Complementary symbiont contributions to plant decomposition in a fungus-farming termite

Termites normally rely on gut symbionts to decompose organic matter but the Macrotermitinae domesticated Termitomyces fungi to produce their own food. This transition was accompanied by a shift in the composition of the gut microbiota, but the complementary roles of these bacteria in the symbiosis have remained enigmatic. We obtained high-quality annotated draft genomes of the termite Macrotermes natalensis, its Termitomyces symbiont, and gut metagenomes from workers, soldiers, and a queen. We show that members from 111 of the 128 known glycoside hydrolase families are represented in the symbiosis, that Termitomyces has the genomic capacity to handle complex carbohydrates, and that worker gut microbes primarily contribute enzymes for final digestion of oligosaccharides. This apparent division of labor is consistent with the Macrotermes gut microbes being most important during the second passage of comb material through the termite gut, after a first gut passage where the crude plant substrate is inoculated with Termitomyces asexual spores so that initial fungal growth and polysaccharide decomposition can proceed with high efficiency. Complex conversion of biomass in termite mounds thus appears to be mainly accomplished by complementary cooperation between a domesticated fungal monoculture and a specialized bacterial community. In sharp contrast, the gut microbiota of the queen had highly reduced plant decomposition potential, suggesting that mature reproductives digest fungal material provided by workers rather than plant substrate.Interspecific mutualism usually allows partner species preferential access to complementary resources. Some hosts internalized microbial symbionts, leading to vertical transmission and varying degrees of genome loss (1), whereas others domesticated external partners that maintained independent reproduction (2). Understanding how such ectosymbioses remain evolutionarily stable is challenging (3) because prokaryote and eukaryote symbionts form interacting communities, which may be difficult for hosts to control when symbionts can achieve higher fitness by pursuing selfish reproductive strategies (4). Digestive symbiotic communities in animal guts provide excellent examples of such ambiguities; recent studies of human microbiotas show that gut communities vary by subject age, geography (5), and diet (6) and that deviating microbiotas can be associated with compromised health (7).Given the continuous flow of food through animal guts, it is intriguing that adaptive microbiotas can normally be maintained (8–10) without invasion by less beneficial or harmful microbes (11). Insect lineages that have relied on nutritional symbioses have existed and adaptively radiated for tens of millions of years, suggesting that the benefits of these symbioses surpass the potential levels-of-selection conflicts that need to be regulated (12). However, beyond examples from humans and some domesticated ungulates, we lack fundamental insight into the genes involved, their expression, and their phenotypic functions. Termites provide a case in point, as they originated >150 Mya and have relied on protist and bacterial gut symbionts for the breakdown of lignocellulose throughout their evolutionary history (13), allowing them to become dominant decomposers in terrestrial ecosystems (13, 14).A single monophyletic subfamily, the Macrotermitinae, realized a major evolutionary transition ca. 30 Mya, when they domesticated the ancestor of the fungal genus Termitomyces (15). They have radiated into 11 termite genera with more than 330 extant species (15, 16) to collectively obtain a massive ecological footprint in the Old World (sub)tropics, matched only by the fungus-growing (attine) ants of the New World (14, 17). Throughout their evolutionary history, the partnership with five major clades of Termitomyces has remained obligate, as no macrotermitine termite is known to have abandoned fungus farming or to rear other fungi than Termitomyces (15) (Fig. 1 A and B). Coinciding with the domestication of Termitomyces, the common ancestor of the Macrotermitinae underwent a major shift in the bacterial gut community (18, 19). The fungus-growing termites thus represent a major metazoan radiation based on a simultaneous tripartite life-history transition: insects becoming farmers, fungi becoming crops, and gut microbiotas adopting largely unknown complementary roles.Open in a separate windowFig. 1.The fungus-growing termite symbiosis and its genomic characteristics. (A) A Macrotermes natalensis colony in South Africa: (i) the underground fungus comb in which Termitomyces is maintained and (ii and iii) the royal chamber with the queen (ii) and the king (iii). (B) Geographic distribution of the Macrotermitinae (gray), with darker areas in southern Africa highlighting the known occurrences of M. natalensis (adapted from ref. 61). (C) The substrate and recurrent Termitomyces inoculation within a colony centered around the termite gut: Asexual Termitomyces spores from fungus comb nodules (i) and plant biomass substrate (ii) are mixed within the termite gut (iii, first gut passage) to become the new fungus comb substrate (iv) within which Termitomyces hyphae grow to maturity so that new nodules with asexual spores are produced (v) until the plant substrate is fully used and the old comb (vi) is consumed by the termites (vii, second gut passage). (D) To characterize the genetic potential of the fungus-growing termite symbiosis, we sequenced M. natalensis and Termitomyces and obtained worker, soldier, and queen gut metagenomes (SI Appendix and GigaScience Database, http://dx.doi.org/10.5524/100055).Fungus-growing termites rely on the external decomposition of plant substrate by their Termitomyces fungus garden symbiont. In Macrotermes species, the fungus comb is managed in a highly structured way, with older workers collecting crude forage material and bringing it back to the nest, where younger workers ingest it together with asexual Termitomyces spores (conidia) provided by fungal nodules from established “fungus-garden combs” to produce primary feces that is deposited as new layers of comb (17, 20) (Fig. 1C). This new substrate quickly develops dense hyphal networks and produces the next cohorts of nodules (2, 20), whereas older termites ultimately consume the old comb (Fig. 1C). This combination of substrate processing and inoculation at first gut passage followed by a second digestive phase makes the termite gut the central operational compartment of the symbiosis. It is here that the entire genetic potential of all members of the symbiosis comes together, presumably shaped by natural selection for optimal collective performance in two sequential digestive phases. To investigate functional complementarity of the three major components of the mutualism, we (i) obtained high-quality draft genome sequences of the fungus-growing termite Macrotermes natalensis, its Termitomyces sp. symbiont, and several caste-specific gut microbiotas; (ii) analyzed the genomic potential for lignocellulolytic enzyme potential to assess functional contributions across partners; and (iii) compared gut microbiotas across sterile and reproductive castes to evaluate functional gut specialization across termite family members.     

