Species-dependent Differences Research Articles

Generation of a human iPSC-derived cardiomyocyte/fibroblast engineered heart tissue model

Animal models have proven integral to broadening our understanding of complex cardiac diseases but have been hampered by significant species-dependent differences in cellular physiology. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have shown great promise in the modelling of cardiac diseases despite limitations in functional and structural maturity. 3D stem cell-derived cardiac models represent a step towards mimicking the intricate microenvironment present in the heart as an in vitro model. Incorporation of non-myocyte cell types, such as cardiac fibroblasts, into engineered heart tissue models (EHTs) can help better recapitulate the cell-to-cell and cell-to-matrix interactions present in the human myocardium. Integration of human-induced pluripotent stem cell-derived cardiac fibroblasts (hiPSC-CFs) and hiPSC-CM into EHT models enables the generation of a genetically homogeneous modelling system capable of exploring the abstruse structural and electrophysiological interplay present in cardiac pathophysiology. Furthermore, the construction of more physiologically relevant 3D cardiac models offers great potential in the replacement of animals in heart disease research. Here we describe efficient and reproducible protocols for the differentiation of hiPSC-CMs and hiPSC-CFs and their subsequent assimilation into EHTs. The resultant EHT consists of longitudinally arranged iPSC-CMs, incorporated alongside hiPSC-CFs. EHTs with both hiPSC-CMs and hiPSC-CFs exhibit slower beating frequencies and enhanced contractile force compared to those composed of hiPSC-CMs alone. The modified protocol may help better characterise the interplay between different cell types in the myocardium and their contribution to structural remodelling and cardiac fibrosis.

Generation of a human iPSC-derived cardiomyocyte/fibroblast engineered heart tissue model.

Animal models have proven integral to broadening our understanding of complex cardiac diseases but have been hampered by significant species-dependent differences in cellular physiology. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have shown great promise in the modelling of cardiac diseases despite limitations in functional and structural maturity. 3D stem cell-derived cardiac models represent a step towards mimicking the intricate microenvironment present in the heart as an in vitro model. Incorporation of non-myocyte cell types, such as cardiac fibroblasts, into engineered heart tissue models (EHTs) can help better recapitulate the cell-to-cell and cell-to-matrix interactions present in the human myocardium. Integration of human-induced pluripotent stem cell-derived cardiac fibroblasts (hiPSC-CFs) and hiPSC-CM into EHT models enables the generation of a genetically homogeneous modelling system capable of exploring the abstruse structural and electrophysiological interplay present in cardiac pathophysiology. Furthermore, the construction of more physiologically relevant 3D cardiac models offers great potential in the replacement of animals in heart disease research. Here we describe efficient and reproducible protocols for the differentiation of hiPSC-CMs and hiPSC-CFs and their subsequent assimilation into EHTs. The resultant EHT consists of longitudinally arranged iPSC-CMs, incorporated alongside hiPSC-CFs. EHTs with both hiPSC-CMs and hiPSC-CFs exhibit slower beating frequencies and enhanced contractile force compared to those composed of hiPSC-CMs alone. The modified protocol may help better characterise the interplay between different cell types in the myocardium and their contribution to structural remodelling and cardiac fibrosis.

A conserved microtubule-binding region in Xanthomonas XopL is indispensable for induced plant cell death reactions.

