A blended genome and exome sequencing method captures genetic variation in an unbiased, high-quality, and cost-effective manner.

We deployed the Blended Genome Exome (BGE), a DNA library blending approach that generates low pass whole genome (1-4× mean depth) and deep whole exome (30-40× mean depth) data in a single sequencing run. This technology is cost-effective, empowers most genomic discoveries possible with deep whole genome sequencing, and provides an unbiased method to capture the diversity of common SNP variation across the globe. To evaluate this new technology at scale, we applied BGE to sequence >53,000 samples from the Populations Underrepresented in Mental Illness Associations Studies (PUMAS) Project, which included participants across African, African American, and Latin American populations. We evaluated the accuracy of BGE imputed genotypes against raw genotype calls from the Illumina Global Screening Array. All PUMAS cohorts had R 2 concordance ≥95% among SNPs with MAF≥1%, and never fell below ≥90% R 2 for SNPs with MAF<1%. Furthermore, concordance rates among local ancestries within two recently admixed cohorts were consistent among SNPs with MAF≥1%, with only minor deviations in SNPs with MAF<1%. We also benchmarked the discovery capacity of BGE to access protein-coding copy number variants (CNVs) against deep whole genome data, finding that deletions and duplications spanning at least 3 exons had a positive predicted value of ~90%. Our results demonstrate BGE scalability and efficacy in capturing SNPs, indels, and CNVs in the human genome at 28% of the cost of deep whole-genome sequencing. BGE is poised to enhance access to genomic testing and empower genomic discoveries, particularly in underrepresented populations.

Phage detection by a bacterial NLR-related protein is mediated by DnaJ.

Bacteria encode a wide range of antiphage systems and a subset of these proteins are homologous to components of the human innate immune system. Mammalian nucleotide-binding and leucine-rich repeat containing proteins (NLRs) and bacterial NLR-related proteins use a central NACHT domain to link infection detection with initiation of an antimicrobial response. Bacterial NACHT proteins provide defense against both DNA and RNA phages. Here we determine the mechanism of RNA phage detection by the bacterial NLR-related protein bNACHT25 in E. coli. bNACHT25 was specifically activated by Emesvirus ssRNA phages and analysis of MS2 phage suppressor mutants that evaded detection revealed Coat Protein (CP) was sufficient for activation. bNACHT25 and CP did not physically interact. Instead, we found bNACHT25 requires the host chaperone DnaJ to detect CP. Our data suggest that bNACHT25 detects a wide range of phages by guarding a host cell process rather than binding a specific phage-derived molecule.

Development of a Self-Report Measure of Prediction in Daily Life: The Prediction-Related Experiences Questionnaire.

PURPOSE: Predictions are complex, multisensory, and dynamic processes involving real-time adjustments based on environmental inputs. Disruptions to prediction abilities have been proposed to underlie characteristics associated with autism. While there is substantial empirical literature related to prediction, the field lacks a self-assessment measure of prediction skills related to daily tasks. Such a measure would be useful to better understand the nature of day-to-day prediction-related activities and characterize these abilities in individuals who struggle with prediction. METHODS: An interdisciplinary mixed-methods approach was utilized to develop and validate a self-report questionnaire of prediction skills for adults, the Prediction-Related Experiences Questionnaire (PRE-Q). Two rounds of online field testing were completed in samples of autistic and neurotypical (NT) adults. Qualitative feedback from a subset of these participants regarding question content and quality was integrated and Rasch modeling of the item responses was applied. RESULTS: The final PRE-Q includes 19 items across 3 domains (Sensory, Motor, Social), with evidence supporting the validity of the measure's 4-point response categories, internal structure, and relationship to other outcome measures associated with prediction. Consistent with models of prediction challenges in autism, autistic participants indicated more prediction-related difficulties than the NT group. CONCLUSIONS: This study provides evidence for the validity of a novel self-report questionnaire designed to measure the day-to-day prediction skills of autistic and non-autistic adults. Future research should focus on characterizing the relationship between the PRE-Q and lab-based measures of prediction, and understanding how the PRE-Q may be used to identify potential areas for clinical supports for individuals with prediction-related challenges.

Developing and Validating the Preschool Nutrition Education Practices Survey.