Gut microbiome contributions to altered metabolism in a pig model of undernutrition

The concept that gut microbiome-expressed functions regulate ponderal growth has important implications for infant and child health, as well as animal health. Using an intergenerational pig model of diet restriction (DR) that produces reduced weight gain, we developed a feature-selection algorithm to identify representative characteristics distinguishing DR fecal microbiomes from those of full-fed (FF) pigs as both groups consumed a common sequence of diets during their growth cycle. Gnotobiotic mice were then colonized with DR and FF microbiomes and subjected to controlled feeding with a pig diet. DR microbiomes have reduced representation of genes that degrade dominant components of late growth-phase diets, exhibit reduced production of butyrate, a key host-accessible energy source, and are causally linked to reduced hepatic fatty acid metabolism (β-oxidation) and the selection of alternative energy substrates. The approach described could aid in the development of guidelines for microbiome stewardship in diverse species, including farm animals, in order to support their healthy growth.

Undernutrition afflicts over 200 million children worldwide and accounts for 45% of mortality in children under 5 y (1). Children with acute malnutrition exhibit wasting (impaired ponderal growth), often accompanied by stunting (reduced linear growth), deficits in bone development, neurodevelopment, and immunity, as well as perturbed metabolism (2, 3). Epidemiologic studies indicate that acute malnutrition in children is not due to food insecurity alone and that perturbed gut microbial community development is a contributing factor; children with severe acute malnutrition (SAM) and moderate acute malnutrition (MAM; weight-for-length z-scores are, respectively, 2 to 3 and >3 SDs below World Health Organization mean values) have microbiota that appear “younger” (more immature) compared to those of chronologically aged-matched healthy children (4–6). Studies in gnotobiotic mice colonized with microbiota from healthy and undernourished children have provided evidence that immature microbiota can transmit features of undernutrition (5, 7). These tests of causality inspired development of microbiota-directed complementary foods (MDCFs) designed to repair the microbiota of undernourished children. A controlled feeding study, involving a small group of 12- to 18-mo-old Bangladeshi children with MAM, identified an MDCF formulation that repaired their microbiota; repair was associated with a marked change in their plasma proteome characterized by alterations in levels of key mediators of bone growth, metabolism, immune function, and neurodevelopment toward a healthy state (5). A larger, longer randomized controlled study showed that this MDCF produced a superior effect on ponderal growth compared to a ready-to-use supplementary food even though the caloric density of the MDCF was 20% lower (8).These observations prompted us to examine the influence of the gut microbiome on weight gain in the domestic pig, Sus scrofa domesticus. We focused on this species for several reasons. First, pigs account for ∼35% of global meat intake, second only to poultry (9, 10). Production costs are heavily influenced by how efficiently feed is transformed into body mass, as well as the degree of growth uniformity across animals (11). Second, pigs have been used as a model for studying human nutrition and metabolism because of the many ways in which they are anatomically, physiologically, and metabolically similar to humans (12, 13). Third, most of the commercial pig industry raises animals in highly controlled farming systems engineered to promote efficient and consistent growth phenotypes. These systems typically include phased feeding programs that transition animals from early, more costly, readily digestible, nutrient-rich diets to later, less-expensive diets with less nutrient fortification where energy/nutrient extraction is more dependent on expressed metabolic activities encoded in the gut microbiome. A central premise of the current study is that in order to more fully realize the goal of predictable robust weight gain at affordable prices, additional knowledge is needed regarding codevelopment of the gut microbiome and host; this knowledge could allow diets to be formulated based on greater understanding of which components (features) of the community play key roles in transforming dietary components to products that the animals use to satisfy their growth requirements (14). The environmentally controlled settings for raising pigs provide great opportunities for performing longitudinal studies designed to delineate these interactions between diet, microbiome features, and host physiology. Finally, the need to focus on whether/how the gut microbiome contributes to growth is made more pressing by international mandates to eliminate use of subtherapeutic antibiotics for growth promotion of farm animals because of the spread of antibiotic-resistant organisms (15, 16).In the present study, we developed an algorithm (entropy-based method for microbial ecology research, EMMER), based on the von Neumann entropy calculation from quantum information theory (17, 18), to identify representative characteristics of fecal microbiomes serially sampled from litters of pigs that were or were not subjected to maternal diet restriction (DR) in utero and then provided either ad libitum access to, or restricted amounts of, a sequence of diets commonly given to farm-reared pigs as they complete their growth cycle. A 45% lower weight was attained by DR compared to full-fed (FF) pigs by the third postnatal month and this difference was sustained for the remainder of the 5-mo-long study. DR microbiomes exhibited a significantly reduced representation of genes encoding enzymes involved in the degradation of polysaccharides from dominant components of diets administered after postnatal day 70. These differences in the DR microbiome were associated with diminished fecal levels of butyrate, a major source of host energy, and significant increases in plasma levels of triglycerides, glucogenic amino acids, and urea cycle precursors. Functional features of DR and FF fecal microbiomes, collected during the period of consumption of the corn/soy-rich “finisher” diet (the last given during the feeding program), were subsequently assayed in gnotobiotic mice under controlled feeding conditions where all animals were provided the same amount of the finisher phase pig diet. The results confirmed the reduced capacity of the DR microbiome to generate butyrate. Moreover, mice colonized with the DR microbiome also exhibited reduced fatty acid oxidation in the liver, a metabolic effect that could explain the redirection of amino acids from protein synthesis to replenish hepatic energy reserves in DR pigs. Marrying longitudinal studies of farm animal gut microbiome development and function, conducted in well-engineered farming systems, with gnotobiotic mouse models that incorporate the microbial communities and diets of the farm animals, provides an opportunity to develop an informed set of practices for microbiome husbandry that promotes healthy growth. The results could have substantial economic and societal impact during this time of increasing global food insecurity and when producing sufficient amounts of high-quality protein to feed a rapidly expanding human population is a major challenge (9).     