Pathogenic Xanthomonas bacteria cause disease on more than 400 plant species. These Gram-negative bacteria utilize the type III secretion system to inject type III effector proteins (T3Es) directly into the plant cell cytosol where they can manipulate plant pathways to promote virulence. The host range of a given Xanthomonas species is limited, and T3E repertoires are specialized during interactions with specific plant species. Some effectors, however, are retained across most strains, such as Xanthomonas Outer Protein L (XopL). As an 'ancestral' effector, XopL contributes to the virulence of multiple xanthomonads, infecting diverse plant species. XopL homologs harbor a combination of a leucine-rich-repeat (LRR) domain and an XL-box which has E3 ligase activity. Despite similar domain structure there is evidence to suggest that XopL function has diverged, exemplified by the finding that XopLs expressed in plants often display bacterial species-dependent differences in their sub-cellular localization and plant cell death reactions. We found that XopL from X. euvesicatoria (XopLXe) directly associates with plant microtubules (MTs) and causes strong cell death in agroinfection assays in N. benthamiana. Localization of XopLXe homologs from three additional Xanthomonas species, of diverse infection strategy and plant host, revealed that the distantly related X. campestris pv. campestris harbors a XopL (XopLXcc) that fails to localize to MTs and to cause plant cell death. Comparative sequence analyses of MT-binding XopLs and XopLXcc identified a proline-rich-region (PRR)/α-helical region important for MT localization. Functional analyses of XopLXe truncations and amino acid exchanges within the PRR suggest that MT-localized XopL activity is required for plant cell death reactions. This study exemplifies how the study of a T3E within the context of a genus rather than a single species can shed light on how effector localization is linked to biochemical activity.

You are what you eat-ecological niche and microhabitat influence venom activity and composition in aquatic bugs.

True water bugs (Nepomorpha) are mostly predacious insects that live in aquatic habitats. They use their piercing-sucking mouthparts to inject venomous saliva that facilitates the capture and extra-oral digestion of prey animals, but their venom can also be deployed for defence. In Central Europe, nepomorph species representing different families coexist in the same habitat. However, their feeding ecology, including venom composition and deployment, has not been investigated in detail. We used an integrated proteotranscriptomic and bioactivity-based approach to test whether venom composition and activity differ between four water bug species sharing the same habitat but occupying different ecological niches. We found considerable species-dependent differences in the composition of digestive enzymes and venom components that probably evolved as adaptations to particular food sources, foraging strategies and/or microhabitats. The venom of Corixa punctata differed substantially from that of the three strictly predatory species (Ilyocoris cimicoides, Notonecta glauca and Nepa cinerea), and the abundance of herbivory-associated proteins confirms a mostly plant-based diet. Our findings reveal independent adaptations of the digestive and defensive enzyme repertoires accompanied by the evolution of distinct feeding strategies in aquatic bugs.

Direct inhibition of bisphenols on human and rat 11β-hydroxysteroid dehydrogenase 2: Structure-activity relationship and docking analysis

Bisphenols (BPs) as endocrine-disrupting compounds have drawn attention to their health hazards. Whether a BP interferes with glucocorticoid metabolism remains unclear. 11β-Hydroxysteroid dehydrogenase 2 (11β-HSD2) is a key glucocorticoid-metabolizing enzyme that controls fetal glucocorticoid levels across the placental barrier and mineralocorticoid receptor specificity in the kidney. In this study, 11 BPs were tested to inhibit human placental and rat renal 11β-HSD2 and were analyzed for inhibitory potency, mode action, and docking parameters. BPs had inhibitory potency against human 11β-HSD2: BPFL>BPAP>BPZ>BPB>BPC>BPAF>BPA>TDP and the IC10 values were 0.21, 0.55, 1.04, 2.04, 2.43, 2.57, 14.43, and 22.18 μM, respectively. All BPs are mixed inhibitors except BPAP, which is a competitive inhibitor for human 11β-HSD2. Some BPs also inhibited rat renal 11β-HSD2, with BPB (IC50, 27.74 ± 0.95) > BPZ (42.14 ± 0.59) > BPAF (54.87 ± 1.73) > BPA (77.32 ± 1.20) > other BPs (about 100 μM). Docking analysis showed that all BPs bound to the steroid-binding site, interacting with the catalytic residue Tyr232 of both enzymes and the most potent human 11β-HSD2 inhibitor BPFL acts possibly due to its large fluorene ring that has hydrophobic interaction with residues Glu172 and Val270 and π-stacking interaction with catalytic residue Tyr232. The increase in the size of substituted alkanes and halogenated groups in the methane moiety of the bridge of BPs increases its inhibitory potency. Regressions of the lowest binding energy with inhibition constant indicated that there was an inverse regression. These results indicated that BPs significantly inhibited human and rat 11β-HSD2 activity and that there were species-dependent differences.

Analysis of Cobalamin (Vit B12) in Ripened Cheese by Ultra-High-Performance Liquid Chromatography Coupled with Mass Spectrometry.

