Physiological Osmolarity Research Articles

MICROENVIRONMENTAL DEPENDENCE OF PROPER CHONDROCYTIC MATURATION

Articular cartilage is a relatively hypoxic tissue with a unique extracellular matrix that is enriched with cations, resulting in an elevated interstitial fluid osmolarity. Several biomechanical and physicochemical stimuli are reported to influence chondrocyte metabolism. For regenerative in vitro applications, increasing the extracellular osmolarity above plasma level to more physiological valuesinduces chondrogenic marker expression and the differentiation of chondroprogenitor cells. Calcineurin inhibitor FK506 modulates the differentiation of primary chondrocytes under such conditions and its effect on cell proliferation, extracellular matrix quality, and BMP- and TGF-β signaling will be described. Supraphysiological osmolarity compromises chondrocyte proliferation, while physosmolarity or FK506 did not. Rather, the combination of the latter increased proteoglycan and collagen expression in chondrocytesin vitro and in situ, affecting expression of TGF-β-inducible protein TGFBI and chondrogenic (SOX9, Col2) as well as terminal differentiation markers (e.g., Col10). Surprisingly, expression of particularly minor collagens (e.g., Col9, Col11) was improved. Physiological osmolarity seems to promote terminal chondrogenic differentiation of progenitor cells through sensitization of TGF-β superfamily signaling at the type I receptor. While hyperosmolarity alone facilitates TGF-β superfamily signaling, FK506 seems to enhance signaling by releasing the FKBP12 break from the type I receptor to improve collagenous marker expression. Our data help explaining seemingly contradictory earlier findings and potentially benefit future cell-based cartilage repair strategies.

Characterization of the proteome changes in an in vitro hyperosmotic dry eye model employing human corneal epithelial cells

Aims/Purpose: The detrimental effects of tear hyperosmolarity on human corneal epithelial (HCE) cells is a primary cause of dry eye syndrome (DES). However, the molecular mechanisms underlying inflammatory processes and cellular dysfunction in HCE are largely unknown. This study characterized the proteome changes in an optimized hyperosmolarity‐induced DES model in vitro.Methods: An in vitro hyperosmolar (400–550 mOsm) DES model was established in a simian virus 40 (SV40)‐immortalized HCE cell line. Cells incubated in medium with physiological osmolarity (312 mOsm) were used as control. Cell viability, cell death and intracellular reactive oxygen species (ROS) levels were analysed with MTS, Annexin‐V‐FLUOS, crystal violet and DCFDA assays. Mass spectrometry‐based proteomics analyses characterized the proteome and elucidated the underlying molecular changes.Results: The HCE cell viability was significantly decreased in a concentration‐dependent manner with increasing hyperosmolarity compared to control at 48 hr (450 mOsm, −47%, p = 2.7 × 10−8; 500 mOsm, −71%, p = 1.1 × 10−4; 550 mOsm, −84%, p = 2.6 × 10−5). Notably, cell adherence was significantly decreased (p = 5.8 × 10−9), while levels of ROS (p = 3.6 × 10−2) and apoptosis (p = 3.6 × 10−2) were significantly increased at 450 mOsm compared to control. Proteomics analysis identified ~2000 proteins (FDR <1%) and, as many as 215 proteins were found to be significantly (p < 0.05) differentially abundant in the 450 vs. 312 mOsm. The effects of high hyperosmolarity (450 mOsm)‐induced damage resembling DES on the HCE cell were further demonstrated by the expression of proteins involved in the activation of inflammatory response (p = 5.5 × 10−3), apoptosis (p = 5.3 × 10−4) and generation of ROS (e.g., glutathione S‐transferase P) (p = 7.1 × 10−3). Moreover, many differentially expressed proteins were also implicated in the regulation of cell viability (p = 1.1 × 10−6) and organization of cytoplasm (e.g., cyclase associated actin cytoskeleton regulatory protein 1) (p = 2.4 × 10−2).Conclusions: In summary, an optimized hyperosmolarity‐induced DES cell culture model for proteomics analysis was established, and specific alterations in the proteome were characterized in this model. This in vitro model would be instrumental for testing therapeutic targets for DES in the future.

