Investigation of induced electroosmotic flow in small-scale capillary electrophoresis devices: Strategies for control and reversal.

Electroosmotic flow (EOF) is the bulk flow of solution in a capillary or microchannel induced by an applied electric potential. For capillary and microchip electrophoresis, the EOF enables analysis of both cations and anions in one separation and can be varied to modify separation speed and resolution. The EOF arises from an electrical double layer at the capillary wall and is normally controlled through the pH and ionic strength of the background buffer or with the use of additives. Understanding and controlling the electrical double layer is therefore critical for maintaining acceptable repeatability during method development. Surprisingly, in fused silica capillaries at low pH, studies observe an EOF even though the capillary surface should be neutralized. Previous work has suggested the presence of an "induced electroosmotic flow" from radial electric fields generated across the capillary wall due to the separation voltage and grounded components external to the capillary. Using thin-wall (15 µm) fused silica separation capillaries to facilitate the study of radial fields, we show that the EOF mobility depends on both the separation voltage and the location of external grounds. This is consistent with the induced EOF model, in which radial electric fields embed positive charges at the capillary walls to create an electrical double layer. The magnitude of the effect is characterized and shown to have long-range influences that are difficult to completely null by moving grounded components away from the separation capillary. Instead, active EOF control using externally applied potentials or a passive approach using a negative separation voltage are discussed as two possible methods for controlling the induced EOF. Both methods can reverse the EOF and improve the resolution and peak efficiency in amino acid separations.

Fluoroquinolone-resistant Campylobacter in backyard and commercial broiler production systems in the United States.

Objectives: Campylobacter spp. are one of the leading foodborne pathogens in the world, and chickens are a known reservoir. This is significant considering broiler chicken is the top consumed meat worldwide. In the USA, backyard poultry production is increasing, but little research has been done to investigate prevalence and antimicrobial resistance associated with Campylobacter in these environments. Methods: Our study encompasses a farm-to-genome approach to identify Campylobacter and investigate its antimicrobial resistance phenotypically and genotypically. We travelled to 10 backyard and 10 integrated commercial broiler farms to follow a flock throughout production. We sampled at days 10, 31 and 52 for backyard and 10, 24 and 38 for commercial farms. Bird faecal (n = 10) and various environmental samples (soil n = 5, litter/compost n = 5, and feeder and waterer swabs n = 6) were collected at each visit and processed for Campylobacter. Results: Our results show a higher prevalence of Campylobacter in samples from backyard farms (21.9%) compared to commercial (12.2%). Most of our isolates were identified as C. jejuni (70.8%) and the remainder as C. coli (29.2%). Antimicrobial susceptibility testing reveals phenotypic resistance to ciprofloxacin (40.2%), an important treatment drug for Campylobacter infection, and tetracycline (46.6%). A higher proportion of resistance was found in C. jejuni isolates and commercial farms. Whole-genome sequencing revealed resistance genes, such as tet(O) and gyrA_T86I point mutation, that may confer resistance. Conclusion: Overall, our research emphasizes the need for interventions to curb prevalence of resistant Campylobacter spp. on broiler production systems.

Photothermal Backscatter Interferometry for Enhanced Detection in Capillary Electrophoresis.

Refractive index (RI) detection using backscatter interferometry (BSI) enables universal detection in capillary electrophoresis (CE). BSI detection is a versatile on-capillary approach that is easily integrated with capillary or microfluidic channels, straightforward to miniaturize, and inexpensive. The focused BSI light source can also double as the excitation source for fluorescence, enabling simultaneous universal (BSI) and specific (fluorescence) signals from the same detection volume. To improve BSI detection and expand orthogonal content, we integrate photothermal absorption with BSI detection. Nonradiative relaxation of an excited analyte releases heat into the surroundings, which modifies both the local RI and conductivity (viscosity) of the analyte zone. We recently showed that the BSI signal is sensitive to both RI and conductivity, which makes photothermal absorption a promising route to signal enhancement. Here, we use coaxially delivered BSI and photothermal absorption beams to characterize BSI, photothermal BSI, and fluorescence detection using the separation of test samples. We show that photothermal absorption leads to 3 orders of magnitude improvement in BSI detection limits at the powers studied and provides new opportunities for studying binding interactions with CE.