The analysis of natural cobalamins in dairy products still represents an analytical challenge. The matrix’s complexity, low concentration level, light sensitivity, and binding to proteins are just some of the aspects that make their quantification a difficult goal to achieve. Vitamin B12 plays a fundamental role in human nutrition, and its intake is closely linked to a diet that includes the consumption of food of animal origin. In the current literature, few studies have been carried out on the quantitation of cobalamin in ripened cheeses. A sensitive, selective, and robust ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) method was developed, validated, and applied on ripened cheeses from different species (cow, sheep, and goat) purchased from local Italian markets, highlighting species-dependent differences in vitamin B12 concentrations. The vitamin B12 extraction procedure was performed by converting all cobalamins to the cyanocobalamin form. Furthermore, solid-phase extraction was used for matrix clean-up and analyte preconcentration. The proposed method showed good performance in terms of linearity, sensitivity, reproducibility, and repeatability. The mean vitamin B12 content ranged from <LOQ to 38.9 ng/g. Sheep cheese showed the highest concentrations of vitamin B12, with a mean content of 29.0 ng/g.

Insertion sequences and other mobile elements associated with antibiotic resistance genes in Enterococcus isolates from an inpatient with prolonged bacteraemia.

Insertion sequences (ISs) and other transposable elements are associated with the mobilization of antibiotic resistance determinants and the modulation of pathogenic characteristics. In this work, we aimed to investigate the association between ISs and antibiotic resistance genes, and their role in the dissemination and modification of the antibiotic-resistant phenotype. To that end, we leveraged fully resolved Enterococcus faecium and Enterococcus faecalis genomes of isolates collected over 5 days from an inpatient with prolonged bacteraemia. Isolates from both species harboured similar IS family content but showed significant species-dependent differences in copy number and arrangements of ISs throughout their replicons. Here, we describe two inter-specific IS-mediated recombination events and IS-mediated excision events in plasmids of E. faecium isolates. We also characterize a novel arrangement of the ISs in a Tn1546-like transposon in E. faecalis isolates likely implicated in a vancomycin genotype–phenotype discrepancy. Furthermore, an extended analysis revealed a novel association between daptomycin resistance mutations in liaSR genes and a putative composite transposon in E. faecium , offering a new paradigm for the study of daptomycin resistance and novel insights into its dissemination. In conclusion, our study highlights the role ISs and other transposable elements play in the rapid adaptation and response to clinically relevant stresses such as aggressive antibiotic treatment in enterococci.

Repeated sub-inhibitory doses of cassia essential oil do not increase the tolerance pattern in Listeria monocytogenes cells

Antimicrobial resistance in Listeria monocytogenes biofilms is considered a risk. When using disinfectants, the minimum inhibitory concentration (MIC) must be contemplated to avoid resistance. The objective of the present study was to determine the MIC of four essential oils in four L. monocytogenes strains. Once the MICs were obtained, the effect of subinhibitory doses of the most effective oil was determined. Strains were subsequently subjected to increasing doses of cassia and the MICs were determined again to evaluate differences. The effect of subinhibitory doses for biofilm formation was evaluated using quantitative and observational methodologies. Last, it was studied whether the strains were more sensitive to antibiotics after being in contact with the oil. After continuous exposure to 1/2 MIC, a decrease in the initial MIC (P = 0.013) was observed, specifically for strains belonging to serotype 1/2a (P = 0.041). In contrast, the formation of biofilms did not show differences between the control and exposed groups (P > 0.05). The qualitative study showed that there were no differences in the structure of the biofilms before and after contact with cassia, except for the CECT 935 strain, indicating a strain-dependent trend. Moreover, species-dependent differences were observed in the conformation of the extracellular matrix.