OBJECTIVE: Validate the Preschool Nutrition Education Practices Survey. DESIGN: Iterative approach combining design-based research and Standards for Educational and Psychological Testing. SETTING: Los Angeles, CA and Philadelphia, PA Early Care and Education (ECE) classrooms. PARTICIPANTS: Expert panel members (n = 7); ECE teachers: interviews (n = 8), pilot survey (n = 31), and final survey (n = 136). VARIABLES MEASURED: Early care and education nutrition education practices used in the classroom either during class time or mealtime. ANALYSIS: Qualitative content analysis was implemented for content, response process, and consequences of testing validity evidence. Rasch rating scale analysis was conducted for the response process and internal structure validity and reliability evidence. RESULTS: Qualitative field-testing produced strong content, response process, and consequences of testing validity evidence to inform survey modifications. Quantitative field-testing generated a psychometrically sound, well-targeted 12-item survey on a 4-point frequency scale with excellent item and person reliability (0.97 and 0.93 respectively) and separation (5.36 and 3.77 respectively); good Rasch Principal Components Analysis findings (60.3%); and productive item fit statistics (0.50-1.50 logits). CONCLUSIONS AND IMPLICATIONS: Robust validity (content, response process, consequences of testing, internal structure) and reliability evidence were demonstrated for using the Preschool Nutrition Education Practices Survey to assess ECE teachers' use of nutrition education practices. Future research is needed to examine its relationship to other variables, such as nutrition teaching efficacy, and to determine its ability to detect change in ECE nutrition education practices over time and across groups.

A Multilocus Sequence Typing Scheme for Rapid Identification of Xanthomonas citri Based on Whole-Genome Sequencing Data.

Xanthomonas citri is a plant-pathogenic bacterium associated with a diverse range of host plant species. It has undergone substantial reclassification and currently consists of 14 different subspecies or pathovars that are responsible for a wide range of plant diseases. Whole-genome sequencing (WGS) provides a cutting-edge advantage over other diagnostic techniques in epidemiological and evolutionary studies of X. citri because it has a higher discriminatory power and is replicable across laboratories. WGS also allows for the improvement of multilocus sequence typing (MLST) schemes. In this study, we used genome sequences of Xanthomonas isolates from the NCBI RefSeq database to develop a seven-gene MLST scheme that yielded 19 sequence types (STs) that correlated with phylogenetic clades of X. citri subspecies or pathovars. Using this MLST scheme, we examined 2,911 Xanthomonas species assemblies from NCBI GenBank and identified 15 novel STs from 37 isolates that were misclassified in NCBI. In total, we identified 545 X. citri assemblies from GenBank with 95% average nucleotide identity to the X. citri type strain, and all were classified as one of the 34 STs. All MLST classifications correlated with a phylogenetic position inferred from alignments using 92 conserved genes. We observed several instances where strains from different pathovars formed closely related monophyletic clades and shared the same ST, indicating that further investigation of the validity of these pathovars is required. Our MLST scheme described here is a robust tool for rapid classification of X. citri pathovars using WGS and a powerful method for further comprehensive taxonomic revision of X. citri pathovars.

Xanthomonas rydalmerensis sp. nov., a non-pathogenic member of Group 1 Xanthomonas.

Five bacterial isolates were isolated from Fragaria × ananassa in 1976 in Rydalmere, Australia, during routine biosecurity surveillance. Initially, the results of biochemical characterisation indicated that these isolates represented members of the genus Xanthomonas. To determine their species, further analysis was conducted using both phenotypic and genotypic approaches. Phenotypic analysis involved using MALDI-TOF MS and BIOLOG GEN III microplates, which confirmed that the isolates represented members of the genus Xanthomonas but did not allow them to be classified with respect to species. Genome relatedness indices and the results of extensive phylogenetic analysis confirmed that the isolates were members of the genus Xanthomonas and represented a novel species. On the basis the minimal presence of virulence-associated factors typically found in genomes of members of the genus Xanthomonas, we suggest that these isolates are non-pathogenic. This conclusion was supported by the results of a pathogenicity assay. On the basis of these findings, we propose the name Xanthomonas rydalmerensis, with DAR 34855T = ICMP 24941 as the type strain.

Synthesis and preclinical evaluation of a 89Zr-labelled human single chain antibody for non-invasive detection of hepatic myofibroblasts in acute liver injury.

