Changes in the genetic requirements for microbial interactions with increasing community complexity.

Microbial community structure and function rely on complex interactions whose underlying molecular mechanisms are poorly understood. To investigate these interactions in a simple microbiome, we introduced E. coli into an experimental community based on a cheese rind and identified the differences in E. coli's genetic requirements for growth in interactive and non-interactive contexts using Random Barcode Transposon Sequencing (RB-TnSeq) and RNASeq. Genetic requirements varied among pairwise growth conditions and between pairwise and community conditions. Our analysis points to mechanisms by which growth conditions change as a result of increasing community complexity and suggests that growth within a community relies on a combination of pairwise and higher-order interactions. Our work provides a framework for using the model organism E. coli as a readout to investigate microbial interactions regardless of the genetic tractability of members of the studied ecosystem

Extensive horizontal gene transfer in cheese-associated bacteria.

Acquisition of genes through horizontal gene transfer (HGT) allows microbes to rapidly gain new capabilities and adapt to new or changing environments. Identifying widespread HGT regions within multispecies microbiomes can pinpoint the molecular mechanisms that play key roles in microbiome assembly. We sought to identify horizontally transferred genes within a model microbiome, the cheese rind. Comparing 31 newly sequenced and 134 previously sequenced bacterial isolates from cheese rinds, we identified over 200 putative horizontally transferred genomic regions containing 4733 protein coding genes. The largest of these regions are enriched for genes involved in siderophore acquisition, and are widely distributed in cheese rinds in both Europe and the US. These results suggest that HGT is prevalent in cheese rind microbiomes, and that identification of genes that are frequently transferred in a particular environment may provide insight into the selective forces shaping microbial communities

Scanning laser source and scanning laser detection techniques for different surface crack geometries

Standard test samples typically contain simulated defects such as slots machined normal to the surface. However, real defects will not always propagate in this manner; for example, rolling contact fatigue on rails propagates at around 25º to the surface, and corrosion cracking can grow in a branched manner. Therefore, there is a need to understand how ultrasonic surface waves interact with different crack geometries. We present measurements of machined slots inclined at an angle to the surface normal, or with simple branched geometries, using laser ultrasound. Recently, Rayleigh wave enhancements observed when using the scanning laser source technique, where a generation laser is scanned along a sample, have been highlighted for their potential in detecting surface cracks. We show that the enhancement measured with laser detector scanning can give a more significant enhancement when different crack geometries are considered. We discuss the behaviour of an incident Rayleigh wave in the region of an angled defect, and consider mode-conversions which lead to a very large enhancement when the detector is close to the opening of a shallow defect. This process could be used in characterising defects, as well as being an excellent fingerprint of their presence

Non-contact ultrasonic detection of angled surface defects

Non-destructive testing is an important technique, and improvements are constantly needed. Surface defects in metals are not necessarily confined to orientations normal to the sample surface; however, much of the previous work investigating the interaction of ultrasonic surface waves with surface-breaking defects has assumed cracks inclined at 90° to the surface. This paper explores the interaction of Rayleigh waves with cracks which have a wide range of angles and depths relative to the surface, using a non-contact laser generation and detection system. Additional insight is acquired using a 3D model generated using finite element method software. A clear variation of the reflection and transmission coefficients with both crack angle and length is found, in both the out-of-plane and in-plane components. The 3D model is further used to understand the contributions of different wavemodes to B-Scans produced when scanning a sample, to enable understanding of the reflection and transmission behaviour, and help identify angled defects. Knowledge of these effects is essential to correctly gauge the severity of surface cracking

Direct interaction of FliX and FlbD is required for their regulatory activity in Caulobacter crescentus

Background: The temporal and spatial expression of late flagellar genes in Caulobacter crescentus is activated by the transcription factor FlbD and its partner trans-acting factor FliX. The physical interaction of these two proteins represents an alternative mechanism for regulating the activity of $\sigma^{54}$ transcription factors. This study is to characterize the interaction of the two proteins and the consequences of the interaction on their regulatory activity. Results: FliX and FlbD form stable complexes, which can stand the interference of 2.65 M NaCl. The stability of FliX and FlbD was affected by the co-existence of each other. Five FliX mutants (R71A, L85K, $\Delta$117-118, T130L, and L136K) were created by site-directed mutagenesis in conserved regions of the protein. All mutants were successfully expressed in both wild-type and $\Delta fliX$ Caulobacter strains. All but $FliX_{L85K}$ could rescue the motility and cell division defects of a $\Delta fliX$ mutant strain. The ability of FliX to regulate the transcription of class II and class III/IV flagellar promoters was fully diminished due to the L85K mutation. Co-immunoprecipitation experiment revealed that $FliX_{L85K}$ was unable to physically interact with FlbD. Conclusions: FliX interacts with FlbD and thereby directly regulates the activity of FlbD in response to flagellar assembly. Mutations in highly conserved regions of FliX could severely affect the recognition between FliX and FlbD and hence interrupt the normal progression of flagellar synthesis and other developmental events in Caulobacter.Molecular and Cellular Biolog