Wolbachia bacteria in filarial immunity and disease

Lymphatic filarial nematodes are infected with endosymbiotic Wolbachia bacteria. Lipopolysaccharide from these bacteria is the major activator of innate inflammatory responses induced directly by the parasite. Here, we propose a mechanism by which Wolbachia initiates acute inflammatory responses associated with death of parasites, leading to acute filarial lymphangitis and adverse reactions to antifilarial chemotherapy. We also speculate that repeated exposure to acute inflammatory responses and the chronic release of bacteria, results in damage to infected lymphatics and desensitization of the innate immune system. These events will result in an increased susceptibility to opportunistic infections, which cause acute dermatolymphangitis associated with lymphoedema and elephantiasis. The recognition of the contribution of endosymbiotic bacteria to filarial disease could be exploited for clinical intervention by the targeting of bacteria with antibiotics in an attempt to reduce the development of filarial pathology.     

Complete genome of the uncultured Termite Group 1 bacteria in a single host protist cell

Termites harbor a symbiotic gut microbial community that is responsible for their ability to thrive on recalcitrant plant matter. The community comprises diverse microorganisms, most of which are as yet uncultivable; the detailed symbiotic mechanism remains unclear. Here, we present the first complete genome sequence of a termite gut symbiont-an uncultured bacterium named Rs-D17 belonging to the candidate phylum Termite Group 1 (TG1). TG1 is a dominant group in termite guts, found as intracellular symbionts of various cellulolytic protists, without any physiological information. To acquire the complete genome sequence, we collected Rs-D17 cells from only a single host protist cell to minimize their genomic variation and performed isothermal whole-genome amplification. This strategy enabled us to reconstruct a circular chromosome (1,125,857 bp) encoding 761 putative protein-coding genes. The genome additionally contains 121 pseudogenes assigned to categories, such as cell wall biosynthesis, regulators, transporters, and defense mechanisms. Despite its apparent reductive evolution, the ability to synthesize 15 amino acids and various cofactors is retained, some of these genes having been duplicated. Considering that diverse termite-gut protists harbor TG1 bacteria, we suggest that this bacterial group plays a key role in the gut symbiotic system by stably supplying essential nitrogenous compounds deficient in lignocelluloses to their host protists and the termites. Our results provide a breakthrough to clarify the functions of and the interactions among the individual members of this multilayered symbiotic complex.     

Commensal bacteria play a role in mating preference of Drosophila melanogaster

Development of mating preference is considered to be an early event in speciation. In this study, mating preference was achieved by dividing a population of Drosophila melanogaster and rearing one part on a molasses medium and the other on a starch medium. When the isolated populations were mixed, "molasses flies" preferred to mate with other molasses flies and "starch flies" preferred to mate with other starch flies. The mating preference appeared after only one generation and was maintained for at least 37 generations. Antibiotic treatment abolished mating preference, suggesting that the fly microbiota was responsible for the phenomenon. This was confirmed by infection experiments with microbiota obtained from the fly media (before antibiotic treatment) as well as with a mixed culture of Lactobacillus species and a pure culture of Lactobacillus plantarum isolated from starch flies. Analytical data suggest that symbiotic bacteria can influence mating preference by changing the levels of cuticular hydrocarbon sex pheromones. The results are discussed within the framework of the hologenome theory of evolution.     

