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Purpose: To examine the impact of abutment screw head sizes on preload stability when secured to a standard external hex implant under oblique compressive forces., Materials and Method: Fifteen metal crowns were divided into three equal groups. The first group had five angulated cemented crowns connected to a 3 mm tall straight hexagonal abutment with an external hex abutment screw. The second and third groups each had five straight cemented crowns attached to a tapered abutment with flat slotted and internal hex abutment screws, respectively. Samples were subjected to a static cyclic load until failure. Kruskal-Wallis H, Dunn's, and one-way ANOVA with Tukey HSD tests were performed., Results: Cemented straight crown supported by an angled abutment connected to implants with flat slotted and internal hex abutment screw heads failed at an average of 4.24 x 106 cycles ± 3.31 SD and 12.67 x 106 cycles ± 5.47 SD, respectively. Cemented angled crowns supported by a straight abutment connected to identical implants with an external hex abutment screw survived 18.02 x 106 cycles ± 4.49 SD. Periotest value (PTV) rate of change increased at a higher rate in crowns supported by angled abutments compared to straight abutments (p < 0.05). No cement failure was observed., Conclusion: Under the experimental conditions, larger abutment screw head sizes demonstrated greater stability of the abutment screw joint interface. Based on the in vitro findings, no cement failure was observed between the cemented crown and abutment connection. Future research with standardized comparative setups and larger sample sizes is needed.

Purpose: To compare the effect of taper, length, angle, and number of vertical axial walls on the retentive strength of a cemented crown on a partially customized hybrid abutment in the esthetic zone., Materials and Methods: A total of 35 metal copings were used and divided into two groups. One group had 30 copings cemented to their corresponding 8-degree tapered abutment with lengths from 3 to 8 mm, increasing in 1-mm increments (5 copings per length). The other group had the remaining 5 copings and consisted of a standardized metal coping that matched a hexagonal abutment with 3-mm vertical axial walls and the sequential removal of 1, 2, and 3 contiguous vertical axial walls. Dislodgment tests were performed for all copings in both groups. Maximum retentive forces were measured in retentive strength (kgF), with a conversion factor of 9.807 N &#61; 1 kgF., Results: At each tapered abutment length, the retentive strength increased proportionally and was significantly different, ranging from 31.67 ± 4.10 kgF to 67.68 ± 11.22 kgF (F [5,24] &#61; 20.46, P < .001). An unmodified hexagonal abutment demonstrated the highest retentive strength (70.15 ± 12.97 kgF). The sequential removal of 1, 2, and 3 contiguous vertical axial walls of the hexagonal abutment resulted in retentive strength values of 59.89 ± 10.06 kgF, 57.01 ± 9.62 kgF, and 55.99 ± 9.35 kgF, respectively, with no significant difference (P > .05) in strength., Conclusions: A partially customized abutment with vertical axial walls on one side and a profile reduction on the opposite side can provide comparable retention to cemented copings at one-third the length and at one-sixth the surface area of an 8-mm abutment with an 8-degree taper.

Purpose: To investigate the resistance of an implant crown telescopically engaged to a geometrically defined hexagonal abutment with and without cement under compressive dynamic cyclic fatigue loading., Materials and Methods: 40 crowns, 9 mm in height, were cast from prefabricated plastic copings, each telescopically engaged to a 3-mm-high machined hexagonal abutment. 20 crowns were retained with zinc phosphate cement, and 20 crowns were retained with a screw. A vertical load of 15 kg was applied to all samples under oscilla)on. A Periotest was used to measure the mobility of the implant crown in Periotest values (PTV). Test endpoints were defined by the following: fatigue cycles ≥ 20 million; crown PTV > 10; or if samples became visibly loose or component fracture., Results: Cement-retained crowns failed on average at 2.60 x 106 cycles ± 2.27, while screw-retained crown samples failed at 2.17 x 106 cycles ± 1.27 with no significant difference (P > .05). Implant and abutment screw fractures were the most prevalent mode of failure in the cement-retained group, while in the screw-retained group, failures were caused by the loosening of one or both screw joints. The rate of increase in PTV was higher in the screw-retained group than in the cement-retained group., Conclusions: Under the experimental conditions, an implant crown telescopically engaged vertically to a 3-mm-tall hexagonal abutment, under compressive dynamic cyclic fatigue loading with or without cement, demonstrated no differences in resistance or failure outcomes. A cement-retained implant crown with telescopic engagement to the abutment is more rigid, resulting in more implant and abutment screw fractures than loosening.

The rapid expansion of multi-omics data has transformed biological research, offering unprecedented opportunities to explore complex genomic relationships across diverse organisms. However, the vast volume and heterogeneity of these datasets presents significant challenges for analyses. Here we introduce SocialGene, a comprehensive software suite designed to collect, analyze, and organize multi-omics data into structured knowledge graphs, with the ability to handle small projects to repository-scale analyses. Originally developed to enhance genome mining for natural product drug discovery, SocialGene has been effective across various applications, including functional genomics, evolutionary studies, and systems biology. SocialGene's concerted Python and Nextflow libraries streamline data ingestion, manipulation, aggregation, and analysis, culminating in a custom Neo4j database. The software not only facilitates the exploration of genomic synteny but also provides a foundational knowledge graph supporting the integration of additional diverse datasets and the development of advanced search engines and analyses. This manuscript introduces some of SocialGene's capabilities through brief case studies including targeted genome mining for drug discovery, accelerated searches for similar and distantly related biosynthetic gene clusters in biobank-available organisms, integration of chemical and analytical data, and more. SocialGene is free, open-source, MIT-licensed, designed for adaptability and extension, and available from github.com/socialgene.

Microbial symbionts associate with multicellular organisms on a continuum from facultative associations to mutual codependency. In some of the oldest intracellular symbioses there is exclusive vertical symbiont transmission, and co-diversification of symbiotic partners over millions of years. Such symbionts often undergo genome reduction due to low effective population sizes, frequent population bottlenecks, and reduced purifying selection. Here, we describe multiple independent acquisition events of closely related defensive symbionts followed by genome erosion in a group of Lagriinae beetles. Previous work in Lagria villosa revealed the dominant genome-eroded symbiont of the genus Burkholderia produces the antifungal compound lagriamide and protects the beetle's eggs and larvae from antagonistic fungi. Here, we use metagenomics to assemble 11 additional genomes of lagriamide-producing symbionts from seven different host species within Lagriinae from five countries, to unravel the evolutionary history of this symbiotic relationship. In each host species, we detected one dominant genome-eroded Burkholderia symbiont encoding the lagriamide biosynthetic gene cluster (BGC). Surprisingly, however, we did not find evidence for host-symbiont co-diversification, or for a monophyly of the lagriamide-producing symbionts. Instead, our analyses support at least four independent acquisition events of lagriamide-encoding symbionts and subsequent genome erosion in each of these lineages. By contrast, a clade of plant-associated relatives retained large genomes but secondarily lost the lagriamide BGC. In conclusion, our results reveal a dynamic evolutionary history with multiple independent symbiont acquisitions characterized by high degree of specificity. They highlight the importance of the specialized metabolite lagriamide for the establishment and maintenance of this defensive symbiosis., Competing Interests: Competing Interests The Kwan lab offers their metagenomic binning pipeline Autometa on the paid bioinformatics and computational platform BatchX in addition to distributing it through open source channels.