Proteomic profile of naturally released extracellular vesicles secreted from Leptospira interrogans serovar Pomona in response to temperature and osmotic stresses

Bacterial extracellular vesicles (EVs) are generally formed by pinching off outer membrane leaflets while simultaneously releasing multiple active molecules into the external environment. In this study, we aimed to identify the protein cargo of leptospiral EVs released from intact leptospires grown under three different conditions: EMJH medium at 30 °C, temperature shifted to 37 °C, and physiologic osmolarity (EMJH medium with 120 mM NaCl). The naturally released EVs observed under transmission electron microscopy were spherical in shape with an approximate diameter of 80–100 nm. Quantitative proteomics and bioinformatic analysis indicated that the EVs were formed primarily from the outer membrane and the cytoplasm. The main functional COG categories of proteins carried in leptospiral EVs might be involved in cell growth, survival and adaptation, and pathogenicity. Relative to their abundance in EVs grown in EMJH medium at 30 °C, 39 and 69 proteins exhibited significant changes in response to the temperature shift and the osmotic change, respectively. During exposure to both stresses, Leptospira secreted several multifunctional proteins via EVs, while preserving certain virulence proteins within whole cells. Therefore, leptospiral EVs may serve as a decoy structure for host responses, whereas some virulence factors necessary for direct interaction with the host environment are reserved in leptospiral cells. This knowledge will be useful for understanding the pathogenesis of leptospirosis and developing as one of vaccine platforms against leptospirosis in the future.

Physiological Temperature and Osmotic Changes Drive Dynamic Proteome Alterations in the Leptospiral Outer Membrane and Enhance Protein Export Systems.

Leptospirosis, a remerging zoonosis, has no effective vaccine or an unambiguous early diagnostic reagent. Proteins differentially expressed (DE) under pathogenic conditions will be useful candidates for antileptospiral measures. We employed a multipronged approach comprising high-resolution TMT-labeled LC-MS/MS-based proteome analysis coupled with bioinformatics on leptospiral proteins following Triton X-114 subcellular fractionation of leptospires treated under physiological temperature and osmolarity that mimic infection. Although there were significant changes in the DE proteins at the level of the entire cell, there were notable changes in proteins at the subcellular level, particularly on the outer membrane (OM), that show the significance of subcellular proteome analysis. The detergent-enriched proteins, representing outer membrane proteins (OMPs), exhibited a dynamic nature and upregulation under various physiological conditions. It was found that pathogenic proteins showed a higher proportion of upregulation compared to the nonpathogenic proteins in the OM. Further analysis identified 17 virulent proteins exclusively upregulated in the outer membrane during infection that could be useful for vaccine and diagnostic targets. The DE proteins may aid in metabolic adaptation and are enriched in pathways related to signal transduction and antibiotic biosynthesis. Many upregulated proteins belong to protein export systems such as SEC translocase, T2SSs, and T1SSs, indicating their sequential participation in protein transport to the outer leaflet of the OM. Further studies on OM-localized proteins may shed light on the pathogenesis of leptospirosis and serve as the basis for effective countermeasures.

Vesicle budding caused by lysolipid-induced asymmetry stress

Lysolipids such as lauroyl, myristoyl, and palmitoyl lysophosphatidylcholine (LPC) insert into the outer leaflet of liposomes but do not flip to the inner leaflet over many hours. This way, they create asymmetry stress between the intrinsic areas of the two leaflets. We have studied how this stress is relaxed with particular emphasis on the budding and fission of small (diameter 20–30 nm) daughter vesicles (DVs). Asymmetric flow field-flow fractionation was utilized to quantify the extent of budding from large unilamellar vesicles after exposure to LPC. Budding starts at a low threshold of the order of 2 mol% LPC in the outer (and ≈0 mol% LPC in the inner) leaflet. We see reason to assume that the fractional fluorescence intensity from DVs is a good approximation for the fraction of membrane lipid, POPC, transferred into DVs. Accordingly, budding starts with a “budding power” of ≈6 POPC molecules budding off per LPC added, corresponding to a more than 10-fold accumulation of LPC in the outer leaflet of DVs to ≈24 mol%. As long as budding is possible, little strain is built up in the membranes, a claim supported by the lack of changes in limiting fluorescence anisotropy, rotational correlation time, and fluorescence lifetime of symmetrically and asymmetrically inserted TMA-DPH. At physiological osmolarity, budding is typically limited to 20–30% of budded fraction with some batch-to-batch variation, but independent of the LPC species. We hypothesize that the budding limit is determined by the excess area of the liposomes upon preparation, which is then used up upon budding given the larger area-to-volume ratio of smaller liposomes. As the mother vesicles approach ideal spheres, budding must stop. This is qualitatively supported by increased and decreased budding limits of osmotically predeflated and preinflated vesicles, respectively.