Multiple origins of obligate nematode and insect symbionts by a clade of bacteria closely related to plant pathogens

Obligate symbioses involving intracellular bacteria have transformed eukaryotic life, from providing aerobic respiration and photosynthesis to enabling colonization of previously inaccessible niches, such as feeding on xylem and phloem, and surviving in deep-sea hydrothermal vents. A major challenge in the study of obligate symbioses is to understand how they arise. Because the best studied obligate symbioses are ancient, it is especially challenging to identify early or intermediate stages. Here we report the discovery of a nascent obligate symbiosis in Howardula aoronymphium, a well-studied nematode parasite of Drosophila flies. We have found that H. aoronymphium and its sister species harbor a maternally inherited intracellular bacterial symbiont. We never find the symbiont in nematode-free flies, and virtually all nematodes in the field and the laboratory are infected. Treating nematodes with antibiotics causes a severe reduction in fly infection success. The association is recent, as more distantly related insect-parasitic tylenchid nematodes do not host these endosymbionts. We also report that the Howardula nematode symbiont is a member of a widespread monophyletic group of invertebrate host-associated microbes that has independently given rise to at least four obligate symbioses, one in nematodes and three in insects, and that is sister to Pectobacterium, a lineage of plant pathogenic bacteria. Comparative genomic analysis of this group, which we name Candidatus Symbiopectobacterium, shows signatures of genome erosion characteristic of early stages of symbiosis, with the Howardula symbiont’s genome containing over a thousand predicted pseudogenes, comprising a third of its genome.

Intimate symbioses involving intracellular bacteria have transformed eukaryotic life (1, 2), with mitochondria and chloroplasts as canonical examples. More recent, yet still ancient, acquisitions of obligate bacterial intracellular endosymbionts have enabled colonization and radiation by animals into previously inaccessible niches, such as feeding on plant sap and animal blood (3), and surviving in deep-sea hydrothermal vents (4). Among the most difficult questions to resolve in the study of obligate symbiosis are how do obligate symbioses evolve, and where do obligate symbionts come from? This is particularly challenging because most of the obligate symbioses that have been studied are ancient, making it extremely difficult to identify early or intermediate stages.One of the most common ways to acquire an obligate symbiont is via symbiont replacement (5). As a result of a lifestyle shaped by genetic drift, vertically transmitted obligate symbionts follow a syndrome of accumulation of deleterious mutations, leading to genome degradation and reduction (6). A common pattern is that they are replaced by other less broken symbionts that may then renew the cycle of genomic degradation (7). Here the symbiont, which is often descended from common facultative symbionts or parasites (8, 9), is fitted into an established and well-functioning symbiosis (i.e., with a “symbiont-experienced” host). For example, the symbiont Sodalis has independently given rise to numerous obligate nutritional symbioses in blood-feeding flies and lice, sap-feeding mealybugs, spittlebugs, hoppers, and grain-feeding weevils (9).Less studied are young obligate symbioses in host lineages that did not already house obligate symbionts (i.e., “symbiont-naive” hosts) (10). Some of the best known examples originate through host manipulation by the symbiont via addiction or reproductive control. Addiction or dependence may be a common route for obligate symbiosis (11), and one of the most famous examples occurred in the laboratory, on the timescale of years, where strains of Amoeba evolved to become entirely dependent on intracellular symbionts (12). Many maternally inherited symbionts of terrestrial arthropods induce parthenogenetic (i.e., all female) reproduction in their hosts (13); accumulation of deleterious mutations in genes required for sexual reproduction will result in hosts that are unable to reproduce if cured of their symbiont (14). However, despite advances in microbial surveys, there are still few examples of young obligate symbioses that result in novel host functions. One intriguing example involves spheroid bodies, nitrogen-fixing organelles found in rhopalodiacean diatoms, that originated from a single acquisition of a cyanobacterial symbiont as recently as ∼12 Mya (15, 16).Here we report the discovery of a nascent obligate symbiosis in Howardula aoronymphium, a well-studied nematode parasite of Drosophila (17), most recently in the context of a defensive symbiosis. A common host species, Drosophila neotestacea, harbors a strain of the facultative inherited symbiont Spiroplasma that protects it against nematode-induced sterility (18). The protection provided by Spiroplasma is so strong that symbiont-infected flies are spreading across North America and replacing their uninfected counterparts (19). Surprisingly, we have found that H. aoronymphium itself harbors an intracellular bacterial symbiont that is related to Pectobacterium, a well-studied group of plant pathogens often vectored by insects. We also report that the nematode symbiont, which we name Candidatus Symbiopectobacterium (and hereafter Symbiopectobacterium), is a member of a widespread lineage of invertebrate symbionts that has independently given rise to at least four obligate symbioses, one in nematodes and three in insects, representing an exciting model for the study of obligate symbiosis.     

