, better (p < 0.05) post‐freeze‐thaw motility and LSIA proportions were observed when glycerol was added in split doses compared to a single dose. In conclusion, Tris–egg yolk extender with 5% glycerol added in split doses was found most suitable for cryopreserving mithun sperm.Not Availabl

Insights into Insect Resistance in Pulse Crops: Problems and Preventions

Globally, insect pests cause considerable damage to pulse crops. Hence developing broad-spectrum resistance against insect pests has been a major challenge to pulse growers and scientists. Traditionally, cultural practices and synthetic insecticides are being utilized for effective control of insect pests since ages. Apart from these, other strategies such as host plant resistance, insect-resistant transgenic crops, and IPM are also being used to manage the infestation in pulse crops. Though screening of genetic resources for insect resistance has been promising in some pulse crops, fertility barriers and linkage drag minimize the effective utilization of identified resistance in commercially viable crop breeding programs. In parallel, insect-resistant transgenic plants have been developed using various insecticidal proteins from various sources including Bacillus thuringiensis endotoxin, plant protease inhibitors, chitinases, alpha-amylase inhibitors, secondary metabolites, and vegetative insecticidal proteins (VIPs). Deploying transgenic plants with high levels of toxin expression by gene pyramiding is another practical option to delay the resistance development in insects. Nevertheless, the success achieved so far in managing insect pests is limited mainly due to the complex mechanisms underlying the defense strategies together with the lack of precision in screening techniques. Here, we discuss the recent progress and current status of studies toward developing resistance to the most common insect pests of pulses. This chapter points the lack of detailed molecular studies exploring the insect resistance that can advance our knowledge on plant resistance mechanisms and the genes involved. Therefore, a step forward now will be on exploiting natural variations with novel technologies in combination of eco-safe management practices to develop durable insect-resistant pulse crops. Despite technical and regulatory difficulties, developing insect resistance should be the major priority area for future breeding and genetic engineering studies aiming at pulse crop improvement