Scanning laser techniques for characterisation of different surface breaking defect geometries

Measurements using a laser scanning system consisting of a pulsed Nd:YAG laser to generate surface ultrasonic waves and an interferometer to detect the surface displacement, are presented for different samples and defect geometries. We show, firstly, details of the interaction of Rayleigh waves in thick samples with machined slots inclined at an angle to the surface normal, or with simple branched geometries, scanning the generation source over the defect (SLLS) or scanning the detection point over the defect (SLD). Secondly, we discuss effects of Lamb waves interacting with V-shaped defects in thin samples. The results from these measurements have shown that the signal enhancement found in the near-field in both cases can be used to position the defect and gain an idea of its geometry, and have shown this to be a suitable fingerprint of the presence of the defect

Biotic Interactions Shape the Ecological Distributions of Staphylococcus Species.

Many metagenomic sequencing studies have observed the presence of closely related bacterial species or genotypes in the same microbiome. Previous attempts to explain these patterns of microdiversity have focused on the abiotic environment, but few have considered how biotic interactions could drive patterns of microbiome diversity. We dissected the patterns, processes, and mechanisms shaping the ecological distributions of three closely related Staphylococcus species in cheese rind biofilms. Paradoxically, the most abundant species (S. equorum) is the slowest colonizer and weakest competitor based on growth and competition assays in the laboratory. Through in vitro community reconstructions, we determined that biotic interactions with neighboring fungi help resolve this paradox. Species-specific stimulation of the poor competitor by fungi of the genus Scopulariopsis allows S. equorum to dominate communities in vitro as it does in situ Results of comparative genomic and transcriptomic experiments indicate that iron utilization pathways, including a homolog of the S. aureus staphyloferrin B siderophore operon pathway, are potential molecular mechanisms underlying Staphylococcus-Scopulariopsis interactions. Our integrated approach demonstrates that fungi can structure the ecological distributions of closely related bacterial species, and the data highlight the importance of bacterium-fungus interactions in attempts to design and manipulate microbiomes.ImportanceDecades of culture-based studies and more recent metagenomic studies have demonstrated that bacterial species in agriculture, medicine, industry, and nature are unevenly distributed across time and space. The ecological processes and molecular mechanisms that shape these distributions are not well understood because it is challenging to connect in situ patterns of diversity with mechanistic in vitro studies in the laboratory. Using tractable cheese rind biofilms and a focus on coagulase-negative staphylococcus (CNS) species, we demonstrate that fungi can mediate the ecological distributions of closely related bacterial species. One of the Staphylococcus species studied, S. saprophyticus, is a common cause of urinary tract infections. By identifying processes that control the abundance of undesirable CNS species, cheese producers will have more precise control on the safety and quality of their products. More generally, Staphylococcus species frequently co-occur with fungi in mammalian microbiomes, and similar bacterium-fungus interactions may structure bacterial diversity in these systems

Teaching the Tough Stuff: The Opportunities and Challenges in Teaching Implicit Bias, Diversity and Inclusion to Supervising Attorneys

We entrust supervising attorneys with the critical responsibility of providing law students with a meaningful experiential learning experience. Supervising attorneys ultimately control the nature of work students receive, the delivery of feedback, and the work environment that the student inhabits. Thus, it is vital that we equip our supervising attorneys with the basic skills necessary to navigate student supervision effectively. Among those basic skills is the ability to traverse the cross-cultural complexities inherent in working with students whose cultural identities may differ from the supervising attorney. Equally important are the cross-cultural issues that may arise among students and the clients and other persons with whom they interact during their externship. This session will discuss the significance in providing supervising training that focuses on diversity and implicit bias as it relates to students, supervision and the externship context. As externship faculty and staff, we have an integral role in ensuring that our supervising attorneys create a working environment for our students that is devoid of discrimination and bias. The standard “supervising attorney training” provides the ideal forum to introduce cultural diversity and implicit bias concepts to supervising attorneys. In such a setting, externship faculty and staff can train supervising attorneys in cross-cultural pedagogy, and expose practitioners to best practices regarding cultural complexities that may arise in student supervision. However, the forum is not without its challenges. Supervising attorney training sessions only allow a limited time to present such an essential, multifaceted issue. Additionally, supervisor training typically consists of a diverse group of attendees, with varying degrees of understanding of diversity and inclusion issues, and who may be uncomfortable delving into seemingly controversial cultural conversations with strangers. The co-presenters will share their experiences in planning and delivering such training, exploring the effectiveness of various methodologies