Lithified layers of complex microbial mats known as microbialites are ubiquitous in the fossil record, and modern forms are increasingly identified globally. A key challenge to developing an understanding of microbialite formation and environmental role is how to investigate complex and diverse communities in situ. We selected living, layered microbialites (stromatolites) in a peritidal environment near Schoenmakerskop, Eastern Cape, South Africa to conduct a spatial survey mapping the composition and small molecule production of the microbial communities from environmental samples. Substrate core samples were collected from nine sampling stations ranging from the upper point of the freshwater inflow to the lower marine interface where tidal overtopping takes place. Substrate cores provided material for parallel analyses of microbial community diversity by 16S rRNA gene amplicon sequencing and metabolomics using LC-MS2. Species and metabolite diversities were correlated, and prominent specialized metabolites were targeted for preliminary characterization. A new series of cyclic hexadepsipeptides, named ibhayipeptolides, was most abundant in substrate cores of submerged microbialites. These results demonstrate the detection and identification of metabolites from mass-limited environmental samples and contribute knowledge about microbialite chemistry and biology, which facilitates future targeted studies of specialized metabolite function and biosynthesis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Neuhaus et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Microorganisms from the order Burkholderiales have been the source of a number of important classes of natural products in recent years. For example, study of the beetle-associated symbiont Burkholderia gladioli led to the discovery of the antifungal polyketide lagriamide; an important molecule from the perspectives of both biotechnology and chemical ecology. As part of a wider project to sequence Burkholderiales genomes from our in-house Burkholderiales library we identified a strain containing a biosynthetic gene cluster (BGC) similar to the original lagriamide BGC. Structure prediction failed to identify any candidate masses for the products of this BGC from untargeted metabolomics mass spectrometry data. However, genome mining from publicly available databases identified fragments of this BGC from a culture collection strain of Paraburkholderia . Whole genome sequencing of this strain revealed the presence of a homologue of this BGC with very high sequence identity. Stable isotope feeding of the two strains in parallel using our newly developed IsoAnalyst platform identified the product of this lagriamide-like BGC directly from the crude fermentation extracts, affording a culturable supply of this interesting compound class. Using a combination of bioinformatic, computational and spectroscopic methods we defined the absolute configurations for all 11 chiral centers in this new metabolite, which we named lagriamide B. Biological testing of lagriamide B against a panel of 21 bacterial and fungal pathogens revealed antifungal activity against the opportunistic human pathogen Aspergillus niger , while image-based Cell Painting analysis indicated that lagriamide B also causes actin filament disruption in U2-OS osteosarcoma cells., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Objectives: Obesity and type 2 diabetes mellitus (T2DM) are growing health concerns. Since 2005, Student Health Service (SHS) and Hong Kong Paediatric Society formulated a protocol on urine glucose screening (UGS) for early diagnosis of T2DM in students with obesity in Hong Kong. This study reviews students with T2DM captured by this screening program and compare the data with the Hong Kong Children Diabetes Registry (HKCDR) database, to see if the UGS program facilitates early diagnosis of T2DM., Methods: Students between the ages of 10-18 years old with age- and sex-specific body mass index (BMI) >97th percentile who attended SHS between the school years from 2005/06 to 2017/18 were recruited for UGS. Those tested positive for random urine glucose underwent diagnostic testing for T2DM according to ADA guidelines. Demographic data and investigatory results from UGS and HKCDR within the same time period were compared., Results: A total of 216,526 students completed UGS in the said period; 415 (0.19 %) students were tested positive for urine glucose of which 121 students were diagnosed with T2DM. UGS picked up 23 % of the newly diagnosed T2DM cases. When compared to the HKCDR database, students diagnosed via UGS were significantly younger, less obese, and had fewer diabetic related complications. The negative predictive value of UGS is high and can effectively rule out T2DM., Conclusions: Urine glucose screening is an inexpensive and simple test that allows for early diagnosis of T2DM among obese school students. Other methods including POCT HbA 1c can be explored to improve program effectiveness., (© 2023 Walter de Gruyter GmbH, Berlin/Boston.)

Infection is a major complication associated with orthopedic implants. It often involves the development of biofilms on metal substrates, which act as barriers to the host's immune system and systemic antibiotic treatment. The current standard of treatment is revision surgery, often involving the delivery of antibiotics through incorporation into bone cements. However, these materials exhibit sub-optimal antibiotic release kinetics and revision surgeries have drawbacks of high cost and recovery time. Herein, a new approach is presented using induction heating of a metal substrate, combined with an antibiotic-loaded poly(ester amide) coating undergoing a glass transition just above physiological temperature to enable thermally triggered antibiotic release. At normal physiological temperature, the coating provides a rifampicin depot for >100 days, while heating of the coating accelerates drug release, with >20% release over a 1-h induction heating cycle. Induction heating or antibiotic-loaded coating alone each reduce Staphylococcus aureus (S. aureus) viability and biofilm formation on Ti, but the combination causes synergistic killing of S. aureus as measured by crystal violet staining, determination of bacterial viability (>99.9% reduction), and fluorescence microscopy of bacteria on surfaces. Overall, these materials provide a promising platform enabling externally triggered antibiotic release to prevent and/or treat bacterial colonization of implants., (© 2023 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)

Head and neck cancers (HNCs) account for ~4% of all cancers in North America and encompass cancers affecting the oral cavity, pharynx, larynx, sinuses, nasal cavity, and salivary glands. The anatomical complexity of the head and neck region, characterized by highly perfused and innervated structures, presents challenges in the early diagnosis and treatment of these cancers. The utilization of sub-microliter volumes and the unique phenomenon associated with microscale fluid dynamics have facilitated the development of microfluidic platforms for studying complex biological systems. The advent of on-chip microfluidics has significantly impacted the diagnosis and treatment strategies of HNC. Sensor-based microfluidics and point-of-care devices have improved the detection and monitoring of cancer biomarkers using biological specimens like saliva, urine, blood, and serum. Additionally, tumor-on-a-chip platforms have allowed the creation of patient-specific cancer models on a chip, enabling the development of personalized treatments through high-throughput screening of drugs. In this review, we first focus on how microfluidics enable the development of an enhanced, functional drug screening process for targeted treatment in HNCs. We then discuss current advances in microfluidic platforms for biomarker sensing and early detection, followed by on-chip modeling of HNC to evaluate treatment response. Finally, we address the practical challenges that hinder the clinical translation of these microfluidic advances.