Polynucleobacter necessarius,a model for genome reduction in both free-living and symbiotic bacteria

We present the complete genomic sequence of the essential symbiont Polynucleobacter necessarius (Betaproteobacteria), which is a valuable case study for several reasons. First, it is hosted by a ciliated protist, Euplotes; bacterial symbionts of ciliates are still poorly known because of a lack of extensive molecular data. Second, the single species P. necessarius contains both symbiotic and free-living strains, allowing for a comparison between closely related organisms with different ecologies. Third, free-living P. necessarius strains are exceptional by themselves because of their small genome size, reduced metabolic flexibility, and high worldwide abundance in freshwater systems. We provide a comparative analysis of P. necessarius metabolism and explore the peculiar features of a genome reduction that occurred on an already streamlined genome. We compare this unusual system with current hypotheses for genome erosion in symbionts and free-living bacteria, propose modifications to the presently accepted model, and discuss the potential consequences of translesion DNA polymerase loss.Symbiosis, defined as a close relationship between organisms belonging to different species (1), is a ubiquitous, diverse, and important mechanism in ecology and evolution (e.g., refs. 2–4). In extreme cases, through the establishment of symbiotic relationships, quite unrelated lineages can functionally combine their genomes and generate advantageous emergent features or initiate parasite/host arms races. Ciliates, common unicellular protists of the phylum Ciliophora, are extraordinary receptacles for prokaryotic ecto- and endosymbionts (5, 6) that provide varied examples of biodiversity and ecological roles (6). Nevertheless, most of these symbionts are understudied, partially owing to the scarcity of available molecular data and the absence of sequenced genomes. Yet, thanks to their various biologies and the ease of sampling and cultivating their protist hosts, they are excellent potential models for symbioses between bacteria and heterotrophic eukaryotes. Until recently this field was dominated by studies on endosymbionts of invertebrates, especially insects (e.g., ref. 7), although unicellular systems like amoebas (e.g., refs. 8 and 9) have been shown to be suitable models.Polynucleobacter necessarius was first described as a cytoplasmic endosymbiont of the ciliate Euplotes aediculatus (10, 11). Further surveys detected its presence in a monophyletic group of fresh and brackish water Euplotes species (12, 13). All of the investigated strains of these species die soon after being cured of the endosymbiont (10, 12, 13). In the few cases in which P. necessarius is not present, a different and rarer bacterium apparently supplies the same function (12, 14). No attempt to grow symbiotic P. necessarius outside their hosts has yet been successful (15), strongly suggesting that the relationship is obligate for both partners, in contrast to most other known prokaryote/ciliate symbioses (6).Thus, the findings of many environmental 16S rRNA gene sequences similar to that of the symbiotic P. necessarius (16) but belonging to free-living freshwater bacteria came as a surprise. These free-living strains, which have been isolated and cultivated (17), are ubiquitous and abundant in the plankton of lentic environments (17, 18). They are smaller and do not show the most prominent morphological feature of the symbiotic form: the presence of multiple nucleoids, each containing one copy of the genome (10, 11). It is clear that free-living and endosymbiotic P. necessarius are not different life stages of the same organism (15). Nevertheless, these strikingly different bacteria, occupying separate ecological niches, exhibit >99% 16S rRNA gene sequence identity, and phylogenetic analyses fail to separate them into two distinct groups (15). Rather, several lines of evidence point to multiple, recent origins of symbiotic strains from the free-living bacterial pool (14, 15).Thus, the Euplotes–Polynucleobacter symbiosis provides a promising system for the study of changes promoting or caused by the shift to an intracellular lifestyle. The remarkably small (2.16 Mbp) genome of the free-living strain QLW-P1DMWA-1 has been sequenced and studied, especially for features that would explain the success of this lineage in freshwater systems worldwide (19, 20). Phylogenies based on the 16S rRNA gene (13, 14) and multiple-gene analyses (19, 21, 22) consistently cluster Polynucleobacter with bacteria of the family Burkholderiaceae (Betaproteobacteria), either in a basal position or as the sister group of Ralstonia and Cupriavidus.Here we provide the complete genomic sequence of a symbiotic P. necessarius harbored in the cytoplasm of E. aediculatus and present a comparative analysis of the two sequenced Polynucleobacter genomes, addressing the possible biological basis of the Euplotes–Polynucleobacter symbiosis. We also provide insights into the evolution of the unique two-step genome reduction in this bacterial species: the first step involving streamlining in a free-living ancestor and the second a more recent period of genome erosion confined to the symbiotic lineage.     

