Deterministic and stochastic processes generating alternative states of microbiomes.

The structure of microbiomes is often classified into discrete or semi-discrete types potentially differing in community-scale functional profiles. Elucidating the mechanisms that generate such "alternative states" of microbiome compositions has been one of the major challenges in ecology and microbiology. In a time-series analysis of experimental microbiomes, we here show that both deterministic and stochastic ecological processes drive divergence of alternative microbiome states. We introduced species-rich soil-derived microbiomes into eight types of culture media with 48 replicates, monitoring shifts in community compositions at six time points (8 media × 48 replicates × 6 time points = 2304 community samples). We then confirmed that microbial community structure diverged into a few state types in each of the eight medium conditions as predicted in the presence of both deterministic and stochastic community processes. In other words, microbiome structure was differentiated into a small number of reproducible compositions under the same environment. This fact indicates not only the presence of selective forces leading to specific equilibria of community-scale resource use but also the influence of demographic drift (fluctuations) on the microbiome assembly. A reference-genome-based analysis further suggested that the observed alternative states differed in ecosystem-level functions. These findings will help us examine how microbiome structure and functions can be controlled by changing the "stability landscapes" of ecological community compositions.

Metagenomic analysis of ecological niche overlap and community collapse in microbiome dynamics.

Species utilizing the same resources often fail to coexist for extended periods of time. Such competitive exclusion mechanisms potentially underly microbiome dynamics, causing breakdowns of communities composed of species with similar genetic backgrounds of resource utilization. Although genes responsible for competitive exclusion among a small number of species have been investigated in pioneering studies, it remains a major challenge to integrate genomics and ecology for understanding stable coexistence in species-rich communities. Here, we examine whether community-scale analyses of functional gene redundancy can provide a useful platform for interpreting and predicting collapse of bacterial communities. Through 110-day time-series of experimental microbiome dynamics, we analyzed the metagenome-assembled genomes of co-occurring bacterial species. We then inferred ecological niche space based on the multivariate analysis of the genome compositions. The analysis allowed us to evaluate potential shifts in the level of niche overlap between species through time. We hypothesized that community-scale pressure of competitive exclusion could be evaluated by quantifying overlap of genetically determined resource-use profiles (metabolic pathway profiles) among coexisting species. We found that the degree of community compositional changes observed in the experimental microbiome was correlated with the magnitude of gene-repertoire overlaps among bacterial species, although the causation between the two variables deserves future extensive research. The metagenome-based analysis of genetic potential for competitive exclusion will help us forecast major events in microbiome dynamics such as sudden community collapse (i.e., dysbiosis).

Facilitative interaction networks in experimental microbial community dynamics.

Facilitative interactions between microbial species are ubiquitous in various types of ecosystems on the Earth. Therefore, inferring how entangled webs of interspecific interactions shift through time in microbial ecosystems is an essential step for understanding ecological processes driving microbiome dynamics. By compiling shotgun metagenomic sequencing data of an experimental microbial community, we examined how the architectural features of facilitative interaction networks could change through time. A metabolic modeling approach for estimating dependence between microbial genomes (species) allowed us to infer the network structure of potential facilitative interactions at 13 time points through the 110-day monitoring of experimental microbiomes. We then found that positive feedback loops, which were theoretically predicted to promote cascade breakdown of ecological communities, existed within the inferred networks of metabolic interactions prior to the drastic community-compositional shift observed in the microbiome time-series. We further applied "directed-graph" analyses to pinpoint potential keystone species located at the "upper stream" positions of such feedback loops. These analyses on facilitative interactions will help us understand key mechanisms causing catastrophic shifts in microbial community structure.

Core species and interactions prominent in fish-associated microbiome dynamics.

