Guano exposed: Impact of aerobic conditions on bat fecal microbiota

Bats and their associated guano microbiota provide important terrestrial and subterranean ecosystem services and serve as a reservoir for a wide range of epizootic and zoonotic diseases. Unfortunately, large‐scale studies of bats and their guano microbiotas are limited by the time and cost of sample collection, which requires specially trained individuals to work at night to capture bats when they are most active. Indirectly surveying bat gut microbiota through guano deposits could be a more cost‐effective alternative, but it must first be established whether the postdefecation exposure to an aerobic environment has a large impact on the guano microbial community. A number of recent studies on mammalian feces have shown that the impact of aerobic exposure is highly species specific; therefore, it is difficult to predict how exposure will affect the bat guano microbiota without empirical data. In our study, we collected fresh guano samples from 24 individuals of 10 bat species that are common throughout the arid environments of the American southwest and subjected the samples to 0, 1, and 12 hr of exposure. The biodiversity decreased rapidly after the shift from an anaerobic to an aerobic environment—much faster than previously reported in mammalian species. However, the relative composition of the core guano microbiota remained stable and, using highly sensitive targeted PCR methods, we found that pathogens present in the original, non‐exposed samples could still be recovered after 12 hr of exposure. These results suggest that with careful sample analysis protocols, a more efficient passive collection strategy is feasible; for example, guano could be collected on tarps placed near the roost entrance. Such passive collection methods would greatly reduce the cost of sample collection by allowing more sites or roosts to be surveyed with a fraction of trained personnel, time, and effort investments needed.     

Measurement of thymus weight, lumbar node weight and progesterone levels in syngeneically pregnant, allogeneically pregnant, and pseudopregnant mice.

Female CBA mice were mated to fertile CBA males, to vasectomized CBA males, to fertile C57BL males or to vasectomized C57BL males. After allogeneic or syngeneic mating the extent of thymic involution on the 10th day of pregnancy and pseudopregnancy was similar. Lumbar lymph node weight was not affected by pseudopregnancy but increased similarly in allogeneic and syngeneic pregnancies. Serum progesterone levels on the 10th day of pseudopregnancy were similar to those of non-pregnant females, and significantly lower than those of pregnant females. On the 4th to 7th days progesterone levels in pseudopregnant animals were equal to those in pregnant animals. Progesterone levels and thymic involution were similar in syngeneically and allogeneically pregnant females. Progesterone levels were negatively correlated with thymus weight but reached significance only when the mating was allogeneic. It is suggested that there is an interaction between progesterone concentrations and the degree of thymic involution during pregnancy.     

Identification of a serum-inducible messenger RNA (5B10) as the mouse homologue of calcyclin: tissue distribution and expression in metastatic, ras-transformed NIH 3T3 cells

A mouse mRNA, provisionally designated 5B10, has been cloned based on its inducibility by serum in quiescent murine fibroblasts. Here we report the full-length complementary DNA sequence and a partial characterization. There are about five copies of the gene in the mouse genome. Sequence analysis of the 5B10 coding region reveals 94 and 97% amino acid identity to human and rat calcyclin, respectively. Although the coding region has been highly conserved during evolution of the rodent and human genomes, the untranslated flanking sequences differ significantly. A protein of Mr about 8000 was produced by in vitro translation of the mRNA transcribed in vitro from 5B10 complementary DNA in a riboprobe vector. An antiserum raised against a portion of the predicted human calcyclin protein cross-reacted with this mouse protein. 5B10 mRNA was found in greatest amount in organs containing proliferating cells, e.g., epidermis, skin, stomach, uterus of pregnant mouse, placenta, and decidua. Brain, liver, mature thymus, and skeletal muscle had little or no detectable 5B10 mRNA. 5B10 mRNA levels were higher in cells treated with 7,12-dimethylbenzanthracene and 12-O-tetradecanoylphorbol-13-acetate than in their normal counterparts, suggesting a role in tumorigenesis. In addition, high 5B10 mRNA levels were associated with metastatic ability in a series of ras-transformed cells, in proportion to levels of ras p21 expressed by the cells, implicating 5B10 even more deeply in carcinogenesis.     

Major histocompatibility complex (MHC)-encoded HAM2 is necessary for antigenic peptide loading onto class I MHC molecules.

