The origins and diversification of Holarctic brown bear populations inferred from genomes of past and present populations.

The brown bear (Ursus arctos) is one of the survivors of the Late Quaternary megafauna extinctions. However, despite being widely distributed across the Holarctic, brown bears have experienced extensive range reductions, and even extirpations in some geographical regions. Previous research efforts using genetic data have provided valuable insights into their evolutionary history. However, most studies have been limited to contemporary individuals or mitochondrial DNA, limiting insights into population processes that preceded the present. Here, we present genomic data from two Late Pleistocene brown bears from Honshu, Japan and eastern Siberia, and combine them with published contemporary and ancient genomes from across the Holarctic range of brown bears to investigate the evolutionary relationships among brown bear populations through time and space. By including genomic data from Late Pleistocene and Holocene individuals sampled outside the current distribution range, we uncover diversity not present in contemporary populations. Notably, although contemporary individuals display geographically structured populations most likely driven by isolation-by-distance, this pattern varies among the ancient samples across different regions. The inclusion of ancient brown bears in our analysis provides novel insights into the evolutionary history of brown bears and contributes to understanding the populations and diversity lost during the Late Quaternary.

Incidence, clinicopathological features and genetics of in-situ follicular neoplasia: a comprehensive screening study in a Japanese cohort.

AIMS: In-situ follicular neoplasia (ISFN) is a histologically recognizable neoplastic proliferation of follicular lymphoma (FL)-like B cells confined to the germinal centres. While some ISFNs are associated with overt FL, others are incidentally identified as isolated or pure forms in individuals without evidence of overt FL. The prevalence of incidentally found isolated ISFN is approximately 3% in Europe; however, no screening study has been conducted in Asia. To investigate the incidence and clinicopathological characteristics of ISFNs in the Japanese population, we conducted histopathological screening of the lymph nodes (LNs) resected for solid tumours or inflammatory conditions. METHODS AND RESULTS: We screened for ISFN in 5700 LNs from 340 individuals using immunohistochemistry for BCL2 and identified seven ISFNs, with an incidence of 2.1%. The median age of the individuals with ISFN was 67 years, none of whom developed overt FL, with a median follow-up of 59 months. Next-generation sequencing was performed in five ISFNs, and 10 variants in seven FL-associated genes were identified. The identified variants included HIST1H1E (n = 2), ARID1A (n = 2), KMT2D (n = 1), CARD11 (n = 1), BCL7A (n = 1), CREBBP (n = 1) and TNFRSF14 (n = 1). CONCLUSIONS: The incidence of isolated ISFN in the Japanese population is not significantly different from that in Europe, presumably reflecting the recent increase in FL in Japan. These incidentally found ISFNs have a low potential to transform into overt FL. Although mutations of FL-associated genes are already present in ISFNs, further molecular studies are needed to identify driver genes leading to the transformation of ISFN to overt FL.

Evaluation of reported sediment samples from 20 Ma using a molecular phylogenetic approach: comment on Liu et al. (2017).

Liu et al. reported the cultivation and DNA sequencing of 69 fungal isolates (Ascomycota and Basidiomycota) from ancient subseafloor sediments, suggesting that they represent living fungal populations that have persisted for over 20 million years. Because these findings could bring about a paradigm shift in our understanding of the spatial breadth of the deep subsurface biosphere as well as the longevity of ancient DNA, it is extremely important to verify that their samples represent pure ancient fungi from 20 million years ago without contamination by modern species. For this purpose, we estimated the divergence times of Dikarya (Ascomycota + Basidiomycota) and Mucoromycota fungi assuming that the fungal isolates were actually sampled from 20 Ma (mega-annum) sediments and evaluated the validity of the sample ages. Using this approach, we estimate that the age of the last common ancestor of Dikarya and Mucoromycota fungi greatly exceeds the age of the Earth. Our finding emphasizes the importance of using reliable approaches to confirm the dating of ancient samples.

