Operation of a TCA cycle subnetwork in the mammalian nucleus.

Nucleic acid and histone modifications critically depend on the tricarboxylic acid (TCA) cycle for substrates and cofactors. Although a few TCA cycle enzymes have been reported in the nucleus, the corresponding pathways are considered to operate in mitochondria. Here, we show that a part of the TCA cycle is operational also in the nucleus. Using 13C-tracer analysis, we identified activity of glutamine-to-fumarate, citrate-to-succinate, and glutamine-to-aspartate routes in the nuclei of HeLa cells. Proximity labeling mass spectrometry revealed a spatial vicinity of the involved enzymes with core nuclear proteins. We further show nuclear localization of aconitase 2 and 2-oxoglutarate dehydrogenase in mouse embryonic stem cells. Nuclear localization of the latter enzyme, which produces succinyl-CoA, changed from pluripotency to a differentiated state with accompanying changes in the nuclear protein succinylation. Together, our results demonstrate operation of an extended metabolic pathway in the nucleus, warranting a revision of the canonical view on metabolic compartmentalization.

Prior Signal Acquisition Software Versions for Orbitrap Underestimate Low Isobaric Mass Tag Intensities, Without Detriment to Differential Abundance Experiments.

Tandem mass tags (TMTs) enable simple and accurate quantitative proteomics for multiplexed samples by relative quantification of tag reporter ions. Orbitrap quantification of reporter ions has been associated with a characteristic notch region in intensity distribution, within which few reporter intensities are recorded. This has been resolved in version 3 of the instrument acquisition software Tune. However, 47% of Orbitrap Fusion, Lumos, or Eclipse submissions to PRIDE were generated using prior software versions. To quantify the impact of the notch on existing quantitative proteomics data, we generated a mixed species benchmark and acquired quantitative data using Tune versions 2 and 3. Intensities below the notch are predominantly underestimated with Tune version 2, leading to overestimation of the true differences in intensities between samples. However, when summarizing reporter ion intensities to higher-level features, such as peptides and proteins, few features are significantly affected. Targeted removal of spectra with reporter ion intensities below the notch is not beneficial for differential peptide or protein testing. Overall, we find that the systematic quantification bias associated with the notch is not detrimental for a typical proteomics experiment.

Sloth bear attacks: regional differences and safety messaging.

Sloth bears behave aggressively toward humans when threatened and are among the most dangerous wildlife in India. Safety messaging for those who live in sloth bear country must be accurate to be effective, and messaging may need to be modified to account for regional differences in human-bear relationships. The timing of sloth bear attacks on the Deccan Plateau of Karnataka, both by season and by time of day, deviated enough from those reported in other areas such that it warranted further investigation. We compared data from eight studies of human-sloth bear conflict from across the Indian subcontinent and explored possibilities as to why differences exist. Seasonally all studies reported that human-sloth bear conflict was highest when human activity in the forest was greatest, though the season of highest human activity varied significantly by region (χ2 = 5921, df = 5, P < 0.001). The time of day that the majority of attacks occurred also varied significantly by region (χ2 = 666, df = 5, P < 0.001), though human activity was relatively consistent. We speculated that the rate of day attacks on the Deccan Plateau was lower due to the reduced probability of encountering a sleeping bear as they are concealed and secure in shallow caves. Additionally, the rate of attacks was significantly higher at night on the Deccan Plateau because people often to work into nighttime. We concluded that slight differences, or different emphasis, to bear safety messaging may be necessary on a regional basis to keep the messaging accurate and effective.

Efficacy of aerial forward-looking infrared surveys for detecting polar bear maternal dens.