Synaptophysin is expressed on fibrogenic hepatic myofibroblasts. C1-3 is a single chain human antibody (scAb) that binds specifically to synaptophysin on hepatic myofibroblasts, providing a targeting vector for novel in vivo imaging agents of chronic liver disease. C1-3 and a negative control scAb, CSBD9, were radiolabelled with zirconium-89 via desferrioxamine chelation to enable non-invasive molecular imaging with positron emission tomography (PET). DFO-scAb conjugates were characterised by gel electrophoresis (SDS-PAGE) and MALDI-TOF spectrometry, and 89Zr-labelled with high radiolabelling efficiency (99%). [89Zr]Zr-DFO-C1-3 exhibited high in vitro stability (> 99%) in mouse and human sera over 3 days at 25 and 37 °C. Activated hepatic myofibroblasts incubated with [89Zr]Zr-DFO-C1-3 displayed significantly higher internalised activity (59.46%, P = 0.001) compared to the [89Zr]Zr-DFO-CSBD9 control, indicating synaptophysin-mediated uptake and high binding specificity of [89Zr]Zr-DFO-C1-3. Mice with CCl4-induced acute liver damage exhibited significantly higher liver uptake of [89Zr]Zr-DFO-C1-3, compared to controls, confirmed by both Cerenkov imaging and ex vivo gamma counting (4.41 ± 0.19%ID/g, P < 0.0001). CCl4-induced liver damage and the number of hepatic myofibroblasts was confirmed by αSMA staining of liver sections. These findings indicate that [89Zr]Zr-DFO-C1-3 has promising utility as a PET imaging agent for non-invasive detection of hepatic myofibroblasts following acute liver injury.

High-quality full genome assembly of historic Xylella fastidiosa strains from ICMP collection using a hybrid sequencing approach.

High-quality complete genomes of five Xylella fastidiosa strains were assembled by combining Nanopore and Illumina sequencing data. Among these, International Collection of Micro-organisms from Plants (ICMP) 8731, ICMP 8742 and ICMP 8745 belong to subspecies fastidiosa while ICMP 8739 and ICMP 8740 were determined as subspecies multiplex. The strains were further classified into sequence types.

An Engineered Synthetic Receptor-Aptamer Pair for an Artificial Signal Transduction System.

Cell membrane receptors regulate cellular responses through sensing extracellular environmental signals and subsequently transducing them. Receptor engineering provides a means of directing cells to react to a designated external cue and exert programmed functions. However, rational design and precise modulation of receptor signaling activity remain challenging. Here, we report an aptamer-based signal transduction system and its applications in controlling and customizing the functions of engineered receptors. A previously reported membrane receptor-aptamer pair was used to design a synthetic receptor system that transduces cell signaling depending on exogenous aptamer input. To eliminate the cross-reactivity of the receptor with its native ligand, the extracellular domain of the receptor was engineered to ensure that the receptor was solely activated by the DNA aptamer. The present system features tunability in the signaling output level using aptamer ligands with different receptor dimerization propensities. In addition, the functional programmability of DNA aptamers enables the modular sensing of extracellular molecules without the need for genetic engineering of the receptor.

Bacterial NLR-related proteins protect against phage.

Bacteria use a wide range of immune pathways to counter phage infection. A subset of these genes shares homology with components of eukaryotic immune systems, suggesting that eukaryotes horizontally acquired certain innate immune genes from bacteria. Here, we show that proteins containing a NACHT module, the central feature of the animal nucleotide-binding domain and leucine-rich repeat containing gene family (NLRs), are found in bacteria and defend against phages. NACHT proteins are widespread in bacteria, provide immunity against both DNA and RNA phages, and display the characteristic C-terminal sensor, central NACHT, and N-terminal effector modules. Some bacterial NACHT proteins have domain architectures similar to the human NLRs that are critical components of inflammasomes. Human disease-associated NLR mutations that cause stimulus-independent activation of the inflammasome also activate bacterial NACHT proteins, supporting a shared signaling mechanism. This work establishes that NACHT module-containing proteins are ancient mediators of innate immunity across the tree of life.

Domain specific association between physical activity and affect in adolescents' daily lives: an ecological momentary assessment study.