Metagenomic analyses highlight the symbiotic association between the glacier stonefly Andiperla willinki and its bacterial gut community.

The glacier stonefly Andiperla willinki is the largest metazoan inhabiting the Patagonian glaciers. In this study, we analysed the gut microbiome of the aquatic nymphs by 16S rRNA gene amplicon and metagenomic sequencing. The bacterial gut community was consistently dominated by taxa typical of animal digestive tracts, such as Dysgonomonadaceae and Lachnospiraceae, as well as those generally indigenous to glacier environments, such as Polaromonas. Interestingly, the dominant Polaromonas phylotypes detected in the stonefly gut were almost never detected in the glacier surface habitat. Fluorescence in situ hybridization analysis revealed that the bacterial lineages typical of animal guts colonized the gut wall in a co-aggregated form, while Polaromonas cells were not included in the aggregates. Draft genomes of several dominant bacterial lineages were reconstructed from metagenomic datasets and indicated that the predominant Dysgonomonadaceae bacterium is capable of degrading various polysaccharides derived from host-ingested food, such as algae, and that other dominant bacterial lineages ferment saccharides liberated by the polysaccharide degradation. Our results suggest that the gut bacteria-host association in the glacier stonefly contributes to host nutrition as well as material cycles in the glacier environment.

Nitrogen Cycle Evaluation (NiCE) Chip for Simultaneous Analysis of Multiple N Cycle-Associated Genes.

Various microorganisms play key roles in the nitrogen (N) cycle. Quantitative PCR (qPCR) and PCR amplicon sequencing of N cycle functional genes allow us to analyze the abundance and diversity of microbes responsible for N-transforming reactions in various environmental samples. However, analysis of multiple target genes can be cumbersome and expensive. PCR-independent analysis, such as metagenomics and metatranscriptomics, is useful but expensive, especially when we analyze multiple samples and try to detect N cycle functional genes present at a relatively low abundance. Here, we present the application of microfluidic qPCR chip technology to simultaneously quantify and prepare amplicon sequence libraries for multiple N cycle functional genes as well as taxon-specific 16S rRNA gene markers for many samples. This approach, named the nitrogen cycle evaluation (NiCE) chip, was evaluated by using DNA from pure and artificially mixed bacterial cultures and by comparing the results with those obtained by conventional qPCR and amplicon sequencing methods. Quantitative results obtained by the NiCE chip were comparable to those obtained by conventional qPCR. In addition, the NiCE chip was successfully applied to examine the abundance and diversity of N cycle functional genes in wastewater samples. Although nonspecific amplification was detected on the NiCE chip, this can be overcome by optimizing the primer sequences in the future. As the NiCE chip can provide a high-throughput format to quantify and prepare sequence libraries for multiple N cycle functional genes, this tool should advance our ability to explore N cycling in various samples.IMPORTANCE We report a novel approach, namely, the nitrogen cycle evaluation (NiCE) chip, by using microfluidic qPCR chip technology. By sequencing the amplicons recovered from the NiCE chip, we can assess the diversities of N cycle functional genes. The NiCE chip technology is applicable to analysis of the temporal dynamics of N cycle gene transcription in wastewater treatment bioreactors. The NiCE chip can provide a high-throughput format to quantify and prepare sequence libraries for multiple N cycle functional genes. While there is room for future improvement, this tool should significantly advance our ability to explore the N cycle in various environmental samples.

Demographic analysis of cyanobacteria based on the mutation rates estimated from an ancient ice core.