Cough in the elderly: a novel strategy for preventing aspiration pneumonia

Management of cough in the elderly with a deteriorated physical and mental status has received little focus. Since an aged population is rapidly increasing in developed countries, the research in this population are warranted. Cough reflex sensitivity in the elderly was shown to be hypersensitive, normosensitive and hyposensitive. The hypersensitive cough reflex is mostly due to gastro-esophageal reflux in nursing home patients. Impaired cough reflex sensitivity is assumed to play a crucial role in the development of pneumonia in the elderly. A marked depression of cough reflex sensitivity is reported in elderly patients with aspiration pneumonia. The impairment of the cough reflex in patients with aspiration pneumonia can involve both cortical facilitatory pathways for cough and medullary reflex pathways. We found the urge-to-cough in patients with aspiration pneumonia was also down-regulated, suggesting the involvement of supramedullary dysfunction in the etiology of aspiration pneumonia in the elderly. In order to prevent aspiration pneumonia in the elderly, restoration of cough reflex sensitivity is essential. We found several methods to restore cough reflex sensitivity in the elderly. They also improved the swallowing reflex, another important airway protective reflex, in the elderly. In the treatment of aspiration pneumonia, one of the most challenging steps is the start of eating for patients who usually fast at the time of hospitalization. By combining the methods to restore the cough reflex sensitivity and swallowing reflex, we developed a protocol to start eating in the elderly patients with aspiration pneumonia. Using the protocol, we reduced the incidence of re-aspiration due to start of eating in patients with aspiration pneumonia to one third of the patients without the protocol.     

Proteasome inhibition as novel treatment strategy in leukaemia

Following its success in multiple myeloma (MM), proteasome inhibition has become a topic of interest as novel treatment strategy of cancer. By simultaneously affecting multiple pathways in the cancer cell, such as deregulation of the programmed degradation of many cellular proteins, proteasome inhibition causes rapid apoptosis of these cells. Both in rapidly proliferating leukaemic cell lines and in primary leukaemic cells isolated from patients, proteasome inhibition results in antileukaemic activity. The normal counterparts of these cells are much more resistant to proteasome inhibitors (PI), thereby resulting in a favourable therapeutic index. Importantly, while leukaemic stem cells are sensitive to proteasome inhibition, normal haematopoietic stem cells are still viable after drug exposure. Nowadays, many PIs are being identified; bortezomib is the most well known since obtaining Food and Drug Administration approval for clinical use in MM. This review summarises the biological and clinical aspects of proteasome inhibition and discusses the potential role of these inhibitors in the treatment of leukaemia.     

180例次消化道疾病患者致病菌检出率及其耐药性分析

目的调查消化道疾病住院患者临床标本中致病菌菌种的分布及对常用抗菌药物的耐药性，为合理应用抗生素提供依据，方法对消化科180例次住院患者各种临床标本进行常规细菌培养，共分离出29株致病细菌，应用API系统对细菌进行鉴定，并采用VITEK32系统测定其对临床常用抗生素的敏感性。结果总阳性率为16．11％(29／180)，以呼吸道标本阳性率最高，达39．47％(15／38)，其中以鲍曼复合醋酸钙不动杆菌、肺炎克雷伯菌最常见，粪便及尿液标本以大肠埃希菌最常见，药敏试验显示。G^-杆菌对喹诺酮类及头孢三代明显耐药，但对亚胺培南仍高度敏感；G^ 球菌对青霉素、红霉素、苯唑青毒素等也高度耐药，惟对万古霉素高度敏感。结论消化道疾病住院患者常见致病菌仍以G^-杆菌为主，G^-杆菌及G^ 球菌对大部分常用抗生素均呈高度耐药。     

Generalized antifungal activity and 454-screening of Pseudonocardia and Amycolatopsis bacteria in nests of fungus-growing ants

In many host-microbe mutualisms, hosts use beneficial metabolites supplied by microbial symbionts. Fungus-growing (attine) ants are thought to form such a mutualism with Pseudonocardia bacteria to derive antibiotics that specifically suppress the coevolving pathogen Escovopsis, which infects the ants'' fungal gardens and reduces growth. Here we test 4 key assumptions of this Pseudonocardia-Escovopsis coevolution model. Culture-dependent and culture-independent (tag-encoded 454-pyrosequencing) surveys reveal that several Pseudonocardia species and occasionally Amycolatopsis (a close relative of Pseudonocardia) co-occur on workers from a single nest, contradicting the assumption of a single pseudonocardiaceous strain per nest. Pseudonocardia can occur on males, suggesting that Pseudonocardia could also be horizontally transmitted during mating. Pseudonocardia and Amycolatopsis secretions kill or strongly suppress ant-cultivated fungi, contradicting the previous finding of a growth-enhancing effect of Pseudonocardia on the cultivars. Attine ants therefore may harm their own cultivar if they apply pseudonocardiaceous secretions to actively growing gardens. Pseudonocardia and Amycolatopsis isolates also show nonspecific antifungal activities against saprotrophic, endophytic, entomopathogenic, and garden-pathogenic fungi, contrary to the original report of specific antibiosis against Escovopsis alone. We conclude that attine-associated pseudonocardiaceous bacteria do not exhibit derived antibiotic properties to specifically suppress Escovopsis. We evaluate hypotheses on nonadaptive and adaptive functions of attine integumental bacteria, and develop an alternate conceptual framework to replace the prevailing Pseudonocardia-Escovopsis coevolution model. If association with Pseudonocardia is adaptive to attine ants, alternate roles of such microbes could include the protection of ants or sanitation of the nest.     

