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bioRxiv2022

Establishment of physiologically relevant oxygen gradients in microfluidic organ chips

In vitro models of human organs must accurately reconstitute oxygen concentrations and gradients that are observed in vivo to mimic gene expression, metabolism, and host-microbiome interactions. Here we describe a simple strategy to achieve physiologically relevant oxygen tension in a two-channel human small intestine-on-a-chip (Intestine Chip) lined with primary human duodenal epithelium and intestinal microvascular endothelium in parallel channels separated by a porous membrane while both channels are perfused with oxygenated medium. This strategy was developed using computer simulations that predicted lowering the oxygen permeability of poly-dimethlysiloxane (PDMS) chips in specified locations using a gas impermeable film will allow the cells to naturally decrease the oxygen concentration through aerobic respiration and reach steady-state oxygen levels < 36 mm Hg (< 5%) within the epithelial lumen. The approach was experimentally confirmed using chips with embedded oxygen sensors that maintained this stable oxygen gradient. Furthermore, Intestine Chips cultured with this approach supported formation of a villus epithelium interfaced with a continuous endothelium and maintained intestinal barrier integrity for 72 h. This strategy recapitulates in vivo functionality in an efficient, inexpensive, and scalable format that improves the robustness and translatability of Organ Chip technology for studies on microbiome as well as oxygen sensitivity.

bioengineering↗

bioRxiv2022

Local structural dynamics of alpha-synuclein correlate with aggregation in different physiological conditions

In Parkinsons disease and other synucleinopathies, the intrinsically disordered, presynaptic protein alpha-synuclein misfolds and aggregates. We hypothesise that the exposure of alpha-synuclein to different cellular environments, with different chemical compositions, pH and binding partners, alters its biological and pathological function by inducing changes in molecular conformation. Our custom instrumentation and software enable measurement of the amide hydrogen exchange rates of wild-type alpha-synuclein at amino acid resolution under physiological conditions, mimicking those in the extracellular, intracellular, and lysosomal compartments of cells. We characterised the aggregation kinetics and morphology of the resulting fibrils and correlate these with structural changes in the monomer. Our findings reveal that the C-terminal residues of alpha-synuclein are driving its nucleation and thus its aggregation. Furthermore, the entire NAC region and specific other residues strongly promoted elongation of fibrils. This provides new detail on our current understanding of the relationship between the local chemical environment and monomeric conformations of alpha-synuclein.

molecular biology↗

bioRxiv2022

Comparison of Transcranial Doppler Ultrasound with Computational Fluid Dynamics: Responses to Physiological Stimuli

Few studies have compared transcranial Doppler (TCD) ultrasound with independent techniques such as computational fluid dynamics (CFD) simulations, particularly in response to stimuli. We compared TCD cerebral blood flow velocity in healthy participants with subject-specific CFD simulations to determine differences in techniques. Twelve participants underwent head and neck imaging with 3 Tesla magnetic resonance angiography. Velocity waveforms in the middle cerebral artery (MCA) were measured with TCD while velocity and diameter in the neck arteries were measured with duplex ultrasound at rest, hypercapnia and exercise. Subject-specific CFD simulations were developed for each condition, with velocity waveforms extracted in the same region as TCD. We found that absolute TCD velocities were significantly higher than CFD data, and non-significantly correlated across all conditions (r range 0.030-0.377, all P>0.05). However, relative changes from rest to hypercapnia and exercise generally exhibited significant positive correlations (r range 0.448-0.770), with the strongest correlation being average velocity change from rest to exercise (r=0.770, P<0.01). We have found that although absolute MCA velocity measurements from different sources vary, relative velocity changes yield stronger correlations regardless of source. Our findings indicate relative responses to physiological stimuli, along with absolute data, should be considered for analyzing cerebral blood flow velocity.

bioengineering↗

bioRxiv2022

Emotion schema effects on associative memory differ across emotion categories at the behavioural, physiological and neural level