Reporting performance of prognostic models in cancer: a review

<p>Abstract</p> <p>Background</p> <p>Appropriate choice and use of prognostic models in clinical practice require the use of good methods for both model development, and for developing prognostic indices and risk groups from the models. In order to assess reliability and generalizability for use, models need to have been validated and measures of model performance reported. We reviewed published articles to assess the methods and reporting used to develop and evaluate performance of prognostic indices and risk groups from prognostic models.</p> <p>Methods</p> <p>We developed a systematic search string and identified articles from PubMed. Forty-seven articles were included that satisfied the following inclusion criteria: published in 2005; aiming to predict patient outcome; presenting new prognostic models in cancer with outcome time to an event and including a combination of at least two separate variables; and analysing data using multivariable analysis suitable for time to event data.</p> <p>Results</p> <p>In 47 studies, Cox models were used in 94% (44), but the coefficients or hazard ratios for the variables in the final model were reported in only 72% (34). The reproducibility of the derived model was assessed in only 11% (5) of the articles. A prognostic index was developed from the model in 81% (38) of the articles, but researchers derived the prognostic index from the final prognostic model in only 34% (13) of the studies; different coefficients or variables from those in the final model were used in 50% (19) of models and the methods used were unclear in 16% (6) of the articles. Methods used to derive prognostic groups were also poor, with researchers not reporting the methods used in 39% (14 of 36) of the studies and data derived methods likely to bias estimates of differences between risk groups being used in 28% (10) of the studies. Validation of their models was reported in only 34% (16) of the studies. In 15 studies validation used data from the same population and in five studies from a different population. Including reports of validation with external data from publications up to four years following model development, external validation was attempted for only 21% (10) of models. Insufficient information was provided on the performance of models in terms of discrimination and calibration.</p> <p>Conclusions</p> <p>Many published prognostic models have been developed using poor methods and many with poor reporting, both of which compromise the reliability and clinical relevance of models, prognostic indices and risk groups derived from them.</p

Growth on Chitin Impacts the Transcriptome and Metabolite Profiles of Antibiotic-Producing Vibrio coralliilyticus S2052 and Photobacterium galatheae S2753

Members of the Vibrionaceae family are often associated with chitin-containing organisms, and they are thought to play a major role in chitin degradation. The purpose of the present study was to determine how chitin affects the transcriptome and metabolome of two bioactive Vibrionaceae strains, Vibrio coralliilyticus and Photobacterium galatheae. We focused on chitin degradation genes and secondary metabolites based on the assumption that these molecules in nature confer an advantage to the producer. Growth on chitin caused upregulation of genes related to chitin metabolism and of genes potentially involved in host colonization and/or infection. The expression of genes involved in secondary metabolism was also significantly affected by growth on chitin, in one case being 34-fold upregulated. This was reflected in the metabolome, where the antibiotics andrimid and holomycin were produced in larger amounts on chitin. Other polyketide synthase/ nonribosomal peptide synthetase (PKS-NRPS) clusters in P. galatheae were also strongly upregulated on chitin. Collectively, this suggests that both V. coralliilyticus and P. galatheae have a specific lifestyle for growth on chitin and that their secondary metabolites likely play a crucial role during chitin colonization. IMPORTANCE The bacterial family Vibrionaceae (vibrios) is considered a major player in the degradation of chitin, the most abundant polymer in the marine environment; however, the majority of studies on the topic have focused on a small number of Vibrio species. In this study, we analyzed the genomes of two vibrios to assess their genetic potential for the degradation of chitin. We then used transcriptomics and metabolomics to demonstrate that chitin strongly affects these vibrios at both the transcriptional and metabolic levels. We observed a strong increase in production of secondary metabolites, suggesting an ecological role for these molecules during chitin colonization in the marine environment