Multi-level tuberculosis of the spine identified by 18 F-FDG-PET/CT and concomitant urogenital tuberculosis: a case report from the spinal TB X cohort.

BACKGROUND: Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) and typically infects the lungs. However, extrapulmonary forms of TB can be found in approximately 20% of cases. It is suggested, that up to 10% of extrapulmonary TB affects the musculoskeletal system, in which spinal elements (spinal tuberculosis, STB) are involved in approximately 50% of the cases. STB is a debilitating disease with nonspecific symptoms and diagnosis is often delayed for months to years. In our Spinal TB X Cohort, we aim to describe the clinical phenotype of STB using whole-body 18 F-fluorodeoxyglucose positron emission tomography computed tomography (PET/CT) and to identify a specific gene expression profile for the different stages of dissemination on PET/CT. Here we report on the first patient recruited into our cohort who underwent PET/CT before treatment initiation, at 6-months and at 12-months - time of TB treatment completion. CASE PRESENTATION: A 27-year-old immunocompetent male presented with severe thoracolumbar back pain for 9 months with severe antalgic gait and night sweats. Magnetic resonance imaging (MRI) of the whole spine revealed multilevel spinal disease (T5/6, T11/12, L3/4) in keeping with STB. After informed consent and recruitment into the Spinal TB X Cohort, the patient underwent PET/CT as per protocol, which revealed isolated multilevel STB (T4-7, T11/12, L3/4) with no concomitant lung or urogenital lesion. However, sputum and urine were Xpert MTB/RIF Ultra positive and Mtb was cultured from the urine sample. CT-guided biopsy of the T11/12 lesion confirmed drug-sensitive Mtb on Xpert MTB/RIF Ultra and the patient was started on TB treatment according to local guidelines for 12 months. The 6-month follow-up PET/CT revealed new and existing spinal lesions with increased FDG-uptake despite significant improvement of clinical features and laboratory markers. After 9 months of treatment, the patient developed an acute urethral stricture, most likely due to urogenital TB, and a suprapubic catheter was inserted. The 12-month PET/CT showed significantly decreased PET/CT values of all lesions, however, significant persistent spinal inflammation was present at the end of TB treatment. Clinically, the patient was considered cured by the TB control program and currently awaits urethroplasty. CONCLUSIONS: In our case, PET/CT emerged as a valuable imaging modality for the initial assessment, surpassing MRI by revealing more comprehensive extensive disease. Subsequent PET/CT scans at 6-month uncovered new lesions and increased inflammation in existing ones, while by the end of TB treatment, all lesions exhibited improvement. However, the interpretation of FDG avidity remains ambiguous, whether it correlates with active infection and viable Mtb. or fibro- and osteoblast activity indicative of the healing process. Additionally, the absence of extraspinal TB lesions on PET/CT despite positive microbiology from sputum and urine maybe explained by paucibacillary, subclinical infection of extraspinal organs. The Spinal TB X Cohort endeavours to shed light on whole-body imaging patterns at diagnosis, their evolution midway through TB treatment, and upon treatment completion. Ultimately, this study aims to advance our understanding of the biology of this complex disease.

Widespread prevalence of plasmid-mediated blaCTX-M type extended-spectrum beta-lactamase Escherichia coli in backyard broiler production systems in the United States.