Innovation in the healthcare profession to solve complex human problems has always been emulated and based on solutions proven by nature. The conception of different biomimetic materials has allowed for extensive research that spans several fields, including biomechanics, material sciences, and microbiology. Due to the atypical characteristics of these biomaterials, dentistry can benefit from these applications in tissue engineering, regeneration, and replacement. This review highlights an overview of the application of different biomimetic biomaterials in dentistry and discusses the key biomaterials (hydroxyapatite, collagen, polymers) and biomimetic approaches (3D scaffolds, guided bone and tissue regeneration, bioadhesive gels) that have been researched to treat periodontal and peri-implant diseases in both natural dentition and dental implants. Following this, we focus on the recent novel application of mussel adhesive proteins (MAPs) and their appealing adhesive properties, in addition to their key chemical and structural properties that relate to the engineering, regeneration, and replacement of important anatomical structures in the periodontium, such as the periodontal ligament (PDL). We also outline the potential challenges in employing MAPs as a biomimetic biomaterial in dentistry based on the current evidence in the literature. This provides insight into the possible increased functional longevity of natural dentition that can be translated to implant dentistry in the near future. These strategies, paired with 3D printing and its clinical application in natural dentition and implant dentistry, develop the potential of a biomimetic approach to overcoming clinical problems in dentistry.

Invertebrates, particularly sponges, have been a dominant source of new marine natural products. For example, lasonolide A (LSA) is a potential anticancer molecule isolated from the marine sponge Forcepia sp., with nanomolar growth inhibitory activity and a unique cytotoxicity profile against the National Cancer Institute 60-cell-line screen. Here, we identified the putative biosynthetic pathway for LSA. Genomic binning of the Forcepia sponge metagenome revealed a Gram-negative bacterium belonging to the phylum Verrucomicrobia as the candidate producer of LSA. Phylogenetic analysis showed that this bacterium, here named " Candidatus Thermopylae lasonolidus," only has 88.78% 16S rRNA identity with the closest relative, Pedosphaera parvula Ellin514, indicating that it represents a new genus. The lasonolide A ( las ) biosynthetic gene cluster (BGC) was identified as a trans -acyltransferase (AT) polyketide synthase (PKS) pathway. Compared with its host genome, the las BGC exhibits a significantly different GC content and pentanucleotide frequency, suggesting a potential horizontal acquisition of the gene cluster. Furthermore, three copies of the putative las pathway were identified in the candidate producer genome. Differences between the three las repeats were observed, including the presence of three insertions, two single-nucleotide polymorphisms, and the absence of a stand-alone acyl carrier protein in one of the repeats. Even though the verrucomicrobial producer shows signs of genome reduction, its genome size is still fairly large (about 5 Mbp), and, compared to its closest free-living relative, it contains most of the primary metabolic pathways, suggesting that it is in the early stages of reduction. IMPORTANCE While sponges are valuable sources of bioactive natural products, a majority of these compounds are produced in small quantities by uncultured symbionts, hampering the study and clinical development of these unique compounds. Lasonolide A (LSA), isolated from marine sponge Forcepia sp., is a cytotoxic molecule active at nanomolar concentrations, which causes premature chromosome condensation, blebbing, cell contraction, and loss of cell adhesion, indicating a novel mechanism of action and making it a potential anticancer drug lead. However, its limited supply hampers progression to clinical trials. We investigated the microbiome of Forcepia sp. using culture-independent DNA sequencing, identified genes likely responsible for LSA synthesis in an uncultured bacterium, and assembled the symbiont's genome. These insights provide future opportunities for heterologous expression and cultivation efforts that may minimize LSA's supply problem.

Purpose: To evaluate the effects of vertical compressive cyclic load on abutment screws and the stability of screw-retained splinted fixed dental prostheses supported by bone-level nonengaging and partially engaging abutments., Materials and Methods: Ten standardized two-unit prostheses integrated with nonengaging and partially engaging abutments were 3D-printed and cast in nonprecious metal and attached to two implant analogs with conical connections angled 15 degrees toward the vertical axis in a resin bock. The common path of insertion for the prosthesis with partially engaging abutments to angled implant analogs was created by selectively reducing the abutment surface that obstructed the implant housing. A 500-N average load was applied to each sample while oscillating 30 degrees from the vertical axis at 60 Hz to failure. Prosthesis stability was measured by deflection from the initial position using a linear displacement measuring device. Sample groups were assessed using the independent-samples t test and one-way repeated-measures analysis of variance (ANOVA) with post hoc tests., Results: The mean cycles to failure for nonengaging and partially engaging abutment groups were 27,180 ± 29,420 and 457,890 ± 265,734, respectively. Failed nonengaging samples had 9 out of 10 abutment screws fracture inside the implant housing but with minimal wear to the top of the implant analog. For partially engaging samples, 8 out of 10 implant housings and 7 out of 10 abutment screws fractured at test endpoints. There was no difference in initial deflections between groups. Prostheses supported by nonengaging abutments failed before deflection measurements could be taken after loading. Prosthesis displacement of partially engaged abutments decreased significantly from the initial position after 300,000 load cycles., Conclusion: Under the experimental conditions, screw-retained splinted fixed dental prostheses supported by partially engaging abutments are 17 times more stable than prostheses supported by nonengaging abutments. Abutment screw fractures are the most prevalent mode of failure for nonengaging abutments at significantly lower cycles with minimal wear on implant analogs. Partially engaging abutment groups failed from implant housing and abutment screw fractures at higher cycles. Loading appears to concentrate preferentially on the medial side on all angled implant components. Selective removal of the abutment obstruction allows a common path of insertion for multiple implants and partial engagement to implant housings.

The ongoing coronavirus disease 2019 (COVID-19) pandemic demonstrates the threat posed by novel coronaviruses to human health. Coronaviruses share a highly conserved cell entry mechanism mediated by the spike protein, the sole product of the S gene. The structural dynamics by which the spike protein orchestrates infection illuminate how antibodies neutralize virions and how S mutations contribute to viral fitness. Here, we review the process by which spike engages its proteinaceous receptor, angiotensin converting enzyme 2 (ACE2), and how host proteases prime and subsequently enable efficient membrane fusion between virions and target cells. We highlight mutations common among severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern and discuss implications for cell entry. Ultimately, we provide a model by which sarbecoviruses are activated for fusion competency and offer a framework for understanding the interplay between humoral immunity and the molecular evolution of the SARS-CoV-2 Spike. In particular, we emphasize the relevance of the Canyon Hypothesis (M. G. Rossmann, J Biol Chem 264:14587-14590, 1989) for understanding evolutionary trajectories of viral entry proteins during sustained intraspecies transmission of a novel viral pathogen.