Despite the crucial role of cyanobacteria in various ecosystems, little is known about their evolutionary histories, especially microevolutionary dynamics, because of the lack of knowledge regarding their mutation rates. Here we directly estimated cyanobacterial mutation rates based on ancient DNA analyses of ice core samples collected from Kyrgyz Republic that dates back to ~12,500 cal years before present. We successfully sequenced the 16S rRNA and 16S-23S internal transcribed spacer (ITS) region. Two cyanobacterial operational taxonomic units (OTUs) were detected from the ancient ice core samples, and these OTUs are shared with those from the modern glacier surface. The mutation rate of ITS region was estimated by comparing ancient and modern populations, and were at the magnitude of 10-7substitutions/sites/year. By using a model selection framework, we also demonstrated that the ancient sequences from the ice sample were not contaminated from modern samples. Bayesian demographic analysis based on coalescent theory revealed that cyanobacterial population sizes increased over Asia regions during the Holocene. Thus, our results enhance our understanding of the enigmatic timescale of cyanobacterial microevolution, which has the potential to elucidate the environmental responses of cyanobacteria to the drastic climatic change events of the Quaternary.

Experimental Evidence for in Situ Nitric Oxide Production in Anaerobic Ammonia-Oxidizing Bacterial Granules.

Although nitric oxide (NO) emissions from anaerobic ammonium oxidation (anammox)-based processes were reported previously, the NO production pathways are poorly understood. Here, we investigated the NO production pathways in anammox granules in detail by combining 15N-stable isotope tracer experiments with various inhibitors, microsensor measurements, and transcriptome analysis for key genes of NO2- reduction. NO was emitted from the anammox granules, which account for 0.07% of the N2 emission. 15N-stable isotope-tracer experiments indicated that most of the N2 was produced by anammox bacteria, whereas NO was produced from NO2- reduction by anammox and denitrifying bacteria. The NO emission rate was highest at pH 8.0 and accelerated by increasing NH4+ and NO2- concentrations in the culture media. The microsensor analyses showed the in situ NO production rate was highest in the outer layer of the anammox granule where anammox activity was also highest. The detected in situ NO concentrations of up to 2.7 µM were significantly above physiological thresholds known to affect a wide range of microorganisms present in wastewater. Hence, NO likely plays pivotal roles in the microbial interactions in anammox granules, which needs to be further investigated.

Noviherbaspirillum denitrificans sp. nov., a denitrifying bacterium isolated from rice paddy soil and Noviherbaspirillum autotrophicum sp. nov., a denitrifying, facultatively autotrophic bacterium isolated from rice paddy soil and proposal to reclassify Herbaspirillum massiliense as Noviherbaspirillum massiliense comb. nov.

Thirty-nine denitrifying bacterial strains closely related to one another, represented by strains TSA40T and TSA66T, were isolated from rice paddy soils. Strains TSA40T and TSA66T were Gram-stain-negative, slightly curved rod-shaped, and motile by means of polar flagella. They were able to reduce nitrate, nitrite and nitrous oxide, but unable to fix atmospheric N2. While strain TSA66T was able to grow autotrophically by H2-dependent denitrification, strain TSA40T could not. Phylogenetic analysis suggested that they belong to the family Oxalobacteraceae, the order Burkholderiales in the class Betaproteobacteria. Major components in the fatty acids (C16 : 0, C17 : 0 cyclo, C18 : 1ω7c and summed feature 3) and quinone (Q-8) also supported the affiliation of strains TSA40T and TSA66T to the family Oxalobacteraceae. Based on 16S rRNA gene sequence comparisons, strains TSA40T and TSA66T showed the greatest degree of similarity to Herbaspirillum massiliense JC206T, Noviherbaspirillum malthae CC-AFH3T, Noviherbaspirillum humi U15T, Herbaspirillum seropedicae Z67T and Paucimonas lemoignei LMG 2207T, and lower similarities to the members of other genera. Average nucleotide identity values between the genomes of strain TSA40T, TSA66T and H. massiliense JC206T were 75-77 %, which was lower than the threshold value for species discrimination (95-96 %). Based on the 16S rRNA gene sequence analysis in combination with physiological, chemotaxonomic and genomic properties, strains TSA40T (=JCM 17722T=ATCC TSD-69T) and TSA66T (=JCM 17723T=DSM 25787T) are the type strains of two novel species within the genus Noviherbaspirillum, for which the names Noviherbaspirillum denitrificans sp. nov. and Noviherbaspirillum autotrophicum sp. nov. are proposed, respectively. We also propose the reclassification of Herbaspirillum massiliense as Noviherbaspirillum massiliense comb. nov.