Previous behavioural and neuroimaging studies have consistently reported that our memory is enhanced for associations congruent or incongruent with the structure of our prior knowledge, termed as schemas. However, it remains unclear if similar effects exist if encoded associations are emotional. Do emotional schemas also facilitate learning and subsequent retrieval? Does it depend on the type of experienced emotions? Using a novel face-word pair association paradigm combined with fMRI and eye-tracking techniques, we demonstrated and replicated in two independent studies that congruency with emotion schemas and emotion category interact to affect associative memory. Overall, emotion schemas facilitated memory for associative context, paralleled by the recruitment of left inferior frontal gyrus (IFG) during successful encoding of emotionally congruent vs. incongruent pairs. However, emotion schema effects differed across two negative emotion categories: disgust and fear, with disgust remembered better than fear. The IFG engagement was higher during successful encoding of congruent vs. incongruent pairs, but only in the case of disgust, suggestive of more semantic processing involved in learning disgust-related associations. On the contrary, the encoding of congruent vs. incongruent fear-related pairs was supported by activity in right fusiform gyrus (FG), suggesting greater sensory processing of faces. Successful memory formation for congruent disgust-related pairs was associated with a higher loading of pupil dilation component related to sympathetic activation, longer gaze time on words compared to faces, and more gaze switches between the two. This was reversed for fear-related pairs where the faces attracted more attention, as reflected by longer gaze time (compared to words). Overall, our results at the behavioural, physiological, and neural level converge to suggest that emotional congruency influences memory similar to semantic schemas. However, encoding processes and neural effects vary depending on emotion category, reflecting the differential role of semantic processing and visual attention processes in the modulation of memory by disgust and fear.

neuroscience↗

bioRxiv2022

Physiological alterations in microglial morphology associate with the sleep-wake cycle in a brain region-specific manner

Long-term total sleep deprivation induces changes in cortical - and hippocampal microglial morphology that closely resemble the microglial response to the gram-negative bacterial cell wall component lipopolysaccharide (LPS). A recent study found evidence that microglia could modify vigilance-states/sleep, but only few studies investigated microglial throughout the diurnal behavioral inactivity/activity pattern or the naturally occurring sleep-wake cycle, and those who have, only concentrated on the cortical or hippocampal microglia. As microglia demonstrate regional heterogeneity, we compared microglial diurnal morphological alterations in the somatosensory cortex (SC) and dorsal hippocampus (HC) to the basal forebrain (BF), which is a subcortical brain area involved in the regulation of vigilance states. We collected mouse brain samples every 3h throughout the 24h light-dark-cycle and applied a 3D reconstruction method for the acquired confocal microscopy images for each brain area separately. While microglial regional heterogeneity was evident, stimulation of microglia with LPS caused comparable microglial responses in all brain areas. When comparing microglial features between the 12h light- and dark periods, regional heterogeneity re-appeared. As most of the morphological alterations occurred during the light period-the habitual sleeping period of the mice, we performed polysomnography to study the possible interaction of microglial morphology and sleep. We found that cortical-, but not HC- or BF microglial territory and volume negatively correlated with sleep slow wave activity (SWA), an electroencephalic feature of non-REM sleep (NREMS). Since microglia are sensitive to neuronal activity, we propose that the regional differences reflect vigilance-state specific neuronal activity patterns. Table of contents image O_FIG O_LINKSMALLFIG WIDTH=184 HEIGHT=200 SRC="FIGDIR/small/482976v1_ufig1.gif" ALT="Figure 1"> View larger version (71K): org.highwire.dtl.DTLVardef@112d346org.highwire.dtl.DTLVardef@ecc138org.highwire.dtl.DTLVardef@1817331org.highwire.dtl.DTLVardef@1b1b47c_HPS_FORMAT_FIGEXP M_FIG C_FIG Main PointsMicroglia show morphological differences between the somatosensory cortex (SC), hippocampus (HC) and basal forebrain (BF) under physiological conditions. Cortical-, but not HC- or BF microglial cell volume negatively correlates with non-REM sleep slow wave activity.

neuroscience↗

bioRxiv2022

Porous yet Dense matrices: using ice to shape collagen 3D cell culture systems with increased physiological relevance

Standard in vitro cell culture is one of the pillars of biomedical science. However, there is increasing evidence that 2D systems provide biological responses that are often in disagreement with in vivo observations, partially due to limitations in reproducing the native cellular microenvironment. 3D materials that are able to mimic the native cellular microenvironment to a greater extent tackle these limitations. Here, we report Porous yet Dense (PyD) type I collagen materials obtained by ice-templating followed by topotactic fibrillogenesis. These materials combine extensive macroporosity, favouring the cell migration and nutrients exchange, as well as dense collagen walls, which mimic locally the Extracellular Matrix. When seeded with Normal Human Dermal Fibroblasts (NHDFs), PyD matrices allow for a faster and more extensive colonisation when compared with equivalent Non-Porous matrices. The textural properties of the PyD materials also impact cytoskeletal and nuclear 3D morphometric parameters. Due to the effectiveness in creating a biomimetic 3D environment for NHDFs and the ability to promote cell culture for more than 28 days without subculture, we anticipate that PyD materials could configure an important step towards in vitro systems applicable to other cell types and with higher physiological relevance.