The fossil record indicates that the earliest evidence of extant marine sponges (phylum Porifera) existed during the Cambrian explosion and that their symbiosis with microbes may have begun in their extinct ancestors during the Precambrian period. Many symbionts have adapted to their sponge host, where they perform specific, specialized functions. There are also widely distributed bacterial taxa such as Poribacteria , SAUL , and Tethybacterales that are found in a broad range of invertebrate hosts. Here, we added 11 new genomes to the Tethybacterales order, identified a novel family, and show that functional potential differs between the three Tethybacterales families. We compare the Tethybacterales with the well-characterized Entoporibacteria and show that these symbionts appear to preferentially associate with low-microbial abundance (LMA) and high-microbial abundance (HMA) sponges, respectively. Within these sponges, we show that these symbionts likely perform distinct functions and may have undergone multiple association events, rather than a single association event followed by coevolution. IMPORTANCE Marine sponges often form symbiotic relationships with bacteria that fulfil a specific need within the sponge holobiont, and these symbionts are often conserved within a narrow range of related taxa. To date, there exist only three known bacterial taxa ( Entoporibacteria , SAUL , and Tethybacterales ) that are globally distributed and found in a broad range of sponge hosts, and little is known about the latter two. We show that the functional potential of broad-host range symbionts is conserved at a family level and that these symbionts have been acquired several times over evolutionary history. Finally, it appears that the Entoporibacteria are associated primarily with high-microbial abundance sponges, while the Tethybacterales associate with low-microbial abundance sponges.

Background and Aim: Despite efforts in controlling and managing liver diseases, significant health issues remain. This study aims to evaluate the degree of public awareness and knowledge regarding liver health and diseases in Singapore., Methods: A cross-sectional, self-reported, web-based questionnaire was administered to 500 adult individuals. Questionnaire items pertained to knowledge and awareness of overall liver health, liver diseases and their associated risk factors., Results: Sixty-four percent of respondents were ≥35 years old and 54.0% were male. While majority agreed that regular screening was important for liver health (91.2%), only 65.4% attended health screening within recent 2 years. Hepatitis B had more awareness than hepatitis C among the respondents. About 70% agreed the consequences of viral hepatitis included liver cirrhosis, failure, and/or cancer. Yet, only 15% knew hepatitis C is not preventable by vaccination and more than half mistaken hepatitis B and C are transmissible via contaminated or raw seafood. Despite 75% being aware of non-alcoholic fatty liver disease, many were not aware of the related risk factors and complications. Awareness of specific screening and diagnostic tests for liver health was poor as one-fifth correctly identified the diagnostic tests for viral hepatitis. Preferences for doctor's consultation, TV, or newspapers (online) as information channels contrasted those currently used in the public health education efforts., Conclusions: The levels of understanding of liver diseases, risk factors, and potential complications are suboptimal among the Singapore public. More public education efforts aligned with respondents' information-seeking preferences could facilitate addressing misperceptions and increase knowledge about liver diseases., (© 2021 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Purpose: The aim of this study was to evaluate the clinical use of a high-performance polymer, polyether ether ketone (PEEK), as a material for use in provisional fixed dental prostheses (FDPs) that can be retained by reciprocated guide surfaces of hexagonal-shaped healing abutments during dental implant treatment., Materials and Methods: Patients requiring teeth replacement with dental implants for partially and fully edentulous cases in the maxilla and mandible were included. The provisional FDP was fabricated with hex castable copings and PEEK granules using the lost wax technique. The common path of insertion was established by reducing the coping that is in obstruction to the healing abutment. Retention was provided by the reciprocated guide surfaces of multiple hexagonal-shaped healing abutments., Results: Twenty-three custom provisional PEEK FDPs supported by 166 implants in 20 partially and fully edentulous patients were provided during the treatment period. The mean functional periods of the provisional PEEK FDPs for maxillary and mandibular cases were 6.95 ± 0.84 months and 1.90 ± 0.62 months, respectively. All provisional PEEK FDPs functioned as intended for the entire treatment period. There were a total of 10 complications: 4 cases had resin and denture veneers debond from the PEEK framework; 3 partially edentulous cases required supplementary screw retention; and 3 implants in separate cases did not osseointegrate, requiring extended use of the provisional prosthesis. Two provisional mandibular PEEK FDPs with prolonged usage (over 2 years) were excluded from calculations. No fracture of the PEEK framework was observed., Conclusion: The results from this study suggest that PEEK can be a suitable material for use in provisional FDPs during dental implant treatment. Prosthetic complications were frequent but were resolved intraorally. The reciprocated guide surfaces of multiple hexagonal-shaped healing abutments can provide adequate retention and resistance for edentulous cases during the implant treatment period. However, in partially edentulous cases, a prosthesis without cross-arch stabilization supplementary screw retention was required.

Stromatolites are complex microbial mats that form lithified layers. Fossilized stromatolites are the oldest evidence of cellular life on Earth, dating back over 3.4 billion years. Modern stromatolites are relatively rare but may provide clues about the function and evolution of their ancient counterparts. In this study, we focus on peritidal stromatolites occurring at Cape Recife and Schoenmakerskop on the southeastern South African coastline, the former being morphologically and structurally similar to fossilized phosphatic stromatolites formations. Using assembled shotgun metagenomic analysis, we obtained 183 genomic bins, of which the most dominant taxa were from the Cyanobacteria phylum. We identified functional gene sets in genomic bins conserved across two geographically isolated stromatolite formations, which included relatively high copy numbers of genes involved in the reduction of nitrates and phosphatic compounds. Additionally, we found little evidence of Archaeal species in these stromatolites, suggesting that they may not play an important role in peritidal stromatolite formations, as proposed for hypersaline formations., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

New cyanobacteria-derived bifunctional analogues of doscadenamide A, a LasR-dependent quorum sensing (QS) activator in Pseudomonas aeruginosa , characterized by dual acylation of the pyrrolinone core structure and the pendant side chain primary amine to form an imide/amide hybrid are reported. The identities of doscadenamides B-J were confirmed through total synthesis and a strategic focused library with different acylation and unsaturation patterns was created. Key molecular interactions for binding with LasR and a functional response through mutation studies coupled with molecular docking were identified. The structure-activity relationships (SARs) were probed in various Gram-negative bacteria, including P. aeruginosa and Vibrio harveyi , indicating that the pyrrolinone-N acyl chain is critical for full agonist activity, while the other acyl chain is dispensable or can result in antagonist activity, depending on the bacterial system. Since homoserine lactone (HSL) quorum sensing activators have been shown to act in synergy with TRAIL to induce apoptosis in cancer cells, selected doscadenamides were tested in orthogonal eukaryotic screening systems. The most potent QS agonists, doscadenamides S10-S12, along with doscadenamides F and S4 with partial or complete saturation of the acyl side chains, exhibited the most pronounced synergistic effects with TRAIL in triple negative MDA-MB-231 breast cancer cells. The overall correlation of the SAR with respect to prokaryotic and eukaryotic targets may hint at coevolutionary processes and intriguing host-bacteria relationships. The doscadenamide scaffold represents a non-HSL template for combination therapy with TRAIL pathway stimulators.