Extended-spectrum beta-lactamase (ESBL) Escherichia coli (E. coli) is an emerging pathogen of high concern given its resistance to extended-spectrum cephalosporins. Broiler chicken, which is the number one consumed meat in the United States and worldwide, can be a reservoir of ESBL E. coli. Backyard poultry ownership is on the rise in the United States, yet there is little research investigating prevalence of ESBL E. coli in this setting. This study aims to identify the prevalence and antimicrobial resistance profiles (phenotypically and genotypically) of ESBL E. coli in some backyard and commercial broiler farms in the U.S. For this study ten backyard and ten commercial farms were visited at three time-points across flock production. Fecal (n = 10), litter/compost (n = 5), soil (n = 5), and swabs of feeders and waterers (n = 6) were collected at each visit and processed for E. coli. Assessment of ESBL phenotype was determined through using disk diffusion with 3rd generation cephalosporins, cefotaxime and ceftazidime, and that with clavulanic acid. Broth microdilution and whole genome sequencing were used to investigate both phenotypic and genotypic resistance profiles, respectively. ESBL E. coli was more prevalent in backyard farms with 12.95% of samples testing positive whereas 0.77% of commercial farm samples were positive. All isolates contained a blaCTX-M gene, the dominant variant being blaCTX-M-1, and its presence was entirely due to plasmids. Our study confirms concerns of growing resistance to fourth generation cephalosporin, cefepime, as roughly half (51.4%) of all isolates were found to be susceptible dose-dependent and few were resistant. Resistance to non-beta lactams, gentamicin and ciprofloxacin, was also detected in our samples. Our study identifies prevalence of blaCTX-M type ESBL E. coli in U.S. backyard broiler farms, emphasizing the need for interventions for food and production safety.

Asymmetric magma plumbing system beneath Axial Seamount based on full waveform inversion of seismic data.

The architecture of magma plumbing systems plays a fundamental role in volcano eruption and evolution. However, the precise configuration of crustal magma reservoirs and conduits responsible for supplying eruptions are difficult to explore across most active volcanic systems. Consequently, our understanding of their correlation with eruption dynamics is limited. Axial Seamount is an active submarine volcano located along the Juan de Fuca Ridge, with known eruptions in 1998, 2011, and 2015. Here we present high-resolution images of P-wave velocity, attenuation, and estimates of temperature and partial melt beneath the summit of Axial Seamount, derived from multi-parameter full waveform inversion of a 2D multi-channel seismic line. Multiple magma reservoirs, including a newly discovered western magma reservoir, are identified in the upper crust, with the maximum melt fraction of ~15-32% in the upper main magma reservoir (MMR) and lower fractions of 10% to 26% in other satellite reservoirs. In addition, a feeding conduit below the MMR with a melt fraction of ~4-11% and a low-velocity throat beneath the eastern caldera wall connecting the MMR roof with eruptive fissures are imaged. These findings delineate an asymmetric shallow plumbing system beneath Axial Seamount, providing insights into the magma pathways that fed recent eruptions.

Comparing gene expression profiles of adults with isolated spinal tuberculosis to disseminated spinal tuberculosis identified by 18FDG-PET/CT at time of diagnosis, 6- and 12-months follow-up: classifying clinical stages of spinal tuberculosis and monitoring treatment response (Spinal TB X cohort study).

BACKGROUND: Tuberculosis (TB) is one of the top ten causes of death worldwide, with approximately 10 million cases annually. Focus has been on pulmonary TB, while extrapulmonary TB (EPTB) has received little attention. Diagnosis of EPTB remains challenging due to the invasive procedures required for sample collection. Spinal TB (STB) accounts for 10% of EPTB and often leads to lifelong debilitating disease due to devastating spinal deformation and compression of neural structures. Little is known about the extent of disease, although both isolated STB and a disseminated form of STB have been described. In our Spinal TB X cohort study, we aim to describe the clinical phenotype of STB using whole-body 18FDG-PET/CT, identify a specific gene expression profile for different stages of dissemination and compare findings to previously described gene expression signatures for latent and active pulmonary TB. METHODS: A single-centre, prospective cohort study will be established to describe the distributional pattern of STB detected by whole-body 18FDG-PET/CT and gene expression profile of patients with suspected STB on magnetic resonance imaging (MRI) at point of diagnosis, six months, and 12 months. Blood biobanking will be performed at these time points. Specimens for microbiology will be obtained from sputum/urine, from easily accessible sites of disease (e.g., lymph nodes, abscess) identified in the first 18FDG-PET/CT, from CT-guided biopsy and/or surgery. Clinical parameters and functional scores will be collected at every physical visit. Data will be entered into RedCap® database; data cleaning, validation and analysis will be performed by the study team. The University of Cape Town Ethics Committee approved the protocol (243/2022). DISCUSSION: The Spinal TB X cohort study is the first prospective cohort study using whole-body 18FDG-PET/CT scans in patients with microbiologically confirmed spinal tuberculosis. Dual imaging techniques of the spine using 18FDG-PET/CT and magnetic resonance imaging as well as tissue diagnosis (microbiology and histopathology) will allow us to develop a virtual biopsy model. If successful, a distinct gene-expression profile will aid in blood-based diagnosis (point of care testing) as well as treatment monitoring and would lead to earlier diagnosis of this devastating disease. TRIAL REGISTRATION: The study has been registered on ClinicalTrials.gov (NCT05610098).