bioengineering↗

bioRxiv2022

Heterocyclic aromatic amines (HAAs) target mitochondrial physiology

Heterocyclic aromatic amines (HAA) may be found naturally in plants or are formed through the Maillard reaction when meat is cooked at high temperatures. Previous studies have indicated HAAs are especially toxic to dopaminergic neurons, whereas specific exposures may also affect other neuronal populations such as cholinergic neurons. Biochemical mechanisms of neurotoxic action implicate elevated oxidative stress and afflicted mitochondria. Notably, these mechanisms are of importance in Alzheimers disease (AD) and Parkinsons disease (PD). Most neurodegenerative disease cases are sporadic, where environmental and dietary factors may modulate risk. To further investigate HAA neurotoxicity, we tested the effect of common HAA (harmane, harmine, norharmane, PhIP, and HONH-PhIP) exposure on mitochondrial physiology, a key pathogenic target in AD and PD. Upon assessing mitochondrial bioenergetics, we observed a significant reduction in basal respiration, ATP production, maximal respiratory capacity, and non-mitochondrial respiration, indicating that HAA negatively impacts mitochondrial respiration. Followed by more specific studies on individual mitochondrial complexes, it was found that harmane, harmine, norharmane specifically inhibit complex I enzyme activity, whereas HONH-PhiP, a reactive metabolite of PhIP, inhibits Complex III enzyme activity. Our findings provide significant advancement with respect to underlying mechanisms of toxicity, though also validating HAA exposure as a potential risk factor for AD and PD.

neuroscience↗

bioRxiv2022

Liguleless narrow and narrow odd dwarf act in overlapping pathways to regulate maize development & physiology

narrow odd dwarf (nod) and Liguleless narrow (Lgn) are pleiotropic maize mutants that both encode plasma membrane proteins, cause similar developmental patterning defects, and constitutively induce stress signaling pathways. To investigate how these mutants coordinate maize development and physiology, we screened for protein interactors of NOD by affinity purification. LGN was identified by this screen as a strong candidate interactor, and we confirmed the NOD-LGN molecular interaction through orthogonal experiments. We further demonstrated that LGN, a receptor-like kinase, can phosphorylate NOD in vitro, hinting that they could act in intersecting signal transduction pathways. To test this hypothesis, we generated Lgn-R;nod mutants in two backgrounds (B73 and A619) and found that these mutations enhance each other, causing more severe developmental defects than either single mutation on its own, with phenotypes including very narrow leaves, increased tillering, and failure of the main shoot. Transcriptomic and metabolomic analyses of the single and double mutants in the two genetic backgrounds revealed widespread induction of pathogen defense genes and a shift in resource allocation away from primary metabolism in favor of specialized metabolism. These effects were similar in each single mutant and heightened in the double mutant, leading us to conclude that NOD and LGN act cumulatively in overlapping signaling pathways to coordinate growth-defense tradeoffs in maize.

plant biology↗

bioRxiv2022

The genomics and physiology of abiotic stressors associated with global elevation gradients in Arabidopsis thaliana

Phenotypic and genetic diversity in Arabidopsis thaliana may be associated with adaptation along its wide elevational range. We took a multi-regional view of elevational adaptation and in a diverse panel of ecotypes measured plant responses to high elevation stressors: low partial CO2 pressure, high light, and night freezing. We conducted genome-wide association studies (GWAS) and found evidence of contrasting locally adaptive clines between regions. Western Mediterranean ecotypes showed low {delta}13C/early flowering at low elevations to high {delta}13C/late flowering at high elevations, while Asian ecotypes showed the opposite pattern. We mapped different candidate genes for each region, and trait-associated SNPs often showed elevational clines likely maintained by selection. Antioxidants and pigmentation showed regional differentiation but rarely elevational clines. GWAS for antioxidants identified an ascorbate transporter PHT4;4 (AT4G00370), which we show alters non-photochemical quenching kinetics under high light and may be involved in local adaptation to Moroccan mountains. The low-antioxidant PHT4;4 GWAS allele was associated with lower PHT4;4 expression and this haplotype was characterized by binding sites of a transcription factor family, DOF, involved in light response. Our results highlight how physiological and genomic elevational clines in different regions can be unique, underlining the complexity of local adaptation in widely distributed species.