Many animal phyla have no representatives within the catalog of whole metazoan genome sequences. This dataset fills in one gap in the genome knowledge of animal phyla with a draft genome of Bugula neritina (phylum Bryozoa). Interest in this species spans ecology and biomedical sciences because B. neritina is the natural source of bioactive compounds called bryostatins. Here we present a draft assembly of the B. neritina genome obtained from PacBio and Illumina HiSeq data, as well as genes and proteins predicted de novo and verified using transcriptome data, along with the functional annotation. These sequences will permit a better understanding of host-symbiont interactions at the genomic level, and also contribute additional phylogenomic markers to evaluate Lophophorate or Lophotrochozoa phylogenetic relationships. The effort also fits well with plans to ultimately sequence all orders of the Metazoa.

Symbiotic mutualisms of bacteria and animals are ubiquitous in nature, running a continuum from facultative to obligate from the perspectives of both partners. The loss of functions required for living independently but not within a host gives rise to reduced genomes in many symbionts. Although the phenomenon of genome reduction can be explained by existing evolutionary models, the initiation of the process is not well understood. Here, we describe the microbiome associated with the eggs of the beetle Lagria villosa , consisting of multiple bacterial symbionts related to Burkholderia gladioli , including a reduced-genome symbiont thought to be the exclusive producer of the defensive compound lagriamide. We show that the putative lagriamide-producing symbiont is the only member of the microbiome undergoing genome reduction and that it has already lost the majority of its primary metabolism and DNA repair pathways. The key step preceding genome reduction in the symbiont was likely the horizontal acquisition of the putative lagriamide lga biosynthetic gene cluster. Unexpectedly, we uncovered evidence of additional horizontal transfers to the symbiont's genome while genome reduction was occurring and despite a current lack of genes needed for homologous recombination. These gene gains may have given the genome-reduced symbiont a selective advantage in the microbiome, especially given the maintenance of the large lga gene cluster despite ongoing genome reduction. IMPORTANCE Associations between microorganisms and an animal, plant, or fungal host can result in increased dependence over time. This process is due partly to the bacterium not needing to produce nutrients that the host provides, leading to loss of genes that it would need to live independently and to a consequent reduction in genome size. It is often thought that genome reduction is aided by genetic isolation-bacteria that live in monocultures in special host organs, or inside host cells, have less access to other bacterial species from which they can obtain genes. Here, we describe exposure of a genome-reduced beetle symbiont to a community of related bacteria with nonreduced genomes. We show that the symbiont has acquired genes from other bacteria despite going through genome reduction, suggesting that isolation has not yet played a major role in this case of genome reduction, with horizontal gene gains still offering a potential route for adaptation., (Copyright © 2020 Waterworth et al.)

Quorum sensing (QS) plays a critical role in the regulation of bacterial pathogenesis. Doscadenamide A ( 1a ) was isolated from a marine cyanobacterium, its structure elucidated by NMR, and its activity linked to QS induction. The total synthesis of 1a was developed, and the absolute configuration confirmed through comparison of the isolated natural product with synthetic diastereomers. Our preliminary investigation indicated that 1a could activate QS signaling in a LasR-dependent manner.

Shotgun metagenomics is a powerful, high-resolution technique enabling the study of microbial communities in situ. However, species-level resolution is only achieved after a process of 'binning' where contigs predicted to originate from the same genome are clustered. Such culture-independent sequencing frequently unearths novel microbes, and so various methods have been devised for reference-free binning. As novel microbiomes of increasing complexity are explored, sometimes associated with non-model hosts, robust automated binning methods are required. Existing methods struggle with eukaryotic contamination and cannot handle highly complex single metagenomes. We therefore developed an automated binning pipeline, termed 'Autometa', to address these issues. This command-line application integrates sequence homology, nucleotide composition, coverage and the presence of single-copy marker genes to separate microbial genomes from non-model host genomes and other eukaryotic contaminants, before deconvoluting individual genomes from single metagenomes. The method is able to effectively separate over 1000 genomes from a metagenome, allowing the study of previously intractably complex environments at the level of single species. Autometa is freely available at https://bitbucket.org/jason&#95;c&#95;kwan/autometa and as a docker image at https://hub.docker.com/r/jasonkwan/autometa under the GNU Affero General Public License 3 (AGPL 3)., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Five novel modified linear peptides named brintonamides A-E (1-5) were discovered from a marine cyanobacterial sample collected from Brinton Channel, Florida Keys. The total synthesis of 1-5 in addition to two other structurally related analogues (6 and 7) was achieved, which provided more material to allow rigorous biological evaluation and SAR studies. Compounds were subjected to cancer-focused phenotypic cell viability and migration assays and orthogonal target-based pharmacological screening platforms to identify their protease and GPCR modulatory activity profiles. The cancer related serine protease kallikrein 7 (KLK7) was inhibited to similar extents with an IC 50 near 20 μM by both representative members 1 and 4, which differed in the presence or lack of the N-terminal unit. In contrast to the biochemical protease profiling study, clear SAR was observed in the functional GPCR screens, where five GPCRs in antagonist mode (CCR10, OXTR, SSTR3, TACR2) and agonist mode (CXCR7) were modulated by compounds 1-7 to varying extents. Chemokine receptor type 10 (CCR10) was potently modulated by brintonamide D (4) with an IC 50 of 0.44 μM. We performed in silico modeling to understand the structural basis underlying the differences in the antagonistic activity among brintonamides toward CCR10. Because of the significance of KLK7 and CCR10 in cancer progression and metastasis, we demonstrated the ability of brintonamide D (4) at 10 μM to significantly target downstream cellular substrates of KLK7 (Dsg-2 and E-cad) in vitro and to inhibit CCL27-induced CCR10-mediated proliferation and the migration of highly invasive breast cancer cells.