Identification of the amino acid residues involved in the species-dependent differences in the pyridoxine transport function of SLC19A3

Recent studies have shown that human solute carrier SLC19A3 (hSLC19A3) can transport pyridoxine (vitamin B6) in addition to thiamine (vitamin B1), its originally identified substrate, whereas rat and mouse orthologs of hSLC19A3 can transport thiamine but not pyridoxine. This finding implies that some amino acid residues required for pyridoxine transport, but not for thiamine transport, are specific to hSLC19A3. Here, we sought to identify these residues to help clarify the unique operational mechanism of SLC19A3 through analyses comparing hSLC19A3 and mouse Slc19a3 (mSlc19a3). For our analyses, hSLC19A3 mutants were prepared by replacing selected amino acid residues with their counterparts in mSlc19a3, and mSlc19a3 mutants were prepared by substituting selected residues with their hSLC19A3 counterparts. We assessed pyridoxine and thiamine transport by these mutants in transiently transfected human embryonic kidney 293 cells. Our analyses indicated that the hSLC19A3-specific amino acid residues of Gln86, Gly87, Ile91, Thr93, Trp94, Ser168, and Asn173 are critical for pyridoxine transport. These seven amino acid residues were found to be mostly conserved in the SLC19A3 orthologs that can transport pyridoxine but not in orthologs that are unable to transport pyridoxine. In addition, these residues were also found to be conserved in several SLC19A2 orthologs, including rat, mouse, and human orthologs, which were all found to effectively transport both pyridoxine and thiamine, exhibiting no species-dependent differences. Together, these findings provide a molecular basis for the unique functional characteristics of SLC19A3 and also of SLC19A2.

Structural arrangement of auditory brainstem nuclei in the bats Phyllostomus discolor and Carollia perspicillata.

The structure of the mammalian auditory brainstem is evolutionarily highly plastic, and distinct nuclei arrange in a species-dependent manner. Such anatomical variability is present in the superior olivary complex (SOC) and the nuclei of the lateral lemniscus (LL). Due to the structure-function relationship in the auditory brainstem, the identification of individual nuclei supports the understanding of sound processing. Here, we comparatively describe the nucleus arrangement and the expression of functional markers in the auditory brainstem of the two bat species Phyllostomus discolor and Carollia perspicillata. Using immunofluorescent labeling, we describe the arrangement and identity of the SOC and LL nuclei based on the expression of synaptic markers (vesicular glutamate transporter 1 and glycine transporter 2), calcium-binding proteins, as well as the voltage-gated ion channel subunits Kv1.1 and HCN1. The distribution of excitatory and inhibitory synaptic labeling appears similar between both species and matches with that of other mammals. The detection of calcium-binding proteins indicates species-dependent differences and deviations from other mammals. Kv1.1 and HCN1 show largely the same expression pattern in both species, which diverges from other mammals, indicating functional adaptations in the cellular physiology of bat neurons.

Partitioning of Space Among Trees in an Old-Growth Spruce Forest in Subarctic Fennoscandia

The distribution of space among forest trees is linked to the availability of resources, among-tree competition, and hence forest dynamics. We studied partitioning of horizontal space among trees and related spatial structures in an old-growth Picea abies (L.) Karst -dominated forest in northeastern subarctic Fennoscandia, where Betula pubescens (Ehrh.) is an important co-dominant. Specifically, we asked (1) how does growing space occupied by trees vary by tree species and size in an old-growth forest with open canopy structure, and (2) at what scales does the variation in tree growing space occur? We mapped an 8.8 ha forest plot with 4,884 live trees. We used Voronoi polygons to quantify the horizontal space potentially available to each tree. We modeled the Voronoi polygon area as a function of tree size and species by using generalized additive models (GAM). We used i-to-any L-functions to study the scale-dependence of tree densities around focal trees, and mark correlation functions to study the relative sizes of trees close to each other. The GAM models showed that tree growing space increased non-linearly with tree size before saturating, and that overall growing space was larger for B. pubescens than for P. abies. Mean space occupied by trees roughly doubled from the smallest diameter class (0–5 cm) to the largest (&amp;gt;25 cm), from 13.7 to 26.7 m2. Depending on diameter class, shade-intolerant B. pubescens occupied on average 5–10 m2 more space than shade-tolerant P. abies. Trees close to each other were smaller than average. Size- and species-dependent differences in local tree densities accumulated mostly at the scale of a few meters but showed also broader-scale variation possibly related to edaphic variation within the study plot. The tree species- and size-related variation in the trees’ growing space suggests that among-tree competition, together with clustering of trees, shape the spatial assembly of the forest.