Yes-associated protein nuclear translocation promotes anabolic activity in human articular chondrocytes

Yes-associated protein (YAP) has been widely studied as a mechanotransducer in many cell types, but its function in cartilage is controversial. The aim of this study was to identify the effect of YAP phosphorylation and nuclear translocation on the chondrocyte response to stimuli relevant to osteoarthritis (OA). Cultured normal human articular chondrocytes from 81 donors were treated with increased osmolarity media as an in vitro model of mechanical stimulation, fibronectin fragments (FN-f) or IL-1β as catabolic stimuli, and IGF-1 as an anabolic stimulus. YAP function was assessed with gene knockdown and inhibition by verteporfin. Nuclear translocation of YAP and its transcriptional co-activator TAZ and site-specific YAP phosphorylation were determined by immunoblotting. Immunohistochemistry and immunofluorescence to detect YAP were performed on normal and OA human cartilage with different degrees of damage. Chondrocyte YAP/TAZ nuclear translocation increased under physiological osmolarity (400mOsm) and IGF-1 stimulation, which was associated with YAP phosphorylation at Ser128. In contrast, catabolic stimulation decreased the levels of nuclear YAP/TAZ through YAP phosphorylation at Ser127. Following YAP inhibition, anabolic gene expression and transcriptional activity decreased. Additionally, YAP knockdown reduced proteoglycan staining and levels of type II collagen. Total YAP immunostaining was greater in OA cartilage, but YAP was sequestered in the cytosol in cartilage areas with more severe damage. YAP chondrocyte nuclear translocation is regulated by differential phosphorylation in response to anabolic and catabolic stimuli. Decreased nuclear YAP in OA chondrocytes may contribute to reduced anabolic activity and promotion of further cartilage loss.

Extensive prokaryotic maintenance respiration in the sea influenced by osmoregulation

Microbial respiration is the major process consuming oxygen in the biosphere. The relative energy demand from growth of biomass or maintenance activities determines the regulation of respiration with impact on how the development of hypoxia and CO2 emissions is controlled. This coupling is crucial for understanding the life history and associated ecological interactions of microorganisms. However, the knowledge of rate and regulating factors of maintenance respiration in the biosphere is limited. In this study, we demonstrated significant relationships in marine field samples where the prokaryotic specific growth rate predicts cell-specific respiration, in accordance with theory from culture models, over a 10-fold salinity range. This enables the first reported direct estimates of maintenance respiration in nature to show a 6-fold variation between 0.12-0.62 fmol O2 cell-1 d-1, comprising 29-72% of prokaryotic specific respiration. The lowest maintenance respiration occurred at salinity close to physiological osmolarity, suggesting osmoregulation as one of the more energy-consuming maintenance activities. A conservative global estimate of maintenance respiration accounted for 66% of the total prokaryotic respiration in the ocean´s mixed layer. This means that maintenance activities dominate the use of the energy generated by prokaryotic respiration in the sea, where osmoregulation is one significant energy consumer. Consequently, maintenance respiration and its regulation must be included in ecological and biogeochemical models to accurately project and manage the development of hypoxia and CO2 emissions from the ocean.

Physosmotic Induction of Chondrogenic Maturation Is TGF-β Dependent and Enhanced by Calcineurin Inhibitor FK506.