Microbial symbionts are often a source of chemical novelty and can contribute to host defense against antagonists. However, the ecological relevance of chemical mediators remains unclear for most systems. Lagria beetles live in symbiosis with multiple strains of Burkholderia bacteria that protect their offspring against pathogens. Here, we describe the antifungal polyketide lagriamide, and provide evidence supporting that it is produced by an uncultured symbiont, Burkholderia gladioli Lv-StB, which is dominant in field-collected Lagria villosa. Interestingly, lagriamide is structurally similar to bistramides, defensive compounds found in marine tunicates. We identify a gene cluster that is probably involved in lagriamide biosynthesis, provide evidence for horizontal acquisition of these genes, and show that the naturally occurring symbiont strains on the egg are protective in the soil environment. Our findings highlight the potential of microbial symbionts and horizontal gene transfer as influential sources of ecological innovation.

Bacteria have supplied us with many bioactive molecules for use in medicine and agriculture. However, rates of discovery have decreased as the biosynthetic capacity of the culturable biosphere has been continuously mined for many decades. The as-yet-uncultured biosphere is likely to hold far greater biosynthetic potential, especially where ecological niches favor the selection of therapeutically useful bioactivities. I outline here how metagenomics and other systems biology approaches can be used to gain insight into small-molecule biosynthesis and the selective forces which shape it. I also argue that we need a greater understanding of the function of small molecules in complex microbiomes and rational synthetic biology methods to functionally reconstruct large biosynthetic pathways in heterologous hosts., Competing Interests: Conflict of Interest Disclosures: J.C.K. has nothing to disclose. Conflict of Interest Disclosures: J.C.K. has nothing to disclose.

A symbiotic lifestyle frequently results in genome reduction in bacteria; the isolation of small populations promotes genetic drift and the fixation of deletions and deleterious mutations over time. Transitions in lifestyle, including host restriction or adaptation to an intracellular habitat, are thought to precipitate a wave of sequence degradation events and consequent proliferation of pseudogenes. We describe here a verrucomicrobial symbiont of the tunicate Lissoclinum sp. that appears to be undergoing such a transition, with low coding density and many identifiable pseudogenes. However, despite the overall drive toward genome reduction, this symbiont maintains seven copies of a large polyketide synthase (PKS) pathway for the mandelalides ( mnd ), cytotoxic compounds that likely constitute a chemical defense for the host. There is evidence of ongoing degradation in a small number of these repeats-including variable borders, internal deletions, and single nucleotide polymorphisms (SNPs). However, the gene dosage of most of the pathway is increased at least 5-fold. Correspondingly, this single pathway accounts for 19% of the genome by length and 25.8% of the coding capacity. This increased gene dosage in the face of generalized sequence degradation and genome reduction suggests that mnd genes are under strong purifying selection and are important to the symbiotic relationship. IMPORTANCE Secondary metabolites, which are small-molecule organic compounds produced by living organisms, provide or inspire drugs for many different diseases. These natural products have evolved over millions of years to provide a survival benefit to the producing organism and often display potent biological activity with important therapeutic applications. For instance, defensive compounds in the environment may be cytotoxic to eukaryotic cells, a property exploitable for cancer treatment. Here, we describe the genome of an uncultured symbiotic bacterium that makes such a cytotoxic metabolite. This symbiont is losing genes that do not endow a selective advantage in a hospitable host environment. Secondary metabolism genes, however, are repeated multiple times in the genome, directly demonstrating their selective advantage. This finding shows the strength of selective forces in symbiotic relationships and suggests that uncultured bacteria in such relationships should be targeted for drug discovery efforts.

Cone snails are biomedically important sources of peptide drugs, but it is not known whether snail-associated bacteria affect venom chemistry. To begin to answer this question, we performed 16S rRNA gene amplicon sequencing of eight cone snail species, comparing their microbiomes with each other and with those from a variety of other marine invertebrates. We show that the cone snail microbiome is distinct from those in other marine invertebrates and conserved in specimens from around the world, including the Philippines, Guam, California, and Florida. We found that all venom ducts examined contain diverse 16S rRNA gene sequences bearing closest similarity to Stenotrophomonas bacteria. These sequences represent specific symbionts that live in the lumen of the venom duct, where bioactive venom peptides are synthesized. IMPORTANCE In animals, symbiotic bacteria contribute critically to metabolism. Cone snails are renowned for the production of venoms that are used as medicines and as probes for biological study. In principle, symbiotic bacterial metabolism could either degrade or synthesize active venom components, and previous publications show that bacteria do indeed contribute small molecules to some venoms. Therefore, understanding symbiosis in cone snails will contribute to further drug discovery efforts. Here, we describe an unexpected, specific symbiosis between bacteria and cone snails from around the world., (Copyright © 2017 American Society for Microbiology.)

Genome mining has become an increasingly powerful, scalable, and economically accessible tool for the study of natural product biosynthesis and drug discovery. However, there remain important biological and practical problems that can complicate or obscure biosynthetic analysis in genomic and metagenomic sequencing projects. Here, we focus on limitations of available technology as well as computational and experimental strategies to overcome them. We review the unique challenges and approaches in the study of symbiotic and uncultured systems, as well as those associated with biosynthetic gene cluster (BGC) assembly and product prediction. Finally, to explore sequencing parameters that affect the recovery and contiguity of large and repetitive BGCs assembled de novo , we simulate Illumina and PacBio sequencing of the Salinispora tropica genome focusing on assembly of the salinilactam ( slm ) BGC., Competing Interests: The authors declare no conflict of interest.

The uncultured bacterial symbiont "Candidatus Endobugula sertula" is known to produce cytotoxic compounds called bryostatins, which protect the larvae of its host, Bugula neritina The symbiont has never been successfully cultured, and it was thought that its genome might be significantly reduced. Here, we took a shotgun metagenomics and metatranscriptomics approach to assemble and characterize the genome of "Ca Endobugula sertula." We found that it had specific metabolic deficiencies in the biosynthesis of certain amino acids but few other signs of genome degradation, such as small size, abundant pseudogenes, and low coding density. We also identified homologs to genes associated with insect pathogenesis in other gammaproteobacteria, and these genes may be involved in host-symbiont interactions and vertical transmission. Metatranscriptomics revealed that these genes were highly expressed in a reproductive host, along with bry genes for the biosynthesis of bryostatins. We identified two new putative bry genes fragmented from the main bry operon, accounting for previously missing enzymatic functions in the pathway. We also determined that a gene previously assigned to the pathway, bryS, is not expressed in reproductive tissue, suggesting that it is not involved in the production of bryostatins. Our findings suggest that "Ca Endobugula sertula" may be able to live outside the host if its metabolic deficiencies are alleviated by medium components, which is consistent with recent findings that it may be possible for "Ca Endobugula sertula" to be transmitted horizontally., Importance: The bryostatins are potent protein kinase C activators that have been evaluated in clinical trials for a number of indications, including cancer and Alzheimer's disease. There is, therefore, considerable interest in securing a renewable supply of these compounds, which is currently only possible through aquaculture of Bugula neritina and total chemical synthesis. However, these approaches are labor-intensive and low-yielding and thus preclude the use of bryostatins as a viable therapeutic agent. Our genome assembly and transcriptome analysis for "Ca Endobugula sertula" shed light on the metabolism of this symbiont, potentially aiding isolation and culturing efforts. Our identification of additional bry genes may also facilitate efforts to express the complete pathway heterologously., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Direct, untargeted sequencing of environmental samples (metagenomics) and de novo genome assembly enable the study of uncultured and phylogenetically divergent organisms. However, separating individual genomes from a mixed community has often relied on the differential-coverage analysis of multiple, deeply sequenced samples. In the metagenomic investigation of the marine bryozoan Bugula neritina, we uncovered seven bacterial genomes associated with a single B. neritina individual that appeared to be transient associates, two of which were unique to one individual and undetectable using certain "universal" 16S rRNA primers and probes. We recovered high quality genome assemblies for several rare instances of "microbial dark matter," or phylogenetically divergent bacteria lacking genomes in reference databases, from a single tissue sample that was not subjected to any physical or chemical pre-treatment. One of these rare, divergent organisms has a small (593 kbp), poorly annotated genome with low GC content (20.9%) and a 16S rRNA gene with just 65% sequence similarity to the closest reference sequence. Our findings illustrate the importance of sampling strategy and de novo assembly of metagenomic reads to understand the extent and function of bacterial biodiversity.