CB1R, CB2R and TRPV1 expression and modulation in in vivo, animal glaucoma models: A systematic review.

The endocannabinoid system (ECS) is a complex biological regulatory system. Its expression and functionality have been widely investigated in ocular tissues. Recent data have reported its modulation to be valid in determining an ocular hypotensive and a neuroprotective effect in preclinical animal models of glaucoma. This study aimed to explore the available literature on cannabinoid receptor 1 (CB1R), cannabinoid receptor 2 (CB2R), and transient receptor potential vanilloid 1 (TRPV1) expression in the trabecular meshwork (TM), ciliary body (CB), and retina as well as their ocular hypotensive and neuroprotective effects in preclinical, in vivo, animal glaucoma models. The study adhered to both PRISMA and SYRCLE guidelines. Sixty-nine full-length articles were included in the final analysis. Preclinical studies indicated a widespread distribution of CB1R, CB2R, and TRPV1 in the TM, CB, and retina, although receptor-, age-, and species-dependent differences were observed. CB1R and CB2R modulation have been shown to exert ocular hypotensive effects in preclinical models via the regulation of inflow and outflow pathways. Retinal cell neuroprotection has been achieved in several experimental models, mediated by agonists and antagonists of CB1R, CB2R, and TRPV1. Despite the growing body of preclinical data regarding the expression and modulation of ECS in ocular tissues, the mechanisms responsible for the hypotensive and neuroprotective efficacy exerted by this system remain largely elusive. Research on this topic is advocated to further substantiate the hypothesis that the ECS is a new potential therapeutic target in the context of glaucoma.

Species-dependent differences in the inhibition of various potassium currents and in their effects on repolarization in cardiac ventricular muscle

Even though rodents are accessible model animals, their electrophysiological properties are deeply different from those of humans, making the translation of rat studies to humans rather difficult. We compared the mechanisms of ventricular repolarization in various animal models to those of humans by measuring cardiac ventricular action potentials from ventricular papillary muscle preparations using conventional microelectrodes and applying selective inhibitors of various potassium transmembrane ion currents. Inhibition of the IK1 current (10µmol/L barium chloride) significantly prolonged rat ventricular repolarization, but only slightly prolonged it in dogs, and did not affect it in humans. On the contrary, IKr inhibition (50nmol/L dofetilide) significantly prolonged repolarization in humans, rabbits, and dogs, but not in rats. Inhibition of the IKur current (1µmol/L XEN-D0101) only prolonged rat ventricular repolarization and had no effect in humans or dogs. Inhibition of the IKs (500nmol/L HMR-1556) and Ito currents (100µmol/L chromanol-293B) elicited similar effects in all investigated species. We conclude that dog ventricular preparations have the strongest translational value and rat ventricular preparations have the weakest translational value in cardiac electrophysiological experiments.

Species-dependent interaction of Gd-based contrast agents with humic substances

In this study, model experiments regarding species-dependent differences in the interaction of gadolinium-based contrast agents (GBCAs) with humic acids as potential binding partners in the aquatic environment are conducted. For this, the Gd content of different weight fractions obtained via ultracentrifugation of incubation solutions of humic acids with a linear (gadodiamide) and a macrocyclic GBCA (gadobutrol) were analyzed via inductively coupled plasma-mass spectrometry (ICP-MS). This enabled the fractionation of Gd-humic acid adducts and intact GBCAs, since Gd bound to macromolecules would be present in the macromolecular fraction of the filter residue while the low molecular weight Gd species can pass the filter with the filtrate. The Gd concentration in the different weight fractions was determined and a different reaction behavior for the examined GBCAs was observed. 73% of the total Gd amount was present in the macromolecular fraction of the linear GBCA compared to 0.41% in case of the macrocyclic GBCA. Speciation analysis of the macromolecular fractions by size exclusion chromatography-UV-ICP-MS confirmed that Gd-humic acid adducts were formed in case of the linear gadodiamide, but not with the macrocyclic gadobutrol. The findings of this study suggest that humic substance was able to react with the linear GBCA while the macrocyclic GBCA remained stable. Since free Gd ions are toxic, the question remains whether the humic acid bound Gd can be remobilized or if subsequent reactions with other molecules can take place. Furthermore, the persistence of macrocyclic GBCAs towards the humic substances indicates the potential accumulation of these compounds in the environment. However, more experiments regarding other binding partners and long term studies are needed to assess their ultimate fate after their release into the environment.