The nitrogen cycle in cryoconites: naturally occurring nitrification-denitrification granules on a glacier.

Cryoconites are microbial aggregates commonly found on glacier surfaces where they tend to take spherical, granular forms. While it has been postulated that the microbes in cryoconite granules play an important role in glacier ecosystems, information on their community structure is still limited, and their functions remain unclear. Here, we present evidence for the occurrence of nitrogen cycling in cryoconite granules on a glacier in Central Asia. We detected marker genes for nitrogen fixation, nitrification and denitrification in cryoconite granules by digital polymerase chain reaction (PCR), while digital reverse transcription PCR analysis revealed that only marker genes for nitrification and denitrification were abundantly transcribed. Analysis of isotope ratios also indicated the occurrence of nitrification; nitrate in the meltwater on the glacier surface was of biological origin, while nitrate in the snow was of atmospheric origin. The predominant nitrifiers on this glacier belonged to the order Nitrosomonadales, as suggested by amoA sequences and 16S ribosomal RNA pyrosequencing analysis. Our results suggest that the intense carbon and nitrogen cycles by nitrifiers, denitrifiers and cyanobacteria support abundant and active microbes on the Asian glacier.

Microfluidic quantitative PCR for simultaneous quantification of multiple viruses in environmental water samples.

To secure food and water safety, quantitative information on multiple pathogens is important. In this study, we developed a microfluidic quantitative PCR (MFQPCR) system to simultaneously quantify 11 major human viral pathogens, including adenovirus, Aichi virus, astrovirus, enterovirus, human norovirus, rotavirus, sapovirus, and hepatitis A and E viruses. Murine norovirus and mengovirus were also quantified in our MFQPCR system as a sample processing control and an internal amplification control, respectively. River water contaminated with effluents from a wastewater treatment plant in Sapporo, Japan, was collected and used to validate our MFQPCR system for multiple viruses. High-throughput quantitative information was obtained with a quantification limit of 2 copies/µl of cDNA/DNA. Using this MFQPCR system, we could simultaneously quantify multiple viral pathogens in environmental water samples. The viral quantities obtained using MFQPCR were similar to those determined by conventional quantitative PCR. Thus, the MFQPCR system developed in this study can provide direct and quantitative information for viral pathogens, which is essential for risk assessments.

Spread of antibiotic resistance genes to Antarctica by migratory birds.

Recent studies have highlighted the presence of antibiotic resistance genes (ARGs) in Antarctica, which are typically indicative of human activity. However, these studies have concentrated in the Antarctic Peninsula region, and relatively less is known about ARG prevalence in East Antarctica, where human activity levels are lower compared to the Antarctic Peninsula. In addition, the mechanisms of ARG transmission to Antarctica through natural or anthropogenic pathways remain unclear. In this study, we analyzed the fecal samples of Adélie penguins and South polar skuas by using high-throughput sequencing and microfluidic quantitative PCR to detect potential pathogens and ARGs at their breeding colonies near Syowa Station in East Antarctica. These results revealed the presence of several potential pathogens in the fecal matter of both bird species. However, the HF183 marker, which indicates human fecal contamination, was absent in all samples, as well as seawater sampled near the breeding colonies. This suggests that the human fecal contamination was negligible in our study area. In addition to pathogens, we found a significant number of ARGs and metal resistance genes in the feces of both Adélie penguins and South polar skuas, with higher detection rates in skuas than in penguins. To better understand how these birds acquire and transmit these genes, we analyzed the migratory patterns of Adélie penguins and South polar skuas by geolocator tracking. We found that the skuas migrate to the tropical and subtropical regions of the Indian Ocean during the austral winter. On the other hand, Adélie penguins exhibited a more localized migration pattern, mainly staying within Antarctic waters. Because the Indian Ocean is considered one of the major reservoirs of ARGs, South polar skuas might be exposed to ARGs during their winter migration and transfer these genes to Antarctica.