Unlabelled: Diversity-generating metabolism leads to the evolution of many different chemicals in living organisms. Here, by examining a marine symbiosis, we provide a precise evolutionary model of how nature generates a family of novel chemicals, the cyanobactins. We show that tunicates and their symbiotic Prochloron cyanobacteria share congruent phylogenies, indicating that Prochloron phylogeny is related to host phylogeny and not to external habitat or geography. We observe that Prochloron exchanges discrete functional genetic modules for cyanobactin secondary metabolite biosynthesis in an otherwise conserved genetic background. The module exchange leads to gain or loss of discrete chemical functional groups. Because the underlying enzymes exhibit broad substrate tolerance, discrete exchange of substrates and enzymes between Prochloron strains leads to the rapid generation of chemical novelty. These results have implications in choosing biochemical pathways and enzymes for engineered or combinatorial biosynthesis., Importance: While most biosynthetic pathways lead to one or a few products, a subset of pathways are diversity generating and are capable of producing thousands to millions of derivatives. This property is highly useful in biotechnology since it enables biochemical or synthetic biological methods to create desired chemicals. A fundamental question has been how nature itself creates this chemical diversity. Here, by examining the symbiosis between coral reef animals and bacteria, we describe the genetic basis of chemical variation with unprecedented precision. New compounds from the cyanobactin family are created by either varying the substrate or importing needed enzymatic functions from other organisms or via both mechanisms. This natural process matches successful laboratory strategies to engineer the biosynthesis of new chemicals and teaches a new strategy to direct biosynthesis., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Current approaches in tissue engineering are geared toward generating tissue-specific stem cells. Given the complexity and heterogeneity of tissues, this approach has its limitations. An alternate approach is to induce terminally differentiated cells to dedifferentiate into multipotent proliferative cells with the capacity to regenerate all components of a damaged tissue, a phenomenon used by salamanders to regenerate limbs. 5-Azacytidine (AZA) is a nucleoside analog that is used to treat preleukemic and leukemic blood disorders. AZA is also known to induce cell plasticity. We hypothesized that AZA-induced cell plasticity occurs via a transient multipotent cell state and that concomitant exposure to a receptive growth factor might result in the expansion of a plastic and proliferative population of cells. To this end, we treated lineage-committed cells with AZA and screened a number of different growth factors with known activity in mesenchyme-derived tissues. Here, we report that transient treatment with AZA in combination with platelet-derived growth factor-AB converts primary somatic cells into tissue-regenerative multipotent stem (iMS) cells. iMS cells possess a distinct transcriptome, are immunosuppressive, and demonstrate long-term self-renewal, serial clonogenicity, and multigerm layer differentiation potential. Importantly, unlike mesenchymal stem cells, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner and, unlike embryonic or pluripotent stem cells, do not form teratomas. Taken together, this vector-free method of generating iMS cells from primary terminally differentiated cells has significant scope for application in tissue regeneration.

Background: Storage of donor hearts in cardioplegic solutions supplemented with agents that mimic the ischaemic preconditioning response enhanced their post-reperfusion function. The present study examines the minimisation of cell death and activation of pro-survival signalling directed towards maintenance of mitochondrial homeostasis in hearts arrested and stored in two such agents, glyceryl-trinitrate, a nitric oxide donor and cariporide, (a sodium-hydrogen exchange inhibitor)., Methods: After baseline functional measurement, isolated working rat hearts were arrested and stored for 6h at 4°C in either Celsior(®), Celsior(®) containing 0.1mg/ml glyceryl-trinitrate, 10μM cariporide or both agents. After reperfusion, function was remeasured. Hearts were then processed for immunoblotting or histology., Results: Necrotic and apoptotic markers present in the Celsior(®) group post-reperfusion were abolished by glyceryl-trinitrate, cariporide or both. Increased phosphorylation of ERK and Bcl2, after reperfusion in groups stored in glyceryl-trinitrate, cariporide or both along with increased phospho-STAT3 levels in the glyceryl-trinitrate/cariporide group correlated with functional recovery. Inhibition of STAT3 phosphorylation blocked recovery. No phospho-Akt increase was seen in any treatment., Conclusions: Activation of signalling pathways that favour mitophagy activation (ERK and Bcl2 phosphorylation) and maintenance of mitochondrial transition pore closure after reperfusion (STAT3 and ERK phosphorylation) were crucial for functional recovery of the donor heart., (Copyright © 2014 Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) and the Cardiac Society of Australia and New Zealand (CSANZ). Published by Elsevier B.V. All rights reserved.)

Ascidians contain abundant, diverse secondary metabolites, which are thought to serve a defensive role and which have been applied to drug discovery. It is known that bacteria in symbiosis with ascidians produce several of these metabolites, but very little is known about factors governing these 'chemical symbioses'. To examine this phenomenon across a wide geographical and species scale, we performed bacterial and chemical analyses of 32 different ascidians, mostly from the didemnid family from Florida, Southern California and a broad expanse of the tropical Pacific Ocean. Bacterial diversity analysis showed that ascidian microbiomes are highly diverse, and this diversity does not correlate with geographical location or latitude. Within a subset of species, ascidian microbiomes are also stable over time (R=-0.037, P-value=0.499). Ascidian microbiomes and metabolomes contain species-specific and location-specific components. Location-specific bacteria are found in low abundance in the ascidians and mostly represent strains that are widespread. Location-specific metabolites consist largely of lipids, which may reflect differences in water temperature. By contrast, species-specific bacteria are mostly abundant sequenced components of the microbiomes and include secondary metabolite producers as major components. Species-specific chemicals are dominated by secondary metabolites. Together with previous analyses that focused on single ascidian species or symbiont type, these results reveal fundamental properties of secondary metabolic symbiosis. Different ascidian species have established associations with many different bacterial symbionts, including those known to produce toxic chemicals. This implies a strong selection for this property and the independent origin of secondary metabolite-based associations in different ascidian species. The analysis here streamlines the connection of secondary metabolite to producing bacterium, enabling further biological and biotechnological studies.