BACKGROUND: In aquatic ecosystems, the health and performance of fish depend greatly on the dynamics of microbial community structure in the background environment. Nonetheless, finding microbes with profound impacts on fish's performance out of thousands of candidate species remains a major challenge. METHODS: We examined whether time-series analyses of microbial population dynamics could illuminate core components and structure of fish-associated microbiomes in the background (environmental) water. By targeting eel-aquaculture-tank microbiomes as model systems, we reconstructed the population dynamics of the 9605 bacterial and 303 archaeal species/strains across 128 days. RESULTS: Due to the remarkable increase/decrease of constituent microbial population densities, the taxonomic compositions of the microbiome changed drastically through time. We then found that some specific microbial taxa showed a positive relationship with eels' activity levels even after excluding confounding effects of environmental parameters (pH and dissolved oxygen level) on population dynamics. In particular, a vitamin-B12-producing bacteria, Cetobacterium somerae, consistently showed strong positive associations with eels' activity levels across the replicate time series of the five aquaculture tanks analyzed. Network theoretical and metabolic modeling analyses further suggested that the highlighted bacterium and some other closely-associated bacteria formed "core microbiomes" with potentially positive impacts on eels. CONCLUSIONS: Overall, these results suggest that the integration of microbiology, ecological theory, and network science allows us to explore core species and interactions embedded within complex dynamics of fish-associated microbiomes.  Video Abstract.

Alternative stable states, nonlinear behavior, and predictability of microbiome dynamics.

BACKGROUND: Microbiome dynamics are both crucial indicators and potential drivers of human health, agricultural output, and industrial bio-applications. However, predicting microbiome dynamics is notoriously difficult because communities often show abrupt structural changes, such as "dysbiosis" in human microbiomes. METHODS: We integrated theoretical frameworks and empirical analyses with the aim of anticipating drastic shifts of microbial communities. We monitored 48 experimental microbiomes for 110 days and observed that various community-level events, including collapse and gradual compositional changes, occurred according to a defined set of environmental conditions. We analyzed the time-series data based on statistical physics and non-linear mechanics to describe the characteristics of the microbiome dynamics and to examine the predictability of major shifts in microbial community structure. RESULTS: We confirmed that the abrupt community changes observed through the time-series could be described as shifts between "alternative stable states" or dynamics around complex attractors. Furthermore, collapses of microbiome structure were successfully anticipated by means of the diagnostic threshold defined with the "energy landscape" analysis of statistical physics or that of a stability index of nonlinear mechanics. CONCLUSIONS: The results indicate that abrupt microbiome events in complex microbial communities can be forecasted by extending classic ecological concepts to the scale of species-rich microbial systems. Video Abstract.

Adult giant hemangioma of the larynx: a case report.

We report a giant hemangioma that extended from the mesopharynx to the larynx in an adult. The patient was a 39-year-old woman who had demonstrated an asymptomatic pharyngeal hemangioma since she was 1 year-old. She developed vertigo and consulted a hospital at the age of 39. During screening by head MRI for the diagnosis of the vertigo, hemangioma of the laryngopharynx was detected. She was referred to Osaka National Hospital for further examination. Her subjective symptoms were dyspnoea in the lower left lateral-position. On observation by nasopharyngolaryngovideoscope, a giant hemangioma was diagnosed in the left mesopharynx expanding into the larynx. On MRI study, hemangioma widely extended to the right posterior wall at the mesopharynx level. Since we were concerned about risk of choking if the hemangioma of the larynx were left untreated, we recommended surgery. Under the general anesthesia, hemangioma of the larynx was light-coagulated by KTP laser through tracheotomy. The postoperative-course has been good and at 9 months postoperatively, there was no progression of hemangioma.

Long-term histological outcomes of injected autologous fat into human vocal folds after secondary laryngectomy.

OBJECTIVE: To histologically evaluate the long-term outcomes of autologous fat grafts after injection laryngoplasty in the human larynx. STUDY DESIGN AND SETTING: We injected liposuctioned fat for vocal fold augmentation in patients with vocal fold paralysis. We suctioned autologous fat from the low abdomen with an 18-G disposable needle and a 20-mL disposable syringe under negative pressure. This is different from the conventional liposuction technique and avoids the use of special equipment. In this article, we report the histological evaluation of 2 patients (patient 1: 12 months, patient 2: 41 months) who required total laryngectomy after autologous fat injection into the vocal folds. RESULTS: Histological examination revealed normal-appearing viable adipocytes with minimal inflammatory response in both patients. CONCLUSIONS: Our liposuctioned autologous fat injection histologically offered long-term improvement in patients with impaired glottal closure from vocal fold paralysis.