OBJECTIVE: Little is known about the domain specific associations between moderate-to-vigorous physical activity (MVPA) and affect. This study aimed to determine whether the association between MVPA and affect differed by domain in adolescents. DESIGN: Overall, 119 adolescents (mean age = 14.7 years) provided information about their affective states multiple times a day over a four-day period using ecological momentary assessment (EMA). Additionally, minutes of MVPA were measured using accelerometers and participants self-reported whether they were participating in recreational physical activity, active travel, or household physical activity at the time of an EMA prompt. MAIN OUTCOME MEASURES: The main outcomes that were measured were affective valence, energetic arousal and tense arousal. RESULTS: Participants who engaged in more recreational MVPA on average reported more positive valence, more energetic arousal, and less tense arousal. Additionally, participants reported more energetic arousal when they participated in greater levels of recreational MVPA than usual, but also more tense arousal. Active travel and household physical activity were not associated with valence, energetic arousal or tense arousal. CONCLUSION: These findings highlight the importance of encouraging participation in recreational physical activities to promote positive affective outcomes.

The influence of degree of labelling upon cellular internalisation of antibody-cell penetrating peptide conjugates.

Antibody-based agents are increasingly used as therapeutics and imaging agents, yet are generally restricted to cell surface targets due to inefficient cellular internalisation and endosomal entrapment. Enhanced cell membrane translocation of antibodies can be achieved by the covalent attachment of cell-penetrating peptides, including the HIV-1-derived transactivator of transcription (TAT) peptide. This study evaluated the cellular internalisation properties of five anti-HER2 Herceptin-TAT conjugates with degrees of TAT labelling (DOLTAT) ranging from one to five. Herceptin-TAT conjugates were synthesised via a strain-promoted alkyne-azide cycloaddition reaction, characterised by UV-vis spectroscopy, MALDI-TOF, and gel electrophoresis, then radiolabelled with zirconium-89 to permit measurement of cellular internalisation by gamma counting. [89Zr]Zr-DFO-Her-TAT(0-5) conjugates were isolated in high radiochemical purity (>99%) and exhibited high stability in murine and human serum over 7 days at 37 °C. Significant increases in cellular internalisation were observed for [89Zr]Zr-DFO-Her-TAT conjugates with DOLTAT values of 2 and above in SKBR3 (high HER2) cells over 48 h, in contrast to low-level non-specific uptake in MDA-MB-468 (low HER2) cells that did not increase over time. Notably, [89Zr]Zr-DFO-Her-TAT conjugates with DOLTAT of 3, 4, and 5 reached uptake values in SKBR3 cells of 5, 6, and 8% of the applied dose at 48 h respectively, representing 9, 10, 14-fold increases relative to the TAT-free control conjugate, [89Zr]Zr-DFO-Her-TAT(0).

Salmonella enterica Infections Are Disrupted by Two Small Molecules That Accumulate within Phagosomes and Differentially Damage Bacterial Inner Membranes.

Gram-negative bacteria have a robust cell envelope that excludes or expels many antimicrobial agents. However, during infection, host soluble innate immune factors permeabilize the bacterial outer membrane. We identified two small molecules that exploit outer membrane damage to access the bacterial cell. In standard microbiological media, neither compound inhibited bacterial growth nor permeabilized bacterial outer membranes. In contrast, at micromolar concentrations, JAV1 and JAV2 enabled the killing of an intracellular human pathogen, Salmonella enterica serovar Typhimurium. S. Typhimurium is a Gram-negative bacterium that resides within phagosomes of cells from the monocyte lineage. Under broth conditions that destabilized the lipopolysaccharide layer, JAV2 permeabilized the bacterial inner membrane and was rapidly bactericidal. In contrast, JAV1 activity was more subtle: JAV1 increased membrane fluidity, altered reduction potential, and required more time than JAV2 to disrupt the inner membrane barrier and kill bacteria. Both compounds interacted with glycerophospholipids from Escherichia coli total lipid extract-based liposomes. JAV1 preferentially interacted with cardiolipin and partially relied on cardiolipin production for activity, whereas JAV2 generally interacted with lipids and had modest affinity for phosphatidylglycerol. In mammalian cells, neither compound significantly altered mitochondrial membrane potential at concentrations that killed S. Typhimurium. Instead, JAV1 and JAV2 became trapped within acidic compartments, including macrophage phagosomes. Both compounds improved survival of S. Typhimurium-infected Galleria mellonella larvae. Together, these data demonstrate that JAV1 and JAV2 disrupt bacterial inner membranes by distinct mechanisms and highlight how small, lipophilic, amine-substituted molecules can exploit host soluble innate immunity to facilitate the killing of intravesicular pathogens. IMPORTANCE Innovative strategies for developing new antimicrobials are needed. Combining our knowledge of host-pathogen interactions and relevant drug characteristics has the potential to reveal new approaches to treating infection. We identified two compounds with antibacterial activity specific to infection and with limited host cell toxicity. These compounds appeared to exploit host innate immunity to access the bacterium and differentially damage the bacterial inner membrane. Further, both compounds accumulated within Salmonella-containing and other acidic vesicles, a process known as lysosomal trapping, which protects the host and harms the pathogen. The compounds also increased host survival in an insect infection model. This work highlights the ability of host innate immunity to enable small molecules to act as antibiotics and demonstrates the feasibility of antimicrobial targeting of the inner membrane. Additionally, this study features the potential use of lysosomal trapping to enhance the activities of compounds against intravesicular pathogens.

