Changes in the Active, Dead, and Dormant Microbial Community Structure across a Pleistocene Permafrost Chronosequence.

Permafrost hosts a community of microorganisms that survive and reproduce for millennia despite extreme environmental conditions, such as water stress, subzero temperatures, high salinity, and low nutrient availability. Many studies focused on permafrost microbial community composition use DNA-based methods, such as metagenomics and 16S rRNA gene sequencing. However, these methods do not distinguish among active, dead, and dormant cells. This is of particular concern in ancient permafrost, where constant subzero temperatures preserve DNA from dead organisms and dormancy may be a common survival strategy. To circumvent this, we applied (i) LIVE/DEAD differential staining coupled with microscopy, (ii) endospore enrichment, and (iii) selective depletion of DNA from dead cells to permafrost microbial communities across a Pleistocene permafrost chronosequence (19,000, 27,000, and 33,000 years old). Cell counts and analysis of 16S rRNA gene amplicons from live, dead, and dormant cells revealed how communities differ between these pools, how they are influenced by soil physicochemical properties, and whether they change over geologic time. We found evidence that cells capable of forming endospores are not necessarily dormant and that members of the class Bacilli were more likely to form endospores in response to long-term stressors associated with permafrost environmental conditions than members of the Clostridia, which were more likely to persist as vegetative cells in our older samples. We also found that removing exogenous "relic" DNA preserved within permafrost did not significantly alter microbial community composition. These results link the live, dead, and dormant microbial communities to physicochemical characteristics and provide insights into the survival of microbial communities in ancient permafrost.IMPORTANCE Permafrost soils store more than half of Earth's soil carbon despite covering ∼15% of the land area (C. Tarnocai et al., Global Biogeochem Cycles 23:GB2023, 2009, https://doi.org/10.1029/2008GB003327). This permafrost carbon is rapidly degraded following a thaw (E. A. G. Schuur et al., Nature 520:171-179, 2015, https://doi.org/10.1038/nature14338). Understanding microbial communities in permafrost will contribute to the knowledge base necessary to understand the rates and forms of permafrost C and N cycling postthaw. Permafrost is also an analog for frozen extraterrestrial environments, and evidence of viable organisms in ancient permafrost is of interest to those searching for potential life on distant worlds. If we can identify strategies microbial communities utilize to survive in permafrost, it may yield insights into how life (if it exists) survives in frozen environments outside of Earth. Our work is significant because it contributes to an understanding of how microbial life adapts and survives in the extreme environmental conditions in permafrost terrains.

SPECT/CT versus MRI in patients with nonspecific pain of the hand and wrist - a pilot study.

BACKGROUND: Hand and wrist pain is a diagnostic challenge for hand surgeons and radiologists due to the complex anatomy of the involved small structures. The American College of Radiology recommends MRI as the study of choice in patients with chronic wrist pain if radiographs are negative. Lately, state-of-the-art SPECT/CT systems have been introduced and may help in the diagnosis of this selected indication. MATERIALS AND METHODS: This retrospective study included 21 patients with nonspecific pain of the hand/wrist. The diagnosis of nonspecific wrist pain was made by the referring hand surgeon based on patient history, clinical examination, plain radiography and clinical guidelines. All patients received planar early-phase imaging and late-phase SPECT/CT imaging as well as MRI. Lesions were divided into major (causative) and minor (not causative) pathologies according to clinical follow-up. Furthermore, oedema-like bone marrow changes seen on MRI were compared with focally increased tracer uptake seen on SPECT/CT images. RESULTS: MRI yielded a quite high sensitivity (0.86), but a low specificity (0.20). In contrast, SPECT/CT yielded a high specificity (1.00) and a low sensitivity (0.71). Oedema-like bone marrow changes were detected in 15 lesions in 11 patients. In ten lesions with bone marrow oedema on MRI, foci of elevated tracer uptake were detected on SPECT/CT. Overall, MRI was more sensitive, but SPECT/CT was more specific in the evaluation of causative pathologies. CONCLUSION: In this initial comparison, SPECT/CT showed higher specificity than MRI in the evaluation of causative pathologies in patients with nonspecific wrist pain. However, MRI was more sensitive. Thus, SPECT/CT was shown to be a useful problem-solving tool in the diagnostic work-up of these patients.

Microbial abundance, composition, and function in nectar are shaped by flower visitor identity.

Microbial dispersal is essential for establishment in new habitats, but the role of vector identity is poorly understood in community assembly and function. Here, we compared microbial assembly and function in floral nectar visited by legitimate pollinators (hummingbirds) and nectar robbers (carpenter bees). We assessed effects of visitation on the abundance and composition of culturable bacteria and fungi and their taxonomy and function using shotgun metagenomics and nectar chemistry. We also compared metagenome-assembled genomes (MAGs) of Acinetobacter, a common and highly abundant nectar bacterium, among visitor treatments. Visitation increased microbial abundance, but robbing resulted in 10× higher microbial abundance than pollination. Microbial communities differed among visitor treatments: robbed flowers were characterized by predominant nectar specialists within Acetobacteraceae and Metschnikowiaceae, with a concurrent loss of rare taxa, and these resulting communities harbored genes relating to osmotic stress, saccharide metabolism and specialized transporters. Gene differences were mirrored in function: robbed nectar contained a higher percentage of monosaccharides. Draft genomes of Acinetobacter revealed distinct amino acid and saccharide utilization pathways in strains isolated from robbed versus pollinated flowers. Our results suggest an unrecognized cost of nectar robbing for pollination and distinct effects of visitor type on interactions between plants and pollinators. Overall, these results suggest vector identity is an underappreciated factor structuring microbial community assembly and function.