Increasing extracellular osmolarity 100 mOsm/kg above plasma level to the physiological levels for cartilage induces chondrogenic marker expression and the differentiation of chondroprogenitor cells. The calcineurin inhibitor FK506 has been reported to modulate the hypertrophic differentiation of primary chondrocytes under such conditions, but the molecular mechanism has remained unclear. We aimed at clarifying its role. Chondrocyte cell lines and primary cells were cultured under plasma osmolarity and chondrocyte-specific in situ osmolarity (+100 mOsm, physosmolarity) was increased to compare the activation of nuclear factor of activated T-cells 5 (NFAT5). The effects of osmolarity and FK506 on calcineurin activity, cell proliferation, extracellular matrix quality, and BMP- and TGF-β signaling were analyzed using biochemical, gene, and protein expression, as well as reporter and bio-assays. NFAT5 translocation was similar in chondrocyte cell lines and primary cells. High supraphysiological osmolarity compromised cell proliferation, while physosmolarity or FK506 did not, but in combination increased proteoglycan and collagen expression in chondrocytes in vitro and in situ. The expression of the TGF-β-inducible protein TGFBI, as well as chondrogenic (SOX9, Col2) and terminal differentiation markers (e.g., Col10) were affected by osmolarity. Particularly, the expression of minor collagens (e.g., Col9, Col11) was affected. The inhibition of the FK506-binding protein suggests modulation at the TGF-β receptor level, rather than calcineurin-mediated signaling, as a cause. Physiological osmolarity promotes terminal chondrogenic differentiation of progenitor cells through the sensitization of the TGF-β superfamily signaling at the type I receptor. While hyperosmolarity alone facilitates TGF-β superfamily signaling, FK506 further enhances signaling by releasing the FKBP12 break from the type I receptor to improve collagenous marker expression. Our results help explain earlier findings and potentially benefit future cell-based cartilage repair strategies.

Topical Pirfenidone-Loaded Liposomes Ophthalmic Formulation Reduces Haze Development after Corneal Alkali Burn in Mice.

Corneal chemical burns (CCBs) frequently result in corneal fibrosis or haze, an opacity of the cornea that obstructs vision and induces corneal blindness. Diverse strategies have been employed to prevent or reduce CCB-related corneal haze. In this study, we evaluated the physicochemical characteristics and biologic effects of a topical pirfenidone (PFD)-loaded liposomal formulation (PL) on a corneal alkali burn mice model. We found that PL was appropriate for ocular application due to its physiologic tear pH, osmolarity and viscosity suitable for topical ophthalmic use. Regarding its therapeutic activity, PL-treated mice had significantly reduced haze size and density, corneal edema, corneal thickness, and corneal inflammatory infiltration, in contrast to PFD in aqueous solution (p < 0.01). Importantly, the antifibrotic activity of PL (reduction of corneal haze) was associated with modulation of transforming growth factor (TGF)-β and Interleukin (IL)-1β genes. PL suppressed TGF-β expression and restored normal IL-1β expression in corneal tissue more efficiently in contrast to PFD in aqueous solution. In conclusion, PFD showed essential anti-inflammatory and anti-fibrotic effects in the treatment of alkali burns. Noteworthy, a new formulation of PFD-loaded liposomes remarkably improved these effects, standing out as a promising treatment for corneal haze.

Rational formulation design of injectable thermosensitive chitosan-based hydrogels for cell encapsulation and delivery

This work reports a rational design of injectable thermosensitive chitosan systems for cell encapsulation and delivery. Using mixtures of two phosphate salts, beta-glycerophosphate and ammonium hydrogen phosphate, we demonstrate that the pH and the osmolarity can be adjusted separately by varying the molar ratios between the salts and the d-glucosamine monomers. We found the existence of a critical temperature above which gelation time decays following a power-law. This gelation kinetics can be finely tuned through the pH and salt-glucosamine ratios. Formulations having physiological pH and osmolarity were produced for chitosan concentrations ranging from 0.4 to 0.9 wt%. They remain liquid for more than 2 h at 20 °C and form a macroporous gel within 2 min at 37 °C. In vitro encapsulation of pre-osteoblastic cells and gingival fibroblasts showed homogeneous cell distribution and good cell viability up to 24 h. Such an approach provides a valuable platform to design thermosensitive cell-laden systems.

Strain Hardening in Highly Acetylated Chitosan Gels.