Bacterial diversity and biopotentials of Hamtah glacier cryoconites, Himalaya.

Cryoconite is a granular structure present on the glaciers and ice sheets found in polar regions including the Himalayas. It is composed of organic and inorganic matter which absorb solar radiations and reduce ice surface albedo, therefore impacting the melting and retreat of glaciers. Though climate warming has a serious impact on Himalayan glaciers, the biodiversity of sub-glacier ecosystems is poorly understood. Moreover, cryoconite holes are unique habitats for psychrophile biodiversity hotspots in the NW Himalayas, but unfortunately, studies on the microbial diversity of such habitats remain elusive. Therefore, the current study was designed to explore the bacterial diversity of the Hamtah Glacier Himalaya using both culturable and non-culturable approaches. The culturable bacterial count ranged from 2.0 × 103 to 8.8 × 105 colony-forming units (CFUs)/g at the different locations of the glacier. A total of 88 bacterial isolates were isolated using the culturable approach. Based on the 16S ribosomal RNA gene (16S rRNA), the identified species belong to seven genera, namely, Cryobacterium, Duganella, Janthinobacterium, Pseudomonas, Peribacillus, Psychrobacter, and Sphingomonas. In the non-culturable approach, high-throughput sequencing of 16S rRNA genes (using MiSeq) showed unique bacterial community profiles and represented 440 genera belonging to 20 phyla, namely, Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes, Chloroflexi, Acidobacteria, Planctomycetes, Cyanobacteria, Verrucomicrobia, Spirochaetes, Elusimicrobia, Armatimonadetes, Gemmatimonadetes, Deinococcus-Thermus, Nitrospirae, Chlamydiae, Chlorobi, Deferribacteres, Fusobacteria, Lentisphaerae, and others. High relative abundances of Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes were observed in the samples. Phototrophic (Cyanobacteria and Chloroflexi) and nitrifier (Nitrospirae) in bacterial populations indicated sustenance of the micro-ecosystem in the oligotrophic glacier environment. The isolates varied in their phenotypic characteristics, enzyme activities, and antibiotic sensitivity. Furthermore, the fatty acid profiles of bacterial isolates indicate the predominance of branched fatty acids. Iso-, anteiso-, unsaturated and saturated fatty acids together constituted a major proportion of the total fatty acid composition. High cold-adapted enzyme activities such as lipase and cellulase expressed by Cryobacterium arcticum (KY783365) and protease and cellulase activities by Pseudomonas sp. strains (KY783373, KY783377-79, KY783382) provide evidence of the possible applications of these organisms. Additionally, antibiotic tests indicated that most isolates were sensitive to antibiotics. In conclusion, the present study contributed for the first time to bacterial diversity and biopotentials of cryoconites of Hamtah Glacier, Himalayas. Furthermore, the cold-adapted enzymes and polyunsaturated fatty acids (PUFAs) may provide an opportunity for biotechnology in the Himalayas. Inductively coupled plasma mass spectrometry (ICPMS) analyses showed the presence of several elements in cryoconites, providing a clue for the accelerating melting and retreating of the Hamtah glacier.

Abnormalities in C-type lectin-like receptor 2 in a patient with Gorham-Stout disease: the first case report.