Climate, climate change and the global diversity of human houses.

Globally, human house types are diverse, varying in shape, size, roof type, building materials, arrangement, decoration and many other features. Here we offer the first rigorous, global evaluation of the factors that influence the construction of traditional (vernacular) houses. We apply macroecological approaches to analyse data describing house features from 1900 to 1950 across 1000 societies. Geographic, social and linguistic descriptors for each society were used to test the extent to which key architectural features may be explained by the biophysical environment, social traits, house features of neighbouring societies or cultural history. We find strong evidence that some aspects of the climate shape house architecture, including floor height, wall material and roof shape. Other features, particularly ground plan, appear to also be influenced by social attributes of societies, such as whether a society is nomadic, polygynous or politically complex. Additional variation in all house features was predicted both by the practices of neighouring societies and by a society's language family. Collectively, the findings from our analyses suggest those conditions under which traditional houses offer solutions to architects seeking to reimagine houses in light of warmer, wetter or more variable climates.

Site- and species-specific metal concentrations, mobility, and bioavailability in sediment, flora, and fauna of a southeastern United States salt marsh.

Salt marshes are highly productive and valuable coastal ecosystems that act as filters for nutrients and pollutants at the land-sea interface. The salt marshes of the mid-Atlantic United States often exhibit geochemical behavior that varies significantly from other estuaries around the world, but our understanding of metal mobility and bioavailability remains incomplete for these systems. We sampled abiotic (water and sediment) and native biotic (three halophyte and two bivalve species) compartments of a southeastern United States salt marsh to understand the site- and species-specific metal concentrations, fractionation, and bioavailability for 16 metals and metalloids, including two naturally occurring radionuclides. Location on the marsh platform greatly influenced metal concentrations in sediment and metal bioaccumulation in halophytes, with sites above the mean high-water mark (i.e., high marsh zone) having lower concentrations in sediment but plants exhibiting greater biota sediment accumulation factors (BSAFs). Transition metal concentrations in the sediment were an average of 6× higher in the low marsh zone compared to the high marsh zone and heavy metals were on average 2× higher. Tissue- and species-specific preferential accumulation in bivalves provide opportunities for tailored biomonitoring programs. For example, mussel byssal threads accumulated ten of the sixteen studied elements to significantly greater concentrations compared to soft tissues and oysters had remarkably high soft tissue zinc concentrations (~5000 mg/kg) compared to all other species and element combinations studied. Additionally, some of our results have important implications for understanding metal mobility and implementing effective remediation (specifically phytoremediation) strategies, including observations that (1) heavy metals exhibit distinct concentration spatial distributions and metal fractionation patterns which vary from the transition metals and (2) sediment organic matter fraction appears to play an important role in controlling sediment metal concentrations, fractionation, and plant bioavailability.

Higher Sensitivity of Xpert MTB/RIF Ultra Over Tuberculosis Culture for the Diagnosis of Spinal Tuberculosis With Open or Computed Tomography-Guided Biopsies.