Largazole is a potent class I selective histone deacetylase (HDAC) inhibitor. The majority of largazole analogues to date have modified the thiazole-thiazoline and the warhead moiety. In order to elucidate class I-specific structure-activity relationships, a series of analogues with modifications in the valine or the linker region were prepared and evaluated for their class I isoform selectivity. The inhibition profile showed that the C2 position of largazole has an optimal steric requirement for efficient HDAC inhibition and that substitution of the trans-alkene in the linker with an aromatic group results in complete loss of activity. This data will aid the design of class I isoform selective HDAC inhibitors., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Natural products (secondary metabolites) found in marine invertebrates are often thought to be produced by resident symbiotic bacteria, and these products appear to play a major role in the symbiotic interaction of bacteria and their hosts. In these animals, there is extensive variation, both in chemistry and in the symbiotic bacteria that produce them. Here, we sought to answer the question of what factors underlie chemical variation in the ocean. As a model, we investigated the colonial tunicate Lissoclinum patella because of its rich and varied chemistry and its broad geographic range. We sequenced mitochondrial cytochrome c oxidase 1 (COXI) genes, and found that animals classified as L. patella fall into three phylogenetic groups that may encompass several cryptic species. The presence of individual natural products followed the phylogenetic relationship of the host animals, even though the compounds are produced by symbiotic bacteria that do not follow host phylogeny. In sum, we show that cryptic populations of animals underlie the observed chemical diversity, suggesting that the host controls selection for particular secondary metabolite pathways. These results imply novel approaches to obtain chemical diversity from the oceans, and also demonstrate that the diversity of marine natural products may be greatly impacted by cryptic local extinctions.

Natural products made by marine cyanobacteria are often highly modified peptides and depsipeptides that have the potential to act as inhibitors for proteases. In the interests of finding new protease inhibition activity and selectivity, grassypeptolide A (1) was screened against a panel of proteases and found to inhibit DPP8 selectively over DPP4. Grassypeptolides were also found to inhibit IL-2 production and proliferation in activated T-cells, consistent with a putative role of DPP8 in the immune system. These effects were also observed in Jurkat cells, and DPP activity in Jurkat cell cytosol was shown to be inhibited by grassypeptolides. In silico docking suggests two possible binding modes of grassypeptolides-at the active site of DPP8 and at one of the entrances to the internal cavity. Collectively these results suggest that grassypeptolides might be useful tool compounds in the study of DPP8 function., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Intracellular symbiosis is known to be widespread in insects, but there are few described examples in other types of host. These symbionts carry out useful activities such as synthesizing nutrients and conferring resistance against adverse events such as parasitism. Such symbionts persist through host speciation events, being passed down through vertical transmission. Due to various evolutionary forces, symbionts go through a process of genome reduction, eventually resulting in tiny genomes where only those genes essential to immediate survival and those beneficial to the host remain. In the marine environment, invertebrates such as tunicates are known to harbor complex microbiomes implicated in the production of natural products that are toxic and probably serve a defensive function. Here, we show that the intracellular symbiont Candidatus Endolissoclinum faulkneri is a long-standing symbiont of the tunicate Lissoclinum patella, that has persisted through cryptic speciation of the host. In contrast to the known examples of insect symbionts, which tend to be either relatively recent or ancient relationships, the genome of Ca. E. faulkneri has a very low coding density but very few recognizable pseudogenes. The almost complete degradation of intergenic regions and stable gene inventory of extant strains of Ca. E. faulkneri show that further degradation and deletion is happening very slowly. This is a novel stage of genome reduction and provides insight into how tiny genomes are formed. The ptz pathway, which produces the defensive patellazoles, is shown to date to before the divergence of Ca. E. faulkneri strains, reinforcing its importance in this symbiotic relationship. Lastly, as in insects we show that stable symbionts can be lost, as we describe an L. patella animal where Ca. E. faulkneri is displaced by a likely intracellular pathogen. Our results suggest that intracellular symbionts may be an important source of ecologically significant natural products in animals.

In the oceans, secondary metabolites often protect otherwise poorly defended invertebrates, such as shell-less mollusks, from predation. The origins of these metabolites are largely unknown, but many of them are thought to be made by symbiotic bacteria. In contrast, mollusks with thick shells and toxic venoms are thought to lack these secondary metabolites because of reduced defensive needs. Here, we show that heavily defended cone snails also occasionally contain abundant secondary metabolites, γ-pyrones known as nocapyrones, which are synthesized by symbiotic bacteria. The bacteria, Nocardiopsis alba CR167, are related to widespread actinomycetes that we propose to be casual symbionts of invertebrates on land and in the sea. The natural roles of nocapyrones are unknown, but they are active in neurological assays, revealing that mollusks with external shells are an overlooked source of secondary metabolite diversity., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Secondary metabolites are ubiquitous in bacteria, but by definition, they are thought to be nonessential. Highly toxic secondary metabolites such as patellazoles have been isolated from marine tunicates, where their exceptional potency and abundance implies a role in chemical defense, but their biological source is unknown. Here, we describe the association of the tunicate Lissoclinum patella with a symbiotic α-proteobacterium, Candidatus Endolissoclinum faulkneri, and present chemical and biological evidence that the bacterium synthesizes patellazoles. We sequenced and assembled the complete Ca. E. faulkneri genome, directly from metagenomic DNA obtained from the tunicate, where it accounted for 0.6% of sequence data. We show that the large patellazoles biosynthetic pathway is maintained, whereas the remainder of the genome is undergoing extensive streamlining to eliminate unneeded genes. The preservation of this pathway in streamlined bacteria demonstrates that secondary metabolism is an essential component of the symbiotic interaction.

Drug resistant infectious diseases are quickly becoming a global health crisis. While Streptomyces spp. have been a major source of antibiotics over the past 50 years, efficient methods are needed to identify new antibiotics and greatly improve the rate of discovery. LCMS-based metabolomics were applied to analyze extracts of 50 Streptomyes spp. Using this methodology, we discovered bottromycin D and used whole genome sequencing to determine its biosynthesis by a ribosomal pathway.