Background: Gorham-Stout disease (GSD) is a form of lymphangiomatosis of unknown etiology, characterized by abnormal distribution of lymphatic vessels. Platelets and lymphangiogenesis are closely related via C-type lectin-like receptor 2 (CLEC-2)/podoplanin. Key Clinical Question: Despite similarities between abnormal lymphatic vessels in CLEC-2-deficient mice and patients with GSD, whether CLEC-2 on platelets is involved in GSD pathogenesis is unknown. Clinical Approach: We examined CLEC-2 expression in platelets of a patient with lethal GSD. Most of the patient's platelets expressed aberrant CLEC-2 that was not detectable by certain monoclonal antibodies for human CLEC-2. Further, this population was not activated by a CLEC-2-activating snake venom, rhodocytin. Possible causes of abnormal CLEC-2 including anti-CLEC-2 autoantibodies, podoplanin binding to CLEC-2, and pathogenic CLEC1B gene alteration were excluded. Conclusions: We believe that this is the first report of a patient with structurally and functionally abnormal CLEC-2. CLEC-2 abnormality may be associated with dysregulated lymphangiogenesis in GSD.

Validation and application of high-throughput quantitative PCR for the simultaneous detection of microbial source tracking markers in environmental water.

No single microbial source tracking (MST) marker can be applied to determine the sources of fecal pollution in all water types. This study aimed to validate a high-throughput quantitative polymerase chain reaction (HT-qPCR) method for the simultaneous detection of multiple MST markers. A total of 26 fecal-source samples that had been previously collected from human sewage (n = 6) and ruminant (n = 3), dog (n = 6), pig (n = 6), chicken (n = 3), and duck (n = 2) feces in the Kathmandu Valley, Nepal, were used to validate 10 host-specific MST markers, i.e., Bacteroidales (BacHum, gyrB, BacR, and Pig2Bac), mitochondrial DNA (mtDNA) (swine, bovine, and Dog-mtDNA), and viral (human adenovirus, porcine adenovirus, and chicken/turkey parvovirus) markers, via HT-qPCR. Only Dog-mtDNA showed 100 % accuracy. All the tested bacterial markers showed a sensitivity of 100 %. Nine of the 10 markers were further used to identify fecal contamination in groundwater sources (n = 54), tanker filling stations (n = 14), drinking water treatment plants (n = 5), and river water samples (n = 6). The human-specific Bacteroidales marker BacHum and ruminant-specific Bacteroidales marker BacR was detected at a high ratio in river water samples (83 % and 100 %, respectively). The results of HT-qPCR were in agreement with the standard qPCR. The comparable performances of HT-qPCR and standard qPCR as well as the successful detection of MST markers in the fecal-source and water samples demonstrated the potential applicability of these markers for detecting fecal contamination sources via HT-qPCR.

Simultaneous quantification of multiple food- and waterborne pathogens by use of microfluidic quantitative PCR.

The direct quantification of multiple pathogens has been desired for diagnostic and public health purposes for a long time. In this study, we applied microfluidic quantitative PCR (qPCR) technology to the simultaneous detection and quantification of multiple food- and waterborne pathogens. In this system, multiple singleplex qPCR assays were run under identical detection conditions in nanoliter-volume chambers that are present in high densities on a chip. First, we developed 18 TaqMan qPCR assays that could be run in the same PCR conditions by using prevalidated TaqMan probes. Specific and sensitive quantification was achieved by using these qPCR assays. With the addition of two previously validated TaqMan qPCR assays, we used 20 qPCR assays targeting 10 enteric pathogens, a fecal indicator bacterium (general Escherichia coli), and a process control strain in the microfluidic qPCR system. We preamplified the template DNA to increase the sensitivity of the qPCR assays. Our results suggested that preamplification was effective for quantifying small amounts of the template DNA without any major impact on the sensitivity, efficiency, and quantitative performance of qPCR. This microfluidic qPCR system allowed us to detect and quantify multiple pathogens from fecal samples and environmental water samples spiked with pathogens at levels as low as 100 cells/liter. These results suggest that the routine monitoring of multiple pathogens in food and water samples is now technically feasible. This method may provide more reliable information for risk assessment than the current fecal contamination indicator approach.