Exposed red leaves display adaptive adjustments in chlorophyll and photosystem ratios compatible with the shade imposed by anthocyanin accumulation

Foliar anthocyanins shape a peculiar shade in a red leaf's interior leading to uneven energy distribution between the two photosystems. Accordingly, a readjustment of PSII/PSI stoichiometry could restore excitation balance. To test this hypothesis, 77 K fluorescence emission spectra of thylakoids from green and red leaves of seven species with different pigment profiles were compared. The ratio of F686/F736 served as an indication of the PSII/PSI functional ratio. To avoid possible species-dependent differences in the measured parameters, plants showing intra-individual, intra-species, or intra-leaf variation in the expression of the anthocyanic character were used. Red leaves or red leaf areas displayed higher PSII/PSI ratio, irrespectively of species and anthocyanin accumulation pattern. PSII/PSI ratio declined in parallel with anthocyanin decrease. In five species, red leaves displayed also a lower Chl a/b ratio. We conclude that red leaves growing in full sunlight develop adaptive adjustments in their chlorophyll and photosystem ratios, compatible with the shade-acclimation syndrome.

Epigenetic-structural changes in X chromosomes promote Xic pairing during early differentiation of mouse embryonic stem cells.

X chromosome inactivation center (Xic) pairing occurs during the differentiation of embryonic stem (ES) cells from female mouse embryos, and is related to X chromosome inactivation, the circadian clock, intra-nucleus architecture, and metabolism. However, the mechanisms underlying the identification and approach of X chromosome pairs in the crowded nucleus are unclear. To elucidate the driving force of Xic pairing, we developed a coarse-grained molecular dynamics model of intranuclear chromosomes in ES cells and in cells 2 days after the onset of differentiation (2-day cells) by considering intrachromosomal epigenetic-structural feature-dependent mechanics. The analysis of the experimental data showed that X-chromosomes exhibit the rearrangement of their distributions of open/closed chromatin regions on their surfaces during cell differentiation. By simulating models where the excluded volume effects of closed chromatin regions are stronger than those of open chromatin regions, such rearrangement of open/closed chromatin regions on X-chromosome surfaces promoted the mutual approach of the Xic pair. These findings suggested that local intrachromosomal epigenetic features may contribute to the regulation of cell species-dependent differences in intranuclear architecture.

Comparative lipid profiling of murine and human atherosclerotic plaques using high-resolution MALDI MSI

The distribution of atherosclerotic lesions in the aorta and its branches of ApoE knockout (ApoE−/−) mice is like that of patients with atherosclerosis. By using high-resolution MALDI mass spectrometry imaging (MSI), we aimed at characterizing universally applicable physiological biomarkers by comparing the murine lipid marker profile with that of human atherosclerotic arteries. Therefore, the aorta or carotid artery of male ApoE−/− mice at different ages, human arteries with documented atherosclerotic changes originated from amputated limbs, and corresponding controls were analysed. Obtained data were subjected to multivariate statistical analysis to identify potential biomarkers. Thirty-one m/z values corresponding to individual lipid species of cholesterol esters, lysophosphatidylcholines, lysophosphatidylethanolamines, and cholesterol derivatives were found to be specific in aortic atherosclerotic plaques of old ApoE−/− mice. The lipid composition at related vessel positions of young ApoE−/− mice was more comparable with wild-type mice. Twenty-six m/z values of the murine lipid markers were found in human atherosclerotic peripheral arteries but also control vessels and showed a more patient-dependent diverse distribution. Extensive data analysis without marker preselection based on mouse data revealed lysophosphatidylcholine and glucosylated cholesterol species, the latter not being detected in the murine atherosclerotic tissue, as specific potential novel human atherosclerotic vessel markers. Despite the heterogeneous lipid profile of atherosclerotic peripheral arteries derived from human patients, we identified lipids specifically colocalized to atherosclerotic human tissue and plaques in ApoE−/− mice. These data highlight species-dependent differences in lipid profiles between peripheral artery disease and aortic atherosclerosis.