Within-Person Associations Between Physical and Social Contexts With Movement Behavior Compositions in Adolescents: An Ecological Momentary Assessment Study Using a Compositional Data Analysis Approach.

BACKGROUND: External contexts, including the social and physical contexts, are independent predictors of momentary physical activity and sedentary behaviors. However, no studies to date have examined how external contexts are related to overall momentary movement behavior compositions using compositional data analysis. Therefore, this study aimed to determine differences in momentary movement behavior compositions between different social and physical contexts in adolescents. METHODS: Overall, 119 adolescents (mean age 14.7 y, SD = 1.44) provided details about their momentary physical and social contexts over 4 days using ecological momentary assessment. Sedentary behaviors, light-intensity physical activity, and moderate to vigorous physical activity were assessed using ActiGraph GT3X+ accelerometers. Compositional multivariate multilevel models were estimated to determine if movement behavior compositions differed between contexts. RESULTS: Participants engaged in significantly less sedentary behaviors when outdoors compared with indoors and replaced it with moderate to vigorous physical activity. Participants also engaged in significantly less sedentary behaviors when with friends or friends and family and replaced it with light-intensity physical activity. CONCLUSION: These results highlight the potential of targeting external contexts to increase physical activity and to reduce sedentary behavior in adolescents' daily lives. These factors could be targeted in mobile health and just-in-time adaptive interventions to improve young people's movement behavior compositions.

Synthesis and In Vivo Evaluation of a Site-specifically Labeled Radioimmunoconjugate for Dual-Modal (PET/NIRF) Imaging of MT1-MMP in Sarcomas.

Bone sarcomas are devastating primary bone cancers that mostly affect children, young adults, and the elderly. These aggressive tumors are associated with poor survival, and surgery remains the mainstay of treatment. Surgical planning is increasingly informed by positron emission tomography (PET), and tumor margin identification during surgery is aided by near-infrared fluorescence (NIRF) imaging, yet these investigations are confounded by probes that lack specificity for sarcoma biomarkers. We report the development of a dual-modal (PET/NIRF) immunoconjugate ([89Zr]Zr-DFO-anti-MT1-MMP-IRDye800CW) that targets MT1-MMP, a matrix metalloproteinase overexpressed in high-grade sarcomas. [89Zr]Zr-DFO-anti-MT1-MMP-IRDye800CW was synthesized via site-specific chemoenzymatic glycan modification, characterized, and isolated in high specific activity and radiochemical purity. Saturation binding and immunoreactivity assays indicated only minor perturbation of binding properties. A novel mouse model of dedifferentiated chondrosarcoma based on intrafemoral inoculation of HT1080 WT or KO cells (high and low MT1-MMP expression, respectively) was used to evaluate target binding and biodistribution. Fluorescence and Cerenkov luminescence images of [89Zr]Zr-DFO-anti-MT1-MMP-IRDye800CW showed preferential uptake in HT1080 WT tumors. Ex vivo gamma counting revealed that uptake in MT1-MMP-positive tumors was significantly higher than that in control groups. Taken together, [89Zr]Zr-DFO-anti-MT1-MMP-IRDye800CW is a promising dual-modal sarcoma imaging agent for pre-operative surgical planning and intraoperative surgical guidance.

Using Genomics to Design a Pathovar-Specific Loop-Mediated Isothermal Amplification (LAMP) Assay, for the Improved Detection of Xanthomonas citri pv. citri.