Evolution of snow algae, from cosmopolitans to endemics, revealed by DNA analysis of ancient ice.

Recent studies of microbial biogeography have revealed the global distribution of cosmopolitans and dispersal of regional endemics, but little is known about how these processes are affected by microbial evolution. Here, we compared DNA sequences from snow/glacier algae found in an 8000-year-old ice from a glacier in central Asia with those from modern snow samples collected at 34 snow samples from globally distributed sites at the poles and mid-latitudes, to determine the evolutionary relationship between cosmopolitan and endemic phylotypes of snow algae. We further applied a coalescent theory-based demographic model to the DNA sequences. We found that the genus Raphidonema (Trebouxiophyceae) was distributed over both poles and mid-latitude regions and was detected in different ice core layers, corresponding to distinct time periods. Our results indicate that the modern cosmopolitan phylotypes belonging to Raphidonema were persistently present long before the last glacial period. Furthermore, endemic phylotypes originated from ancestral cosmopolitan phylotypes, suggesting that modern regional diversity of snow algae in the cryosphere is a product of microevolution. These findings suggest that the cosmopolitans dispersed across the world and then derived new localized endemics, which thus improves our understanding of microbial community formation by microevolution in natural environments.

Genetic and immunohistochemical profiling of NK/T-cell lymphomas reveals prognostically relevant BCOR-MYC association.

Extranodal NK/T-cell lymphoma, nasal type (ENKTL) is an Epstein-Barr virus-positive, aggressive lymphoma with a heterogeneous cell of origin and variable clinical course. Several clinical prognostic indices have been proposed for ENKTL; however, there are few pathological biomarkers. This multi-institutional study sought to identify histologically assessable prognostic factors. We investigated mutation profiles by targeted next-generation sequencing (NGS) and immunohistochemical assessments of expression of MYC, Tyr705-phosphorylated (p-)STAT3, and CD30 in 71 ENKTL samples. The median age of the patients was 66 years (range, 6-100). The most frequent mutations were in STAT3 (27%), JAK3 (4%), KMT2D (19%), TP53 (13%), BCOR (10%), and DDX3X (7%). Immunohistochemistry (IHC) revealed that ENKTLs with STAT3 mutations exhibited higher expression of pSTAT3 and CD30. BCOR mutations were associated with increased MYC expression. Univariate analysis in the entire cohort showed that stage (II, III, or IV), BCOR mutations, TP53 mutations, and high MYC expression (defined as ≥40% positive neoplastic cells) were associated with reduced overall survival (OS). Multivariate modeling identified stage (II, III, or IV) and high MYC expression as independent adverse prognostic factors. In a subgroup analysis of patients treated with anthracycline (AC)-free chemotherapy and/or radiotherapy (RT) with curative intent, BCOR but not high MYC expression was an independent adverse prognostic factor. In conclusion, activating STAT3 mutations are common in ENKTLs and are associated with increased CD30 expression. MYC overexpression is, at least in part, associated with deleterious BCOR mutations, and this BCOR-MYC linkage may have prognostic significance, underscoring the potential utility of IHC for MYC in risk stratification of patients with ENKTL.

Temporal dynamics of Campylobacter and Arcobacter in a freshwater lake that receives fecal inputs from migratory geese.