Background: Diagnostic specimens for spinal tuberculosis (STB) are mostly collected via open surgery. Percutaneous computed tomography (CT)-guided biopsies are used in times of limited surgical availability. However, poor diagnostic accuracy of Mycobacterium tuberculosis (Mtb) culture has been reported with this method, due to limited sample volume and the paucibacillary nature of STB. We evaluated Xpert MTB/RIF Ultra on open and CT-guided biopsies as compared with the gold standard Mtb culture and histopathology. Methods: We conducted a prospective diagnostic accuracy study of Xpert Ultra, as compared with tuberculosis culture and histopathology, in adults with signs and symptoms of STB at a tertiary academic hospital in South Africa from November 2020 to December 2021. Diagnostic testing was performed on 31 patients with available samples. Results: Xpert Ultra had a sensitivity of 94.7% (95% CI, 75.3%-99.7%) and specificity of 100% (95% CI, 75.7%-100.0%) against a reference standard of Mtb culture and histopathology. Xpert Ultra had high diagnostic accuracy in open and CT-guided biopsy samples with sensitivity and specificity of 100% and 100% (open) and 89% and 100% (CT), respectively. Mtb culture had limited specificity for CT-guided biopsies (43%; 95% CI, 15.8%-74.9%). HIV-1 coinfection did not affect Mtb abundance measures by Xpert Ultra or culture. Xpert Ultra was also superior to culture for STB diagnosis in patients concurrently treated for pulmonary tuberculosis. Conclusions: Xpert Ultra detected more STB cases than culture for CT-guided biopsy samples. There was also no difference in sensitivity for open biopsies, irrespective of HIV-1 status, making it an important tool for rapid diagnosis, especially during times or in locations where open surgery is not possible or concurrent pulmonary tuberculosis treatment is initiated.

Electric field-enhanced backscatter interferometry detection for capillary electrophoresis.

Backscatter interferometry (BSI) is a refractive index (RI) detection method that is easily integrated with capillary electrophoresis (CE) and is capable of detecting species ranging from inorganic ions to proteins without additional labels or contrast agents. The BSI signal changes linearly with the square of the separation voltage which has been used to quantify sample injection, but has not been explored as a potential signal enhancement mechanism in CE. Here we develop a mathematical model that predicts a signal enhancement at high field strengths, where the BSI signal is dominated by the voltage dependent mechanism. This is confirmed in both simulation and experiment, which show that the analyte peak area grows linearly with separation voltage at high field strengths. This effect can be exploited by adjusting the background electrolyte (BGE) to increase the conductivity difference between the BGE and analyte zones, which is shown to improve BSI performance. We also show that this approach has utility in small bore capillaries where larger separation fields can be applied before excess Joule heating degrades the separation. Unlike other optical detection methods that generally degrade as the optical pathlength is reduced, the BSI signal-to-noise can improve in small bore capillaries as the larger separation fields enhance the signal.

Sourdough starters exhibit similar succession patterns but develop flour-specific climax communities.

The microbial fermentation behind sourdough bread is among our oldest technologies, yet there are many opportunities for sourdough science to learn from traditional bakers. We analyzed 16S rRNA sequences in R to assess the bacterial community structure and performance of 40 starters grown from 10 types of flour over 14 days, and identified six distinct stages of succession. At each stage, bacterial taxa correlate with determinants of bread quality including pH, rise, and aromatic profile. Day 1 starter cultures were dominated by microorganisms commonly associated with plants and flour, and by aromas similar to toasted grain/cereal. Bacterial diversity peaked from days 2-6 as taxa shifted from opportunistic/generalist bacteria associated with flour inputs, toward specialized climax bacterial communities (days 10-14) characterized by acid-tolerant taxa and fruity (p < 3.03e-03), sour (p < 1.60e-01), and fermented (p < 1.47e-05) aromas. This collection of traits changes predictably through time, regardless of flour type, highlighting patterns of bacterial constraints and dynamics that are conserved across systems and scales. Yet, while sourdough climax communities exhibit similar markers of maturity (i.e., pH ≤ 4 and enriched in Lactobacillus (mean abundance 48.1%), Pediococcus (mean abundance 22.7%), and/or Gluconobacter (mean abundance 19.1%)), we also detected specific taxa and aromas associated with each type of flour. Our results address important ecological questions about the relationship between community structure and starter performance, and may enable bakers to deliberately select for specific sourdough starter and bread characteristics.