The ability to swiftly respond to pathogen incursions relies heavily on fast and accurate diagnostics. Current published assays for citrus bacterial canker do not target Xanthomonas citri pv. citri, the causative agent, with high specificity when testing Australian samples. While the current diagnostics are useful in countries where canker is endemic, the detection of canker in Australia requires an emergency response. Close relatives to X. citri pv. citri found in Australia may generate false positives with the current recommended diagnostic assays. Therefore, we developed a more specific detection tool for citrus bacterial canker to provide greater diagnostic confidence for surveillance and eradication efforts. We used genomic comparisons of 161 Xanthomonad genomes and identified and confirmed genomic regions specific for X. citri pv. citri by performing local alignments of unique regions to reference genomes. We then developed loop-mediated isothermal amplification primers and validated them against a panel of 190 isolates to confirm specificity. Our diagnostic assay showed 100% corroboration with the concurrently developed multiplex primers and represents an improved diagnostic method capable of effective citrus bacterial canker identification.

A small molecule that disrupts S. Typhimurium membrane voltage without cell lysis reduces bacterial colonization of mice.

As pathogenic bacteria become increasingly resistant to antibiotics, antimicrobials with mechanisms of action distinct from current clinical antibiotics are needed. Gram-negative bacteria pose a particular problem because they defend themselves against chemicals with a minimally permeable outer membrane and with efflux pumps. During infection, innate immune defense molecules increase bacterial vulnerability to chemicals by permeabilizing the outer membrane and occupying efflux pumps. Therefore, screens for compounds that reduce bacterial colonization of mammalian cells have the potential to reveal unexplored therapeutic avenues. Here we describe a new small molecule, D66, that prevents the survival of a human Gram-negative pathogen in macrophages. D66 inhibits bacterial growth under conditions wherein the bacterial outer membrane or efflux pumps are compromised, but not in standard microbiological media. The compound disrupts voltage across the bacterial inner membrane at concentrations that do not permeabilize the inner membrane or lyse cells. Selection for bacterial clones resistant to D66 activity suggested that outer membrane integrity and efflux are the two major bacterial defense mechanisms against this compound. Treatment of mammalian cells with D66 does not permeabilize the mammalian cell membrane but does cause stress, as revealed by hyperpolarization of mitochondrial membranes. Nevertheless, the compound is tolerated in mice and reduces bacterial tissue load. These data suggest that the inner membrane could be a viable target for anti-Gram-negative antimicrobials, and that disruption of bacterial membrane voltage without lysis is sufficient to enable clearance from the host.

Phylogenetic diversity analysis of shotgun metagenomic reads describes gut microbiome development and treatment effects in the post-weaned pig.

Intensive farming practices can increase exposure of animals to infectious agents against which antibiotics are used. Orally administered antibiotics are well known to cause dysbiosis. To counteract dysbiotic effects, numerous studies in the past two decades sought to understand whether probiotics are a valid tool to help re-establish a healthy gut microbial community after antibiotic treatment. Although dysbiotic effects of antibiotics are well investigated, little is known about the effects of intramuscular antibiotic treatment on the gut microbiome and a few studies attempted to study treatment effects using phylogenetic diversity analysis techniques. In this study we sought to determine the effects of two probiotic- and one intramuscularly administered antibiotic treatment on the developing gut microbiome of post-weaning piglets between their 3rd and 9th week of life. Shotgun metagenomic sequences from over 800 faecal time-series samples derived from 126 post-weaning piglets and 42 sows were analysed in a phylogenetic framework. Differences between individual hosts such as breed, litter, and age, were found to be important contributors to variation in the community composition. Host age was the dominant factor in shaping the gut microbiota of piglets after weaning. The post-weaning pig gut microbiome appeared to follow a highly structured developmental program with characteristic post-weaning changes that can distinguish hosts that were born as little as two days apart in the second month of life. Treatment effects of the antibiotic and probiotic treatments were found but were subtle and included a higher representation of Mollicutes associated with intramuscular antibiotic treatment, and an increase of Lactobacillus associated with probiotic treatment. The discovery of correlations between experimental factors and microbial community composition is more commonly addressed with OTU-based methods and rarely analysed via phylogenetic diversity measures. The latter method, although less intuitive than the former, suffers less from library size normalization biases, and it proved to be instrumental in this study for the discovery of correlations between microbiome composition and host-, and treatment factors.