Incretins as a novel therapeutic strategy in patients with diabetes and heart failure

Heart failure (HF) and diabetes mellitus (DM) commonly co-exist, with a prevalence of DM of up to 40 % in HF patients. Treatment of DM in patients with HF is challenging since many of the contemporary therapies used for the treatment of DM are either contraindicated in HF or are limited in their use due to the high prevalence of co-morbidities such as significant renal dysfunction. This article presents an overview of the physiology of the incretin system and how it can be targeted therapeutically, highlighting implications for the management of patients with DM and HF. Receptors for the incretin glucagon-like peptide-1 (GLP-1) are expressed throughout the cardiovascular system and the myocardium and are up-regulated in HF. GLP-1 therapy improves cardiac function in animal models of HF through augmented glucose uptake in the myocardium mediated through a p38 MAP kinase pathway. Small clinical studies have shown that GLP-1 improves ejection fraction, reduces BNP levels and enhances functional capacity in patients with chronic HF. A number of randomized controlled trials are currently underway to define the utility of targeting the incretin system in HF patients with DM. Incretin-based therapy may represent a novel therapeutic strategy in the treatment of HF patients with diabetes, in particular for their cardioprotective effects independent of those attributable to tight glycemic control.     

Future expectations in digestive endoscopy: competition with other novel imaging techniques

Digestive endoscopy has been evolving from primary diagnostic to extensive therapeutic modalities in the management of gastrointestinal diseases. The present endoscopic imaging includes (A) standard endoscopy alone and /or with adjunct technologies such as point enhancement, e.g. confocal endomicroscopy and field enhancement technologies such as chromoendoscopy, NBI and FICE and (B) endoscopic ultrasound. Other novel imaging technologies including virtual colonoscopy or CT/MR colonography, CT or MRI enterography and capsule endoscopy have also been developed. This article reviews the diagnostic and therapeutic role of digestive endoscopy and future directions of digestive endoscopy are discussed. Digestive endoscopy is also compared with emerging novel imaging techniques in gastrointestinal diseases such as capsule endoscopy and CT colonography. The fact that digestive endoscopy has become a multidisciplinary specialty combining advances in all fields (radiology, bioengineering, surgery and gastroenterology) is highlighted.     

Therapeutic vaccines based on genetically modified Salmonella: a novel strategy in cancer immunotherapy

In the course of evolution, bacteria from the genus Salmonella adapted to survive and multiply in a vertebrate host. Skillful use of bacterial interactions with the host immune system became the basis for the development of modified Salmonella-based therapeutic vaccines. Bacterial genome can be modified to reduce toxicity and to develop or enhance therapeutic activity. Salmonella-based therapeutic vaccines are an attractive and novel alternative for conventional cancer treatment (chemotherapy, radiotherapy, and passive immunotherapy). Live bacteria have the natural ability to sense external environment and penetrate the target tissue. Appropriate strains of Salmonella, infused into experimental animal tumor model, accumulate selectively in solid tumors and inhibit their growth. Moreover, the bacteria can reach tumor areas that are inaccessible for other, passively diffusing therapeutics, e.g., ischemic areas. Thus, bacteria can produce and locally release a natural or recombinant anticancer agent, which enhances their therapeutic effect. S. typhimurium VNP20009 strain is safe, which has been documented in clinical trials. However, the expected therapeutic benefit has not been observed, presumably due to insufficient tumor colonization by bacteria. To enhance colonization of solid tumors, VNP20009 bacteria have been equipped with the ability to express on the surface an antibody fragment specific for carcinoembryonic antigen present on human tumor cells. Additionally, to potentiate antitumor activity, the genetic material of VNP20009 has been engineered to overproduce an endogenous proapoptotic protein, which targets cancer and immune cells promoting tumor growth.     

70S-scanning initiation is a novel and frequent initiation mode of ribosomal translation in bacteria