evolutionary biology↗

bioRxiv2022

Correlative SIP-FISH-SEM-Raman-NanoSIMS links identity, morphology, biochemistry, and physiology of environmental microbes

Microscopic and spectroscopic techniques are commonly applied to study microbial cells but are typically used on separate samples, resulting in population-level datasets that are integrated across different cells with little spatial resolution. To address this shortcoming, we developed a workflow that correlates several microscopic and spectroscopic techniques to generate an in-depth analysis of individual cells. By combining stable isotope probing (SIP), fluorescence in situ hybridization (FISH), scanning electron microscopy (SEM), confocal Raman microspectroscopy (Raman), and nano-scale secondary ion mass spectrometry (NanoSIMS), we illustrate how individual cells can be thoroughly interrogated to obtain information about their taxonomic identity, structure, physiology, and metabolic activity. Analysis of an artificial microbial community demonstrated that our correlative approach was able to resolve the activity of single cells using heavy water SIP in conjunction with Raman and/or NanoSIMS and establish their taxonomy and morphology using FISH and SEM. This workflow was then applied to a sample of yet uncultured multicellular magnetotactic bacteria (MMB). In addition to establishing their identity and activity, backscatter electron microscopy (BSE), NanoSIMS, and energy-dispersive X-ray spectroscopy (EDS) were employed to characterize the magnetosomes within the cells. By integrating these techniques, we demonstrate a cohesive approach to thoroughly study environmental microbes on a single cell level.

microbiology↗

bioRxiv2022

The molecular mechanism and physiological role of silent nociceptor activation

Silent nociceptors are sensory afferents that are insensitive to noxious mechanical stimuli under normal conditions but become sensitized to such stimuli during inflammation. Using RNA-sequencing and quantitative RT-PCR we demonstrate that inflammation selectively upregulates the expression of the transmembrane protein TMEM100 in silent nociceptors and electrophysiology revealed that over-expression of TMEM100 is required and sufficient to un-silence silent nociceptors. Moreover, we show that mice lacking TMEM100 do not develop secondary allodynia - i.e. pain hypersensitivity that spreads beyond the site of inflammation - in a mouse model of knee joint inflammation and that AAV-mediated overexpression of TMEM100 in articular afferents in the absence of inflammation is sufficient to induce allodynia in remote skin regions without causing knee joint pain. Thus, our work identifies TMEM100 as a key regulator of silent nociceptor un-silencing and reveals a physiological role for this hitherto enigmatic afferent subclass in triggering spatially remote secondary allodynia during inflammation.

neuroscience↗

bioRxiv2022

HOPX governs a molecular and physiological switch between cardiomyocyte progenitor and maturation gene programs

This study establishes the homeodomain only protein, HOPX, as a determinant controlling the molecular switch between cardiomyocyte progenitor and maturation gene programs. Time-course single-cell gene expression with genome-wide footprinting reveal that HOPX interacts with and controls core cardiac networks by regulating the activity of mutually exclusive developmental gene programs. Upstream hypertrophy and proliferation pathways compete to regulate HOPX transcription. Mitogenic signals override hypertrophic growth signals to suppress HOPX and maintain cardiomyocyte progenitor gene programs. Physiological studies show HOPX directly governs genetic control of cardiomyocyte cell stress responses, electro-mechanical coupling, proliferation, and contractility. We use human genome-wide association studies (GWAS) to show that genetic variation in the HOPX-regulome is significantly associated with complex traits affecting cardiac structure and function. Collectively, this study provides a mechanistic link situating HOPX between competing upstream pathways where HOPX acts as a molecular switch controlling gene regulatory programs underpinning metabolic, signaling, and functional maturation of cardiomyocytes.

developmental biology↗

bioRxiv2022

Interspecific allometric scaling in eDNA production in fishes reflects physiological and surface area allometry