Strain hardening has recently emerged as a near-universal response of biological tissues to mechanical stimulation as well as a powerful regulator of cell fate. Understanding the mechanistic basis for this nonlinear elasticity is crucial for developing bioinspired materials that mimic extracellular matrix mechanics. Here, we show that covalent networks built from highly acetylated chitosans exhibit strain hardening at physiological pH and osmolarity. While varying the chitosan physical-chemical composition and network connectivity, we provide evidence that temporary nodes arising from the entangling of chains between stable cross-links are at the root of nonlinear elasticity. The contour length (Lc) of the said chains revealed that the larger the chain length between the cross-links, the greater is the entanglement over disentanglement upon network stretching. To this end, we calculated that the minimum number of Khun's segments in Lc that contributes to the onset of strain hardening is 15. Furthermore, we identified a relationship between critical strain marking nonlinear elasticity and the network connectivity, being similar to that found for the cytoskeletal collagen matrix, indicating the potential use of semiflexible (neutral pH-soluble) chitosans in assembling extracellular matrix mimics.

Controlling Nucleopeptide Hydrogel Self-Assembly and Formation for Cell-Culture Scaffold Applications.

Hydrogels made from self-assembling peptides have significant advantages in tissue engineering, namely a biocompatible nature and large molecular repertoire. Short peptides in particular allow for straightforward synthesis, self-assembly, and reproducibility. Applications are currently limited, however, due to potential toxicity of the chemical modifications that drive self-assembly and harsh gelation conditions. Peptides conjugated to nucleobases present one opportunity for a naturally derived species to minimize cytotoxicity. We have developed a hydrogel-formation environment for nucleopeptide gelation modulated entirely by biological buffers and salts. Self-assembly in this system is dependent on buffer and ion identity mediated by pKa and formulation in the former and by valency and ionicity in the latter. Solutions at physiological pH and osmolarity, and in turn compatible with cell culture, initiate hydrogel formation and analytical and computational methods are used to explore pH and salt effects at the molecular and structural level. The mechanism of nucleopeptide self-assembly enables tuning of mechanical properties through the addition of divalent cations and one order of magnitude increase in hydrogel storage modulus. The stability of these constructs therefore provides an opportunity for long-term cell culture, and we demonstrate survival and proliferation of fibroblasts on hydrogel surfaces. This novel, biological buffer-mediated gelation methodology expands opportunities for tissue engineering applications of short peptides and their derivatives.

Importance of Osmolarity and Oxygen Tension for Cartilage Tissue Engineering.

For cartilage repair in vivo or evaluation of new therapeutic approaches in vitro, the generation of functional cartilage tissue is of crucial importance and can only be achieved if the phenotype of the chondrocytes is preserved. Three-dimensional (3D) cell culture is broadly used for this purpose. However, adapting culture parameters like the oxygen tension or the osmolarity to their physiological values is often omitted. Indeed, articular cartilage is an avascular tissue subjected to reduced oxygen tension and presenting and increased osmolarity compared with most other tissues. In this study, we aimed at evaluating the effect of a physiological oxygen tension (3% instead of 21%) and physiological osmolarity (430 vs. 330 mOsm in nonadjusted DMEM) and the combination of both on the cell proliferation, matrix production, and the phenotype of porcine chondrocytes in a scaffold-free 3D culture system. We observed that a physiological osmolarity had no effect on cell proliferation and matrix production but positively influences the chondrocyte phenotype. A physiological oxygen level prevented cell proliferation but resulted in an increased matrix content/million cells and had a positive influence on the chondrocyte phenotype as well. The strongest benefit was reached with the combination of both physiological osmolarity and oxygen levels; with these conditions, type I collagen expression became undetectable. In addition, at 3% O2 the chondrocytes-matrix constructs were found to more closely resemble native cartilage regarding the matrix-to-cell ratio. In conclusion, this study clearly demonstrates the benefit of using physiological oxygen tension and osmolarity in cartilage tissue engineering with the combination of both showing the strongest benefit on the chondrocyte phenotype.

Mechanical Response Changes in Porcine Tricuspid Valve Anterior Leaflet Under Osmotic-Induced Swelling.