According to the standard model of bacterial translation initiation, the small ribosomal 30S subunit binds to the initiation site of an mRNA with the help of three initiation factors (IF1–IF3). Here, we describe a novel type of initiation termed “70S-scanning initiation,” where the 70S ribosome does not necessarily dissociate after translation of a cistron, but rather scans to the initiation site of the downstream cistron. We detailed the mechanism of 70S-scanning initiation by designing unique monocistronic and polycistronic mRNAs harboring translation reporters, and by reconstituting systems to characterize each distinct mode of initiation. Results show that 70S scanning is triggered by fMet-tRNA and does not require energy; the Shine–Dalgarno sequence is an essential recognition element of the initiation site. IF1 and IF3 requirements for the various initiation modes were assessed by the formation of productive initiation complexes leading to synthesis of active proteins. IF3 is essential and IF1 is highly stimulating for the 70S-scanning mode. The task of IF1 appears to be the prevention of untimely interference by ternary aminoacyl (aa)-tRNA•elongation factor thermo unstable (EF-Tu)•GTP complexes. Evidence indicates that at least 50% of bacterial initiation events use the 70S-scanning mode, underscoring the relative importance of this translation initiation mechanism.It is textbook knowledge that 30S subunits initiate protein synthesis in bacteria; they recognize the initiation site of the mRNA composed of the Shine–Dalgarno (SD) sequence, the AUG codon, and fMet-tRNA, together with three initiation factors (IFs) forming the 30S initiation complex (30SIC). Association of the large 50S subunit triggers the release of the IFs, leading to the 70S initiation complex (70SIC) that enters the elongation phase of translation (reviewed in 1). We term this initiation path the “30S-binding mode” of bacterial initiation. After elongation and termination, it is thought that the ribosome dissociates into its subunits, thus providing 30S subunits for the next round of initiation.The functional role of IF2 is well defined. It can bind directly to the 30S, providing a docking site for fMet-tRNA (2), but it can also enter the 30S subunit as ternary complex fMet-tRNA•IF2•GTP (3). Both IF2 and IF3 are essential for viability. IF3 has a binding site at the 30S interface (4), which explains its antiassociation effect (5, 6), as well as its role in dissociation of the terminating 70S ribosome (7). However, the in vivo concentration of IF3 is 100-fold less (8) than required for full dissociation of 70S in vitro (4). Evidence for the presence of IF3 on 70S ribosomes was reported (9), indicating that the functional spectrum of IF3 is possibly not restricted to an antiassociation effect. Both IF3 and IF2 are also responsible for the fidelity of decoding the initiation AUG by fMet-tRNAfMet at the P site of 30S subunits (10).IF1 is universal (11) and essential for viability (12). It is the smallest factor, with 72 amino acid residues in Escherichia coli, and binds to the decoding center at the ribosomal A site (13). Several functions have been described, including stimulation of the formation of the 30SIC and subunit association (14). Interference with the binding of ternary complexes aminoacyl (aa)-tRNA•elongation factor thermo unstable (EF-Tu)•GTP to 30S subunits has also been suggested (15). Omitting IF1 in 30S-binding tests decreased the accuracy of fMet-tRNA selection over the elongator Phe-tRNA about 60-fold, which was suggested to account for the essential nature of IF1 (16). All three factors are thought to dissociate upon 50S arrival or shortly thereafter (1). IF1 is required for proper initiator-tRNA selection on 70S along with IF2 and IF3, in contrast to the 30SIC, where IF2 and IF3 provide tRNA selection (17).In addition to the 30S-binding initiation, a second initiation mode exists that has a niche existence in bacteria: Leaderless mRNA (lmRNA) contains an initiator AUG codon within the first 5 nt at the 5′-end, and thus does not contain an SD sequence. This initiation mode uses 70S ribosomes with the special feature that the ribosomal proteins S1 and S2 are not required, which are otherwise important for the 30S-binding mode (18). Initiation of lmRNA can even occur in the absence of all IFs (19, 20). Additional information about lmRNAs is provided in SI Appendix, Introduction.The existence of a third initiation mode, viz. a 70S type of bacterial initiation, has been conjectured several times previously (21–23), although no in-depth mechanistic evidence has verified this mode thus far. For example:i) The formylation of the initiator Met-tRNAfMet in bacteria was interpreted as an indication of a 70S initiation mode (22). Indeed, only the anticodon loop of a tRNA and a part of the anticodon stem interact with the 30S subunit (24, 25), leaving the fMet residue as a substrate for the peptidyltransferase center on the large subunit within the 70S ribosome.ii) When an AUG codon without a preceding SD sequence follows a stop codon within a distance of <20 nt, a mutational study unexpectedly revealed that efficient protein synthesis can be initiated in vivo at this AUG codon. The interpretation was that ribosomes were sliding down from the stop codon of the preceding cistron, although it was not analyzed whether 70S ribosomes or 30S subunits were involved in sliding or whether factors were required (26). Further evidence for a 70S type of initiation is described in SI Appendix, Introduction and concerns both studies of translational coupling and a consideration of the fact that more than 75% of the intercistronic distances are shorter than 30 nt, which is too short to allow an independent termination of cistron n and initiation of downstream cistron n + 1 (SI Appendix, Fig. S1 A and B).Here, we demonstrate that there is an additional and frequent initiation mode that we term “70S-scanning initiation.” The 70S ribosomes, rather than the 30S subunits, scan the sequence surrounding the termination signal for the presence of an SD sequence after termination. Furthermore, we show that the requirement of IF1 and IF3 for the three initiation modes (30S binding, 70S scanning, and initiation of lmRNAs) is distinct for each mode.