Denned polar bears (Ursus maritimus) are invisible under the snow, therefore winter-time petroleum exploration and development activities in northern Alaska have potential to disturb maternal polar bears and their cubs. Previous research determined forward looking infrared (FLIR) imagery could detect many polar bear maternal dens under the snow, but also identified limitations of FLIR imagery. We evaluated the efficacy of FLIR-surveys conducted by oil-field operators from 2004-2016. Aerial FLIR surveys detected 15 of 33 (45%) and missed 18 (55%) of the dens known to be within surveyed areas. While greater adherence to previously recommended protocols may improve FLIR detection rates, the physical characteristics of polar bear maternal dens, increasing frequencies of weather unsuitable for FLIR detections-caused by global warming, and competing false positives are likely to prevent FLIR surveys from detecting maternal dens reliably enough to afford protections consonant with increasing global threats to polar bear welfare.

Habitat use and social mixing between groups of resident and augmented bighorn sheep.

Monitoring dispersal, habitat use, and social mixing of released ungulates is crucial for successful translocation and species conservation. We monitored 127 female bighorn sheep (Ovis canadensis) released in three populations from 2000 to 2009 to investigate if augmented bighorns expanded and shifted seasonal ranges, used different habitat compared with resident females, and if animals mixed socially. Augmented bighorns in all populations expanded range use compared with residents by shifting utilization distributions. Size of utilization distributions, however, were smaller for augmented females compared with residents in all areas except one. Overlap of seasonal utilization distributions between augmented and resident bighorns and use of slope and elevation differed across populations. In two populations, differences in size and overlap of seasonal utilization distributions and use of slope and elevation supported the hypothesis that habitat use of bighorns in their source area influenced their habitat use after release. Mixing between resident and augmented adult females occurred on average during only 21% of sightings and was similar across populations. Our results clarify how augmented bighorns mix with resident animals and how habitat use is modified following augmentations. Such information is needed to improve bighorn sheep augmentations and can be applied to augmentations of other ungulates.

Combining LOPIT with differential ultracentrifugation for high-resolution spatial proteomics.

The study of protein localisation has greatly benefited from high-throughput methods utilising cellular fractionation and proteomic profiling. Hyperplexed Localisation of Organelle Proteins by Isotope Tagging (hyperLOPIT) is a well-established method in this area. It achieves high-resolution separation of organelles and subcellular compartments but is relatively time- and resource-intensive. As a simpler alternative, we here develop Localisation of Organelle Proteins by Isotope Tagging after Differential ultraCentrifugation (LOPIT-DC) and compare this method to the density gradient-based hyperLOPIT approach. We confirm that high-resolution maps can be obtained using differential centrifugation down to the suborganellar and protein complex level. HyperLOPIT and LOPIT-DC yield highly similar results, facilitating the identification of isoform-specific localisations and high-confidence localisation assignment for proteins in suborganellar structures, protein complexes and signalling pathways. By combining both approaches, we present a comprehensive high-resolution dataset of human protein localisations and deliver a flexible set of protocols for subcellular proteomics.

Trans-acting translational regulatory RNA binding proteins.

The canonical molecular machinery required for global mRNA translation and its control has been well defined, with distinct sets of proteins involved in the processes of translation initiation, elongation and termination. Additionally, noncanonical, trans-acting regulatory RNA-binding proteins (RBPs) are necessary to provide mRNA-specific translation, and these interact with 5' and 3' untranslated regions and coding regions of mRNA to regulate ribosome recruitment and transit. Recently it has also been demonstrated that trans-acting ribosomal proteins direct the translation of specific mRNAs. Importantly, it has been shown that subsets of RBPs often work in concert, forming distinct regulatory complexes upon different cellular perturbation, creating an RBP combinatorial code, which through the translation of specific subsets of mRNAs, dictate cell fate. With the development of new methodologies, a plethora of novel RNA binding proteins have recently been identified, although the function of many of these proteins within mRNA translation is unknown. In this review we will discuss these methodologies and their shortcomings when applied to the study of translation, which need to be addressed to enable a better understanding of trans-acting translational regulatory proteins. Moreover, we discuss the protein domains that are responsible for RNA binding as well as the RNA motifs to which they bind, and the role of trans-acting ribosomal proteins in directing the translation of specific mRNAs. This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes Translation > Translation Regulation Translation > Translation Mechanisms.