Since many soft tissues function in an isotonic in-vivo environment, it is expected that physiological osmolarity will be maintained when conducting experiments on these tissues ex-vivo. In this study, we aimed to examine how not adhering to such a practice may alter the mechanical response of the tricuspid valve (TV) anterior leaflet. Tissue specimens were immersed in deionized (DI) water prior to quantification of the stress–strain responses using an in-plane biaxial mechanical testing device. Following a two-hour immersion in DI water, the tissue thickness increased an average of 107.3% in the DI water group compared to only 6.8% in the control group, in which the tissue samples were submerged in an isotonic phosphate buffered saline solution for the same period of time. Tissue strains evaluated at 85 kPa revealed a significant reduction in the radial direction, from 34.8% to 20%, following immersion in DI water. However, no significant change was observed in the control group. Our study demonstrated the impact of a hypo-osmotic environment on the mechanical response of TV anterior leaflet. The imbalance in ions leads to water absorption in the valvular tissue that can alter its mechanical response. As such, in ex-vivo experiments for which the native mechanical response of the valves is important, using an isotonic buffer solution is essential.

Assessment of Phosphate and Osmolarity Levels in Chronically Administered Eye Drops

Objectives:To assess phosphate and osmolarity levels of chronically administered eye drops commercially available in Turkey.Materials and Methods:A total of 53 topical eye drops including 18 antiglaucoma drugs, 4 nonsteroidal anti-inflammatory drugs (NSAIDs), 10 corticosteroids, 7 antihistaminics, and 14 artificial tears identified using the Vademecum Modern Medications Guideline (2018) were included in the study. Phosphate levels were assessed using Roche Cobas C501 analyzer (Roche Diagnostics GmbH, Mannheim, Germany) and the respective kits. Osmolarity was assessed using Vescor Vapro 5600 vapor pressure osmometer (Sanova Medical Systems, Vienna, Austria). Mean phosphate and osmolarity levels were obtained after averaging three measurements. Eye drops were categorized as isoosmolar, hypoosmolar and hyperosmolar based on physiologic tear osmolarity range (296.5±9.8 mOsm/L).Results:The highest phosphate concentration was found in the antiglaucoma group (20.3±35.4 mmol/L), followed by antihistaminics (17.3±17.9 mmol/L), corticosteroids (15.2±19.1 mmol/L), artificial tears (0.8±1.0), and NSAIDs (0.04±0.08). Percentage of medications in the hyperosmolar category was highest in the NSAI group (75%), followed by antihistaminics (43%), corticosteroids (20%), and antiglaucoma drugs (19%). Nearly all of the artificial tear formulations were in the hypoosmolar (71%) or isoosmolar (21%) categories.Conclusion:Approximately 40% of glaucoma medications and approximately 60% of corticosteroid and antihistaminic medications had a phosphate concentration higher than the physiologic tear phosphate level (1.45 mmol/L).

Increased TGF-β and BMP Levels and Improved Chondrocyte-Specific Marker Expression In Vitro under Cartilage-Specific Physiological Osmolarity.

During standard expansion culture (i.e., plasma osmolarity, 280 mOsm) human articular chondrocytes dedifferentiate, making them inappropriate for autologous chondrocyte implantation to treat cartilage defects. Increasing the osmolarity of culture media to physiological osmolarity levels of cartilage (i.e., 380 mOsm), increases collagen type II (COL2A1) expression of human articular chondrocytes in vitro, but the underlying molecular mechanism is not fully understood. We hypothesized that TGF-β superfamily signaling may drive expression of COL2A1 under physiological osmolarity culture conditions. Human articular chondrocytes were cultured in cytokine-free medium of 280 or 380 mOsm with or without siRNA mediated TGF-β2 knockdown (RNAi). Expression of TGF-β isoforms, and collagen type II was evaluated by RT-qPCR and immunoblotting. TGF-β2 protein secretion was evaluated using ELISA and TGF-β bioactivity was determined using an established reporter assay. Involvement of BMP signaling was investigated by culturing human articular chondrocytes in the presence or absence of BMP inhibitor dorsomorphin and BMP bioactivity was determined using an established reporter assay. Physiological cartilage osmolarity (i.e., physosmolarity) most prominently increased TGF-β2 mRNA expression and protein secretion as well as TGF-β bioactivity. Upon TGF-β2 isoform-specific knockdown, gene expression of chondrocyte marker COL2A1 was induced. TGF-β2 RNAi under physosmolarity enhanced TGF-β bioactivity. BMP bioactivity increased upon physosmotic treatment, but was not related to TGF-β2 RNAi. In contrast, dorsomorphin inhibited COL2A1 mRNA expression in human articular chondrocytes independent of the osmotic condition. Our data suggest a role for TGF-β superfamily member signaling in physosmolarity-induced mRNA expression of collagen type II. As physosmotic conditions favor the expression of COL2A1 independent of our manipulations, contribution of other metabolic, post-transcriptional or epigenetic factors cannot be excluded in the underlying complex and interdependent regulation of marker gene expression. Dissecting these molecular mechanisms holds potential to further improve future cell-based chondral repair strategies.