The mutant murine lymphoma cell line RMA-S is unable to present endogenous antigens due to its inability to efficiently assemble class I major histocompatibility complex molecules and antigenic peptides. Therefore, it has been suggested that RMA-S cells are defective either in peptide generation or in peptide transport into the endoplasmic reticulum, where class I major histocompatibility complex molecule assembly is believed to occur. As proteasomes and the putative peptide transporters HAM1 and HAM2 have been implicated in class I antigen processing, we have investigated their expression in RMA-S and its wild-type counterpart RMA. Both proteasomes and HAM1 proteins are expressed at similar levels and show identical subcellular distributions in the two cell lines. However, only one copy of the HAM2 gene is present in RMA-S cells, and it contains a point mutation that leads to a premature stop codon. Thus, the HAM2 protein is absent from RMA-S cells. These data demonstrate that HAM2 is essential for peptide loading onto class I molecules.     

Climate change affects the early-life history of a freshwater turtle in a severely drying region

Freshwater turtles are one of the most threatened vertebrate groups. Climate change is a major threat to these species, with impacts affecting all life-history stages. There is currently a limited understanding of how changes in climate may alter the environmental triggers for hatching and emergence from the nests of freshwater turtle hatchlings. This precludes making predictions about how climate change may impact freshwater turtle recruitment success. The southwestern snake-necked turtle (Chelodina oblonga) is endemic to south-western Australia, a global biodiversity hotspot that has undergone severe climatic drying. Recruitment failure is thought to be occurring in many populations of the species. However, there is little understanding as to how environmental change may be influencing recruitment. This study aimed to: (1) determine the incubation duration and hatching and hatchling emergence success of C. oblonga, (2) determine if the species exhibits hatching or emergence synchrony and/or delayed emergence and (3) quantify the effects of temperature and rainfall on hatchling emergence. Using this information, the study assesses how climatic drying and warming may be impacting C. oblonga's early life-history. Between 2018 and 2020 nest sites were monitored around a large urban wetland with weekly assessments of egg and hatchling status. Incubation duration and hatching and hatchling emergence success were calculated, and generalized linear models were built to determine how temperature and/or rainfall predicted emergence. Hatchlings either emerged shortly after hatching or overwintered in the nest, and both hatching and emergence were asynchronous. Both emergence periods were positively associated with temperature and rainfall. This study reveals that incubation duration, hatching success, hatchling emergence and survival are all likely to be impacted by recent and projected climate change, and especially drying. Warming and drying are predicted for many temperate regions globally, and it is therefore important that their impacts on the early life history of freshwater turtles be better understood.     

ADP ribosylation adapts an ER chaperone response to short-term fluctuations in unfolded protein load

Gene expression programs that regulate the abundance of the chaperone BiP adapt the endoplasmic reticulum (ER) to unfolded protein load. However, such programs are slow compared with physiological fluctuations in secreted protein synthesis. While searching for mechanisms that fill this temporal gap in coping with ER stress, we found elevated levels of adenosine diphosphate (ADP)-ribosylated BiP in the inactive pancreas of fasted mice and a rapid decline in this modification in the active fed state. ADP ribosylation mapped to Arg470 and Arg492 in the substrate-binding domain of hamster BiP. Mutations that mimic the negative charge of ADP-ribose destabilized substrate binding and interfered with interdomain allosteric coupling, marking ADP ribosylation as a rapid posttranslational mechanism for reversible inactivation of BiP. A kinetic model showed that buffering fluctuations in unfolded protein load with a recruitable pool of inactive chaperone is an efficient strategy to minimize both aggregation and costly degradation of unfolded proteins.     

Multiple Reaction Monitoring Enables Precise Quantification of 97 Proteins in Dried Blood Spots