[Diagnosis of lateral cervical lymph node metastasis of papillary carcinoma of the thyroid by ultrasonography].

No consensus for papillary carcinoma of the thyroid exists on the preoperative diagnosis of lateral cervical lymph node metastasis, indications, or range of neck dissection, so we studied the usefulness and limits of ultrasonography and sufficient dissection by comparing preoperative ultrasonographic and postoperative histopathological diagnosis. Subjects were 45 patients (51 affected sides) with lateral cervical lymph node metastasis of papillary carcinoma of the thyroid who underwent modified neck dissection between July 1997 and July 2003. Preoperative ultrasonographic and postoperative histopathological diagnosis were compared. Specimens excised by neck dissection contained 1,325 lymph nodes. Of these, 198 (15%) detected by preoperative ultrasonography were selected for investigation of diagnostic criteria for metastasis-positive lymph nodes. The best criterion for the diagnosis of metastasis-positive lymph node was 0.5 or greater [minor axis/major axis] with 6 mm or greater minor axis at levels III, IV, or V (7 mm or greater at level II), and sensitivity, specificity, and accuracy were 78%, 100%, and 84% respectively. The lateral cervical lymph node metastasis rate obtained by this diagnostic criterion was 41%. Regional histopathological metastasis positivity was investigated in the lateral cervical region, and high positivity rates were obtained: 57% at level II, 71% at level III, and 84% at level IV. Considering these findings and the preoperative ultrasonographic diagnosis rate of 41%, sufficient dissection at levels II-IV may be necessary for patients in whom lateral cervical metastasis is observed before surgery. The metastasis rate was 10% at level V, but dissection should always be done in lateral cervical metastasis-positive patients because: 1) No trend was observed in age, gender, the number of metastatic lymph nodes, or regional metastasis rate; 2) no anatomical boundary is present between levels II, III, IV and level V; 3) no functional disorder due to preservation of the accessory nerve occurred; 4) the prognosis of patients with advancement to the accessory nerve was poor; and 5) improvement of the prognosis of papillary carcinoma of the thyroid by modified radical neck dissection has been reported.

[A clinical study of ultrasonography for lymph node metastases in head and neck cancer].

Ultrasonography (US) is very useful in evaluating cervical lymph node swelling in head and neck cancers. We studied problems with US in evaluating lymph nodes. Cervical lymph nodes were removed by radical neck dissection or modified radical neck dissection from 79 patients with squamous cell carcinoma in the head and neck. We studied the correlation between preoperative US findings and the histopathological features. Preoperative lymph nodes were measured three-dimensionally. We diagnosed lymph nodes as metastases when they meet two criteria: One is the shortest diameter exceeding 7 mm in level I and II and 6 mm in level III, IV and V. The other is shortest to longest diameter ratio exceeding 0.5. A total of 2004 lymph nodes were removed by neck dissection, and 199 lymph nodes were diagnosed histopathologically as metastases. Of the 199 metastatic lymph nodes, 93 (46%) were diagnosed as metastases by preoperative US findings and 33 (17%) were false negative. Thirty-six cases were diagnosed preoperatively as N0 by US findings, but 15 of these were pN(+) histopathologically. In the 15 cases, 21 lymph nodes were metastases. Of the 21 metastatic lymph nodes, 10 nodes were not detected by US. Thirty-one cases were diagnosed preoperatively as N1 by US findings, but 20 of these were pN2b histopathologically. In the 20 cases, 66 lymph nodes were metastases. Of the 66 metastatic lymph nodes, 46 were not diagnosed as metastases. They often located distant level from the lymph node diagnosed correctly as a metastasis. US is very useful in evaluating cervical lymph node metastasis, but it has the limitations indicated above. If 1 metastatic lymph node is detected by US, there will be multiple metastatic lymph nodes and sometimes they are distant from the original level. Radical neck dissection should be done for positive lymph nodes detected by US findings. If a lymph node is not clearly a metastasis, fine-needle aspiration cytology (FNA) should be done, because it provides more accurate diagnosis for metastatic lymph nodes.