Migratory geese could influence the microbiological water quality; however, their impacts on pathogen dynamics remain largely unknown. In this study, we analyzed the population dynamics of Campylobacter and Arcobacter group bacteria (AGB) in a freshwater lake in Japan over two years. The bacteria were quantified by using both culture-dependent and -independent methods. The potential sources of these bacteria were examined by a high-throughput flaA sequencing approach. Campylobacter was abundantly detected both by culture-dependent and -independent methods in the lake, especially when migratory geese were present in the lake. High-throughput flaA sequencing suggests that geese were the likely source of Campylobacter in the lake. The viable population of Campylobacter exceeds the concentrations that can potentially cause 10-4 infections per person per year when water is used to grow fresh vegetables. The occurrence of AGB, on the other hand, was not directly related to the population of migratory geese. AGB were not detected in geese fecal samples. Diverse AGB flaA genotypes occurred in the lake over multiple seasons. Our results suggest that AGB likely comprise a part of the indigenous microbial population of the lake and grow in response to high nutrient, warm temperature, and low dissolved oxygen concentrations in the lake. Geese therefore can indirectly impact the AGB population by providing nutrients to cause eutrophication and lower the dissolved oxygen concentration. Since geese travel long-distance and disperse their fecal microbiota and nutrients to wide areas, they may have significant impacts on water quality and public health.

Application of a high-throughput quantitative PCR system for simultaneous monitoring of SARS-CoV-2 variants and other pathogenic viruses in wastewater.

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are continuously emerging, highlighting the importance of regular surveillance of SARS-CoV-2 and other epidemiologically significant pathogenic viruses in the current context. Reverse transcription-quantitative PCR (RT-qPCR) is expensive, time-consuming, labor-intensive, requires a large reagent volume, and only tests a few targets in a single run. High-throughput qPCR (HT-qPCR) utilizing the Biomark HD system (Fluidigm) can be used as an alternative. This study applied an HT-qPCR to simultaneously detect SARS-CoV-2, SARS-CoV-2 nucleotide substituted RNA, and other pathogenic viruses in wastewater. Wastewater samples were collected from the coronavirus disease 2019 (COVID-19) quarantine facility between October 2020 and February 2021 (n = 4) and from the combined and separated sewer lines of a wastewater treatment plant (WWTP) in Yokkaichi, Mie Prefecture, Japan, between March and August 2021 (n = 23 each). The samples were analyzed by HT-qPCR using five SARS-CoV-2, nine SARS-CoV-2 spike gene nucleotide substitution-specific, five pathogenic viruses, and three process control assays. All samples from the quarantine facility tested positive for SARS-CoV-2 and the nucleotide substitutions N501Y and S69-70 del (Alpha variant) were detected in the December 2020 sample, coinciding with the first clinical case in Japan. Only three WWTP samples were positive when tested with a single SARS-CoV-2 assay, whereas more than eight samples were positive when tested with all assays, indicating that using multiple assays increases the likelihood of detection. The nucleotide substitution L452R (Delta variant) was detected in the WWTP samples of Mie Prefecture in April 2021, but the detection of Delta variant from patients had not been reported until May 2021. Aichi virus 1 and norovirus GII were prevalent in WWTP samples. This study demonstrated that HT-qPCR may be the most time- and cost-efficient method for tracking COVID-19 and broadly monitoring community health.

Paleogenomics reveals independent and hybrid origins of two morphologically distinct wolf lineages endemic to Japan.

Little is known about the spatiotemporal dynamics of gray wolves in the Pleistocene across low-latitude regions of Eurasia. In Japan, a small-bodied endemic subspecies of Japanese wolves existed and went extinct in the early 1900s. The fossil record indicates that a giant wolf, which reached 70 cm in body height, inhabited Japan during the Pleistocene, but its evolutionary relationship, if any, with the Japanese wolf remains uncertain. Here, to reveal the genetic origin of the Japanese wolf, we analyzed ancient DNA from remains (recovered in Japan) of one Pleistocene wolf that lived 35,000 years ago and one Holocene wolf from 5,000 years ago. The analysis of the mitochondrial DNA revealed that the Pleistocene wolf was not part of the Japanese wolf clade but rather an earlier-diverging lineage. The analysis of the nuclear DNA of the Holocene Japanese wolf revealed that it was an admixture of the Japanese Pleistocene wolf and continental wolf lineages. These findings suggest that the Japanese wolf originated via waves of colonization of multiple Pleistocene wolf populations at 57-35 and 37-14 ka, respectively, followed by interpopulation hybridization.