Relating environmental DNA (eDNA) signal strength to organism abundance requires a fundamental understanding of eDNA production. A number of studies have demonstrated that eDNA production may scale allometrically - that is, larger organisms tend to exhibit lower mass-specific eDNA production rates, likely due to allometric scaling in key processes related to eDNA production (e.g. surface area, excretion/egestion). While most previous studies have examined intra-specific allometry, physiological rates and organism surface area also scale allometrically across species. We therefore hypothesize that eDNA production will similarly exhibit inter-specific allometric scaling. To evaluate this hypothesis, we reanalyzed previously published eDNA data from Stoeckle et al. (2021) which compared metabarcoding read count to organism count and biomass data obtained from trawl surveys. Using a Bayesian model we empirically estimated the value of the allometric scaling coefficient ( b) for bony fishes to be 0.67 (credible interval = 0.58 - 0.77), although our model failed to converge for chondrichthyan species. We found that integrating allometry significantly improved correlations between organism abundance and metabarcoding read count relative to traditional metrics of abundance (density and biomass) for bony fishes. Although substantial unexplained variation remains in the relationship between read count and organism abundance, our study provides evidence that eDNA production tends to scale allometrically across species. Future studies investigating the relationship between eDNA signal strength and metrics of fish abundance could potentially be improved by accounting for allometry - a scaling coefficient value of [~]2/3 appears to be both theoretically and empirically justified.

ecology↗

bioRxiv2022

Generic solving of physiologically-based kinetic models in support of next generation risk assessment due to chemicals

Increasing the confidence in using in vitro and in silico model-based data to aid the chemical risk assessment process is one, if not the most, important challenge currently facing regulatory authorities. A particularly crucial concern is to fully take advantage of scientifically valid Physiologically-Based Kinetic (PBK) models. Nevertheless, risk assessors remain still unwilling in employing PBK models within their daily work. Indeed, PBK models are not often included in current official guidance documents. In addition, most users have limited experience in using modelling in general. So, the complexity of PBK models, together with a lack to evaluation methods of their performances, certainly contributes to their under-use in practical risk assessment. This paper proposes an innovative and unified modelling framework, in both the writing of PBK equations as matrix ordinary differential equations (ODE), and in its exact solving simply expressed with matrix products. This generic PBK solution allows to consider as many as state-variables as needed to quantify chemical absorption, distribution, metabolism and excretion processes within living organisms when exposed to chemical substances. This generic PBK model makes possible any compartmentalisation to be considered, as well as all appropriate inter-connections between compartments and with the external medium. We first introduce our PBK modelling framework, with all intermediate steps from the matrix ODE to the exact solution. Then we apply this framework to bioaccumulation testing, before illustrating its concrete use through complementary case studies in terms of species, compounds and model complexity. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

pharmacology and toxicology↗

bioRxiv2022

Quantitative stable-isotope probing (qSIP) with metagenomics links microbial physiology and activity to soil moisture in Mediterranean-climate grassland ecosystems

The growth and physiology of soil microorganisms, which play vital roles in biogeochemical cycling, are likely dependent on current and prior soil moisture levels. Here, we developed and applied a genome-resolved metagenomic implementation of quantitative stable isotope probing (qSIP) to an H218O labeling experiment to determine which microbial community members, and with what capacities, are growing under in situ conditions. qSIP enabled measurement of taxon-specific growth because isotopic incorporation into microbial DNA requires production of new genome copies. We studied three Mediterranean grassland soils across a rainfall gradient to evaluate the hypothesis that historic precipitation levels are an important factor controlling trait selection. We used qSIP-informed genome-resolved metagenomics to resolve an active subset of soil community members and identify the ecophysiological traits that characterize them. Higher year-round precipitation levels correlated with higher activity and growth rates of flagellar motile microorganisms. In addition to bacteria that were heavily isotopically labeled, we identified abundant isotope-labeled phages suggesting phage-induced cell lysis likely contributed to necromass production at all three sites. Further, there was a positive correlation between phage activity and the activity of putative phage hosts. Contrary to our expectations, the capabilities to decompose the diverse complex carbohydrates common in soil necromass or oxidize methanol and carbon monoxide were broadly distributed across active and inactive bacteria in all three soils, implying that these traits are not highly selected for by historical precipitation.

microbiology↗

bioRxiv2022

PHYSIOLOGICAL CHARACTERISTICS OF BACTERIAL DROPLETS INDICATE A CATASTROPHIC CONSEQUENCE WITH AN INCREASE IN IMPACT VELOCITY