Domestication shapes the pig gut microbiome and immune traits from the scale of lineage to population.

Animal ecology and evolution have long been known to shape host physiology, but more recently, the gut microbiome has been identified as a mediator between animal ecology and evolution and health. The gut microbiome has been shown to differ between wild and domestic animals, but the role of these differences for domestic animal evolution remains unknown. Gut microbiome responses to new animal genotypes and local environmental change during domestication may promote specific host phenotypes that are adaptive (or not) to the domestic environment. Because the gut microbiome supports host immune function, understanding the effects of animal ecology and evolution on the gut microbiome and immune phenotypes is critical. We investigated how domestication affects the gut microbiome and host immune state in multiple pig populations across five domestication contexts representing domestication status and current living conditions: free-ranging wild, captive wild, free-ranging domestic, captive domestic in research or industrial settings. We observed that domestication context explained much of the variation in gut microbiome composition, pathogen abundances and immune markers, yet the main differences in the repertoire of metabolic genes found in the gut microbiome were between the wild and domestic genetic lineages. We also documented population-level effects within domestication contexts, demonstrating that fine scale environmental variation also shaped host and microbe features. Our findings highlight that understanding which gut microbiome and immune traits respond to host genetic lineage and/or scales of local ecology could inform targeted interventions that manipulate the gut microbiome to achieve beneficial health outcomes.

Phylogenomics reveals the history of host use in mosquitoes.

Mosquitoes have profoundly affected human history and continue to threaten human health through the transmission of a diverse array of pathogens. The phylogeny of mosquitoes has remained poorly characterized due to difficulty in taxonomic sampling and limited availability of genomic data beyond the most important vector species. Here, we used phylogenomic analysis of 709 single copy ortholog groups from 256 mosquito species to produce a strongly supported phylogeny that resolves the position of the major disease vector species and the major mosquito lineages. Our analyses support an origin of mosquitoes in the early Triassic (217 MYA [highest posterior density region: 188-250 MYA]), considerably older than previous estimates. Moreover, we utilize an extensive database of host associations for mosquitoes to show that mosquitoes have shifted to feeding upon the blood of mammals numerous times, and that mosquito diversification and host-use patterns within major lineages appear to coincide in earth history both with major continental drift events and with the diversification of vertebrate classes.

Disseminated Histoplasmosis in Central California Seen in an Immunocompromised Patient.

Histoplasma capsulatum is a dimorphic fungus found in certain parts of North, Central, and South America. Transmission is primarily through airborne inoculation from inhaled fungal microconidia. Histoplasmosis is typically a self-limited mycosis; however, in patients with immunodeficiency, disseminated disease can occur and may lead to high disease burden. This report studies a case of disseminated histoplasmosis in a patient newly diagnosed with human immunodeficiency virus. His presentation on admission was consistent with infectious pulmonary granulomatous disease, and further imaging and laboratory results showed evidence of multi-organ involvement. It is likely his presentation in Central California was a reactivation infection after inoculation in Central America many years ago.

Field and laboratory guidelines for reliable bioinformatic and statistical analysis of bacterial shotgun metagenomic data.

Shotgun metagenomics is an increasingly cost-effective approach for profiling environmental and host-associated microbial communities. However, due to the complexity of both microbiomes and the molecular techniques required to analyze them, the reliability and representativeness of the results are contingent upon the field, laboratory, and bioinformatic procedures employed. Here, we consider 15 field and laboratory issues that critically impact downstream bioinformatic and statistical data processing, as well as result interpretation, in bacterial shotgun metagenomic studies. The issues we consider encompass intrinsic properties of samples, study design, and laboratory-processing strategies. We identify the links of field and laboratory steps with downstream analytical procedures, explain the means for detecting potential pitfalls, and propose mitigation measures to overcome or minimize their impact in metagenomic studies. We anticipate that our guidelines will assist data scientists in appropriately processing and interpreting their data, while aiding field and laboratory researchers to implement strategies for improving the quality of the generated results.