Sinapis alba L. and Triticum aestivum L. as biotest model species for evaluating municipal solid waste leachate toxicity

The volume of municipal solid waste (MSW) inputs is rapidly increasing with a growing human population, and its composition is changing due an increased diversity of materials being deposited. There is an associated increase in leachate, a common toxic byproduct of MSW facilities that must be collected and treated prior to its release into the environment. There is growing interest in plant-based methods that are economical and efficient for leachate toxicity assessment such as biological tests that use indicator species. In the present study, the tolerance thresholds of two herbaceous species, Sinapis alba L. (mustard) and Triticum aestivum L. (wheat) to increasing shares of leachate sourced from an MSW facility in the Czech Republic were assessed through a variety of physiological parameters. Soil-based biotests showed a stimulation in the shoot biomass, leaf expansion, primary root elongation and carbon assimilation rate of the selected plant species to leachate concentrations between 20 and 50 %. Higher leachate concentrations led to reductions in most physiological parameters, especially the elongation of seedling roots when growth solutions with >50 % leachate were applied. While S. alba was more sensitive to increasing proportions of leachate in terms of growth parameters of the shoot tissues, photosystem II efficiency and chlorophyll pigment concentrations were more responsive in T. aestivum, indicating species-dependent differences. The present biotests provide further support for the use of both Sinapis alba L and Triticum aestivum L. as indicator species of phytotoxicity.

A microfluidic platform for highly parallel bite by bite profiling of mosquito-borne pathogen transmission

Mosquito bites transmit a number of pathogens via salivary droplets deposited during blood-feeding, resulting in potentially fatal diseases. Little is known about the genomic content of these nanodroplets, including the transmission dynamics of live pathogens. Here we introduce Vectorchip, a low-cost, scalable microfluidic platform enabling high-throughput molecular interrogation of individual mosquito bites. We introduce an ultra-thin PDMS membrane which acts as a biting interface to arrays of micro-wells. Freely-behaving mosquitoes deposit saliva droplets by biting into these micro-wells. By modulating membrane thickness, we observe species-dependent differences in mosquito biting capacity, utilizable for selective sample collection. We demonstrate RT-PCR and focus-forming assays on-chip to detect mosquito DNA, Zika virus RNA, as well as quantify infectious Mayaro virus particles transmitted from single mosquito bites. The Vectorchip presents a promising approach for single-bite-resolution laboratory and field characterization of vector-pathogen communities, and could serve as a powerful early warning sentinel for mosquito-borne diseases.

Photosynthesis and foliar vascular adjustments to growth light intensity in summer annual species with symplastic and apoplastic phloem loading

Concomitant adjustments in photosynthetic capacity and size, composition, and/or density of minor foliar veins in response to growth environment were previously described primarily for winter annuals that load sugars into foliar phloem apoplastically. Here, common trends, differences associated with phloem-loading mechanism, and species-dependent differences are identified for summer annuals (loading sugars either symplastically [cucumber, pumpkin, and basil] or apoplastically [tomato and sunflower]) that were grown in low and high light. Photosynthetic capacity per leaf area was significantly positively correlated with leaf-level volume of phloem-loading cells (LCs), sugar-export conduits (sieve elements), and water conduits (tracheary elements) irrespective of phloem-loading mechanism. The relative contribution to leaf-level volume of LC numbers versus individual LC size was greater in apoplastic loaders than in symplastic loaders. Species-dependent differences included different vein density within each loading group and either greater or lower numbers of cells per minor vein (especially of tracheary elements in the symplastic loaders basil versus cucumber, respectively), which may be due to genetic adaptation to different environmental conditions. These results indicate considerable plasticity in foliar vascular features in summer annuals as well as some loading-mechanism-dependent trends.