Droplet impacts on various surfaces play a profound role in different bio-physiological processes and engineering applications. The current study opens a new realm that investigates the plausible effect of impact velocities on bacteria-laden droplets against a solid surface. We unveiled the alarming consequences of Salmonella Typhimurium (STM) laden drop, carrying out the in vitro and intracellular viability of STM to the impact Weber numbers ranging from 100-750. The specified Weber number range mimics the velocity range occurring during the respiratory processes, especially the airborne dispersion of drops during cough. A thick ring of bacterial deposition was observed in all cases irrespective of impacting velocity and the nutrient content of the bacterial medium. The mechanical properties of the bacterial deposit examined using Atomic Force Microscopy reveals the deformation of bacterial morphology, cushioning effect and adhesion energy to determine the cell-cell interactions. The impact velocity induces the shear stress onto the cell walls of STM, thereby deteriorating the in vitro viability. However, we found that even with compromised in vitro viability, Salmonella retrieved from deposited patterns impacted at higher velocity revealed an increased expression of phoP (the response regulator of the PhopQ two-component system) and uninterrupted intracellular proliferation in macrophages. The inability of STM{Delta} phoP growth in nutrient-rich dried droplets to the subjected impact velocities signifies the predominant role of phoP in maintaining the virulence of Salmonella during desiccation stress. Our findings open a promising avenue for understating the effect of bacteria-laden drop impact and its role in disease spread. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

bioengineering↗

bioRxiv2022

Comparative multi-omic analyses reveal gene regulatory networks controlling physiological and metabolic responses to drought in upland cotton

Drought stress substantially impacts crop physiology resulting in alteration of growth and productivity. Understanding the genetic and molecular crosstalk between stress responses and agronomically important traits such as fiber yield is particularly complicated in the allopolyploid species, upland cotton (Gossypium hirsutum), due to reduced sequence variability between A and D subgenomes. To better understand how drought stress impacts yield, the transcriptomes of 22 genetically and phenotypically diverse upland cotton accessions grown under well-watered and water-limited conditions in the Arizona low desert were sequenced. Gene co-expression analyses were performed, uncovering a group of stress response genes, in particular transcription factors GhDREB2A-A and GhHSFA6B-D, associated with improved yield under water-limited conditions in an ABA-independent manner. DNA affinity purification sequencing (DAP-seq), as well as public cistrome data from Arabidopsis, were used to identify targets of these two TFs. Among these targets were two lint-yield associated genes previously identified through genome-wide association studies (GWAS) -based approaches, GhABP-D and GhIPS1-A. Biochemical and phylogenetic approaches were used to determine that GhIPS1-A is positively regulated by GhHSFA6B-D, and that this regulatory mechanism is specific to Gossypium spp. containing the A (old-world) genome. Finally, a SNP was identified within the GhHSFA6B-D binding site in GhIPS1-A that is positively associated with yield under water limiting conditions. These data lay out a regulatory connection between abiotic stress and fiber yield in cotton that appears conserved in other systems such as Arabidopsis. This regulatory mechanism highlights how sub-genome dynamics contribute to phenotypic stress-response plasticity in cotton.

plant biology↗

bioRxiv2022

Physiology and structure of pathogenic Escherichia coli pOmpT reveal two substrate-binding sites

The pathogenicity of bacteria can be achieved by cleaving antimicrobial peptides (AMPs) through the outer membrane protease family (Omptins) to evade or resist the hosts innate immune response. The OmpT-like proteins in Escherichia coli are members of Omptins, which have highly conserved proteolytic activity. Here, sequence alignment and physiological studies have determined that pOmpT is a virulence factor with atypical proteolytic activity. Comparing pOmpT with cOmpT in terms of structure, proteolytic activity and target substrates, it is found that Asp267 and Ser276 of cOmpT are substrate-binding sites and help its catalytic center to cleave substrates (protamine,synthetic peptide or RNase 7). However, special structural features and the nature of residues Ser267 and Thr276 of pOmpT caused the inability of cleavage protamine, but allows it to specifically cleave the human AMP RNase 7. It is suggested that two sites are related with the substrate specificity. In short, we found that pOmpT presents a new structural basis for the specific recognition of substrates, and providing new clues for the development of antimicrobial drugs.

microbiology↗