The dried blood spot (DBS) methodology provides a minimally invasive approach to sample collection and enables room-temperature storage for most analytes. DBS samples have successfully been analyzed by liquid chromatography multiple reaction monitoring mass spectrometry (LC/MRM-MS) to quantify a large range of small molecule biomarkers and drugs; however, this strategy has only recently been explored for MS-based proteomics applications. Here we report the development of a highly multiplexed MRM assay to quantify endogenous proteins in human DBS samples. This assay uses matching stable isotope-labeled standard peptides for precise, relative quantification, and standard curves to characterize the analytical performance. A total of 169 peptides, corresponding to 97 proteins, were quantified in the final assay with an average linear dynamic range of 207-fold and an average R² value of 0.987. The total range of this assay spanned almost 5 orders of magnitude from serum albumin ({"type":"entrez-protein","attrs":{"text":"P02768","term_id":"113576","term_text":"P02768"}}P02768) at 18.0 mg/ml down to cholinesterase ({"type":"entrez-protein","attrs":{"text":"P06276","term_id":"116353","term_text":"P06276"}}P06276) at 190 ng/ml. The average intra-assay and inter-assay precision for 6 biological samples ranged from 6.1–7.5% CV and 9.5–11.0% CV, respectively. The majority of peptide targets were stable after 154 days at storage temperatures from −20 °C to 37 °C. Furthermore, protein concentration ratios between matching DBS and whole blood samples were largely constant (<20% CV) across six biological samples. This assay represents the highest multiplexing yet achieved for targeted protein quantification in DBS samples and is suitable for biomedical research applications.The dried blood spot (DBS)¹ methodology provides several advantages over traditional plasma or serum samples throughout the entire pre-analytical workflow including sample collection, transportation, and storage (1, 2) These blood samples are typically generated using a small sterile lancet to prick the skin and then spotting a drop onto a collection card. Therefore, DBS sampling is less invasive than venipuncture and does not require a trained phlebotomist. This sampling approach also does not require time-sensitive centrifugation, which is crucial for plasma and serum samples to prevent degradation. Many analytes have been determined to be stable in the DBS format at room temperature, eliminating the cost associated with cold-chain logistics for sample transportation and storage. These considerations are also important for sample collection in remote locations that may be without reliable access to a centrifuge and/or a freezer designated for biohazardous materials. Quantitative bioanalytical methods using the DBS methodology have been developed for genomic, metabolomic, and proteomic applications including newborn screening (3, 4), therapeutic drug monitoring (5, 6), toxicology and drugs of abuse (7, 8), viral disease management (9, 10), and many others (2, 11).Targeted MS, in particular selected/multiple reaction monitoring (SRM/MRM) using internal standards, enables the rapid development of quantitative assays with high specificity, precision, and robustness (12–15). The integration of DBS sampling with MRM is well-established for quantifying a wide range of small molecules (16–18). This is now the standard analytical approach for population-wide screening of newborns for errors in metabolism by targeting amino acids, fatty acid acylcarnitines, and organic acid acylcarnitines (3, 4). DBS with MRM is also emerging as an important analytical tool throughout pre-clinical and clinical small-molecule drug development and monitoring (16, 17, 19–21). Furthermore, Zukunft et al. recently demonstrated the high multiplexing capabilities of MRM by using 2 methods to quantify 188 metabolites in DBS samples, including acylcarnitines, amino acids, biogenic amines, free carnitine, glycerophospholipids, hexoses, lysophosphatidylcholines, phosphatidylcholines, and sphingolipids (22).Although DBS with MRM is well-established in small molecule applications, there are only a handful of reports showing the use of this approach to quantify endogenous proteins (23). Daniel et al. measured the ratio between hemoglobin δ and β to screen for β-thalassemia (24). Boemer et al. measured the relative ratios of several hemoglobin variants (including HbS, HbC, HbE, and others) to help diagnose Sickle Cell disease and other clinically relevant hemoglobinopathies (25). The same group then screened >40,000 newborns in Belgium and successfully detected 16 patients with severe hemoglobin disorders (26). Moats et al. used a similar approach to screen >13,000 newborns in the UK for Sickle Cell disease and correctly identified all seven disease occurrences (27). Because hemoglobin is the most abundant protein in whole blood (∼150 mg/ml), these four studies achieved adequate sensitivity by simply infusing the trypsin digested samples into a triple-quadrupole MS. To move beyond hemoglobin, additional sensitivity can be provided by using liquid chromatography (LC) separations coupled online with MRM. deWilde et al. used LC/MRM-MS to quantify ceruloplasmin as a screen for Wilson''s disease (28). Recently, Cox et al. reported LC/MRM-MS methods for quantifying insulin-like growth facter-1 for the detection of human growth hormone abuse in sports (29, 30).Our group reported the first LC/MRM-MS assay to quantify multiple endogenous proteins in DBS samples (31). In that exploratory study, we selected a small test panel of 60 high-abundance proteins and were ultimately able to quantify 37 proteins using stable isotope-labeled standard (SIS) peptides and standard curves. In this work, we describe method refinement and further evaluation of LC/MRM-MS for quantifying endogenous proteins in human DBS samples. A more comprehensive approach has now been taken to evaluate sensitivity and suitability, as the initial target panel has been increased to 393 proteins. The protocol has also been modified so that all liquid handling steps in the sample preparation protocol are now automated in a 96-well format for improved sample throughput. Standard curves using SIS peptides were produced using a pooled patient sample, and assay precision was determined in biological samples from six different individuals. In addition, we have provided a detailed discussion of the quantification results from multiple peptides per protein, a comparison to measured protein concentrations in whole blood, an analyte stability assessment at various storage temperatures, and an evaluation of volumetric spotting devices. Ultimately, we have developed a multiplexed LC/MRM-MS assay to quantify 97 proteins in DBS samples that is suitable for biomedical research applications.