Hidden diversity: comparative functional morphology of humans and other species.

Gastrointestinal (GI) morphology plays an important role in nutrition, health, and epidemiology; yet limited data on GI variation have been collected since 1885. Here we demonstrate that students can collect reliable data sets on gut morphology; when they do, they reveal greater morphological variation for some structures in the GI tract than has been documented in the published literature. We discuss trait variability both within and among species, and the implications of that variability for evolution and epidemiology. Our results show that morphological variation in the GI tract is associated with each organ's role in food processing. For example, the length of many structures was found to vary significantly with feeding strategy. Within species, the variability illustrated by the coefficients of variation suggests that selective constraints may vary with function. Within humans, we detected significant Pearson correlations between the volume of the liver and the length of the appendix (t-value = 2.5278, df = 28, p = 0.0174, corr = 0.4311) and colon (t-value = 2.0991, df = 19, p = 0.0494, corr = 0.4339), as well as between the lengths of the small intestine and colon (t-value = 2.1699, df = 17, p = 0.0445, corr = 0.4657), which are arguably the most vital organs in the gut for nutrient absorption. Notably, intraspecific variation in the small intestine can be associated with life history traits. In humans, females demonstrated consistently and significantly longer small intestines than males (t-value15 = 2.245, p = 0.0403). This finding supports the female canalization hypothesis, specifically, increased female investment in the digestion and absorption of lipids.

Using FastID to analyze complex SNP mixtures from indoor dust.

Forensically relevant single nucleotide polymorphisms (SNPs) can provide valuable supplemental information to short tandem repeats (STRs) for investigative leads, and genotyping can now be streamlined using massively parallel sequencing (MPS). Dust is an attractive evidence source, as it accumulates on undisturbed surfaces, often is overlooked by perpetrators, and contains sufficient human DNA for analysis. To assess whether SNPs genotyped from indoor dust using MPS could be used to detect known household occupants, 13 households were recruited and provided buccal samples from each occupant and dust from five predefined indoor locations. Thermo Fisher Scientific Precision ID Identity and Ancestry Panels were utilized for SNP genotyping, and sequencing was completed using Illumina® chemistry. FastID, a software developed to permit mixture analysis and identity searching, was used to assess whether known occupants could be detected from associated household dust samples. A modified "subtraction" method was also used in FastID to estimate the percentage of alleles in each dust sample contributed by known and unknown occupants. On average, 72% of autosomal SNPs were recovered from dust samples. When using FastID, (a) 93% of known occupants were detected in at least one indoor dust sample and could not be excluded as contributors to the mixture, and (b) non-contributor alleles were detected in 54% of dust samples (29 ± 11 alleles per dust sample). Overall, this study highlights the potential of analyzing human DNA present in indoor dust to detect known household occupants, which could be valuable for investigative leads.

Predicting diet in brachyuran crabs using external morphology.

Morphological traits have often been used to predict diet and trophic position of species across many animal groups. Variation in gut size of closely related animals is known to be a good predictor of dietary habits. Species that are more herbivorous or that persist on low-quality diets often have larger stomachs than their carnivorous counterparts. This same pattern exists in crabs and in most species, individuals exhibit external markings on the dorsal side of their carapace that appear to align with the position and size of their gut. We hypothesized that these external markings could be used as an accurate estimate of the crab's cardiac stomach size, allowing an approximation of crab dietary strategies without the need to sacrifice and dissect individual animals. We used literature values for mean diet and standardized external gut size markings taken from crab photographs across 50 species to show that percent herbivory in the diet increases non-linearly across species of brachyuran crab with the external estimate of gut size. We also used data from dissections in four species to show that external gut markings were positively correlated with gut sizes, though the strength of this correlation differed across species. We conclude that when rough approximations of diet quality such as percent herbivory will suffice, measuring external carapace markings in crabs presents a quick, free, non-lethal alternative to dissections. Our results also provide important insights into tradeoffs that occur in crab morphology and have implications for crab evolution.