Microbial survival strategies in ancient permafrost: insights from metagenomics.

In permafrost (perennially frozen ground) microbes survive oligotrophic conditions, sub-zero temperatures, low water availability and high salinity over millennia. Viable life exists in permafrost tens of thousands of years old but we know little about the metabolic and physiological adaptations to the challenges presented by life in frozen ground over geologic time. In this study we asked whether increasing age and the associated stressors drive adaptive changes in community composition and function. We conducted deep metagenomic and 16 S rRNA gene sequencing across a Pleistocene permafrost chronosequence from 19 000 to 33 000 years before present (kyr). We found that age markedly affected community composition and reduced diversity. Reconstruction of paleovegetation from metagenomic sequence suggests vegetation differences in the paleo record are not responsible for shifts in community composition and function. Rather, we observed shifts consistent with long-term survival strategies in extreme cryogenic environments. These include increased reliance on scavenging detrital biomass, horizontal gene transfer, chemotaxis, dormancy, environmental sensing and stress response. Our results identify traits that may enable survival in ancient cryoenvironments with no influx of energy or new materials.

Imaging non-specific wrist pain: interobserver agreement and diagnostic accuracy of SPECT/CT, MRI, CT, bone scan and plain radiographs.

PURPOSE: Chronic hand and wrist pain is a common clinical issue for orthopaedic surgeons and rheumatologists. The purpose of this study was 1. To analyze the interobserver agreement of SPECT/CT, MRI, CT, bone scan and plain radiographs in patients with non-specific pain of the hand and wrist, and 2. to assess the diagnostic accuracy of these imaging methods in this selected patient population. MATERIALS AND METHODS: Thirty-two consecutive patients with non-specific pain of the hand or wrist were evaluated retrospectively. All patients had been imaged by plain radiographs, planar early-phase imaging (bone scan), late-phase imaging (SPECT/CT including bone scan and CT), and MRI. Two experienced and two inexperienced readers analyzed the images with a standardized read-out protocol. Reading criteria were lesion detection and localisation, type and etiology of the underlying pathology. Diagnostic accuracy and interobserver agreement were determined for all readers and imaging modalities. RESULTS: The most accurate modality for experienced readers was SPECT/CT (accuracy 77%), followed by MRI (56%). The best performing, though little accurate modality for inexperienced readers was also SPECT/CT (44%), followed by MRI and bone scan (38% each). The interobserver agreement of experienced readers was generally high in SPECT/CT concerning lesion detection (kappa 0.93, MRI 0.72), localisation (kappa 0.91, MRI 0.75) and etiology (kappa 0.85, MRI 0.74), while MRI yielded better results on typification of lesions (kappa 0.75, SPECT/CT 0.69). There was poor agreement between experienced and inexperienced readers in SPECT/CT and MRI. CONCLUSIONS: SPECT/CT proved to be the most helpful imaging modality in patients with non-specific wrist pain. The method was found reliable, providing high interobserver agreement, being outperformed by MRI only concerning the typification of lesions. We believe it is beneficial to integrate SPECT/CT into the diagnostic imaging algorithm of chronic wrist pain.

Hydroxypropyl methylcellulose: A better submucosal fluid cushion for endoscopic mucosal resection.

BACKGROUND: Formation of a submucosal fluid cushion has become integral to endoscopic mucosal and polyp resection. Solutions available for injection into the submucosa of the GI tract create short-lasting submucosal fluid cushions or are costly. The feasibility, durability, and histologic response to submucosal fluid cushions created by injection of hydroxypropyl methylcellulose were studied in the esophagus of pigs. METHODS: Thirty-six esophageal submucosal fluid cushions created with hydroxypropyl methylcellulose were studied in 12 animals divided in 2 groups of 6 animals each. In Group I, 18 submucosal fluid cushions were created by submucosal injection of hydroxypropyl methylcellulose followed by tattooing. In Group II, 18 submucosal fluid cushions were created by submucosal injection of hydroxypropyl methylcellulose, with sites marked by an endoscopically placed suture. In all cases, the duration of the submucosal fluid cushion was measured. After 7 days, submucosal fluid cushion sites were assessed by EGD, necropsy, and histologic evaluation. RESULTS: The mean submucosal fluid cushion duration was 36 minutes (3-45 minutes) and 38 minutes (5-45 minutes) for, respectively, Groups I and II. EGD and necropsy after 1 week showed normal-appearing mucosa at the site of all submucosal fluid cushions in both groups. Some Group I animals additionally had nodules at the tattoo sites. Histologic assessment demonstrated minimal alterations in 33 of 36 (92%) submucosal fluid cushion sites and localized mild inflammatory reaction in 3 of 36 (8%). In Group I, 8 of 18 (44%) tattoo sites exhibited a significant inflammatory reaction, including 3 abscesses. CONCLUSIONS: Hydroxypropyl methylcellulose creates a long-lasting submucosal fluid cushion with minimal tissue reaction and should be considered a low-cost option for creating submucosal fluid cushions. The use of tattooing with carbon black should be carefully considered when histologic study is required to assess potential inflammatory tissue responses to an invasive technique or instillation of foreign material in the GI tract.