Effects of amphipathic profile regularization on structural order and interaction with membrane models of two highly cationic branched peptides with β-sheet propensity

Antimicrobial peptides attracted increasing interest in last decades due to the rising concern of multi-drug resistant pathogens. Dendrimeric peptides are branched molecules with multiple copies of one peptide functional unit bound to the central core. Compared to linear analogues, they usually show improved activity and lower susceptibility to proteases. Knowledge of structure-function relationship is fundamental to tailor their properties. This work is focused on SB056, the smallest example of dendrimeric peptide, whose amino acid sequence is WKKIRVRLSA. Two copies are bound to the α- and ε- nitrogen of one lysine core. An 8-aminooctanamide was added at the C-terminus to improve membrane affinity. Its propensity for β-type structures is also interesting, since helical peptides were already thoroughly studied. Moreover, SB056 maintains activity at physiological osmolarity, a typical limitation of natural peptides. An optimized analogue with improved performance was designed, β-SB056, which differs only in the relative position of the first two residues (KWKIRVRLSA). This produced remarkable differences. Structure order and aggregation behavior were characterized by using complementary techniques and membrane models with different negative charge. Infrared spectroscopy showed different propensity for ordered β-sheets. Lipid monolayers’ surface pressure was measured to estimate the area/peptide and the ability to perturb lipid packing. Fluorescence spectroscopy was applied to compare peptide insertion into the lipid bilayer. Such small change in primary structure produced fundamental differences in their aggregation behavior. A regular amphipathic peptide’s primary structure was responsible for ordered β-sheets in a charge independent fashion, in contrast to unordered aggregates formed by the former analogue.

Proteomic approach and expression analysis revealed the differential expression of predicted leptospiral proteases capable of ECM degradation

Leptospira, the causative agent of leptospirosis is known to have many proteases with potential to degrade extracellular matrix. However, a multipronged approach to identify, classify, characterize and elucidate their role has not been attempted. Our proteomic approach using high-resolution LC-MS/MS analysis of Triton X-114 fractions of Leptospira interrogans resulted in the identification of 104 proteases out of 130 proteases predicted by MEROPS. In Leptospira approximately 3.5% of the genome complements for proteases, which include catalytic types of metallo-, serine-, cysteine-, aspartic-, threonine- and asparagine- peptidases. Comparison of proteases from different serovars revealed that M04, M09B, M14A, M75, M28A, A01 and U73 protease families are exclusively present in pathogenic form. The M23 and S33 protease families are represented with >14 members in Leptospira. The differential expression under physiological temperature (37 °C) and osmolarity (300 mOsM) showed that proteases belonging to the catalytic type of Metallo-peptidases are upregulated significantly in pathogenic conditions. In silico prediction and characterization of the proteases revealed that several proteases are membrane anchored and secretory, classical as well as non-classical system. The study demonstrates the diversity and complexity of proteases, while maintaining conservation across the serovars in Leptospira and their differential expression under pathogenic conditions.

Transcriptional Activator GmrA, Encoded in Genomic Island OI-29, Controls the Motility of Enterohemorrhagic Escherichia coli O157:H7

Enterohemorrhagic Escherichia coli O157:H7 is a major human enteric pathogen capable of causing large outbreaks of severe infections that induce bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. Its genome contains 177 unique O islands (OIs) including those carrying the main virulence elements, Shiga toxin-converting phages (OI-45 and OI-93) and locus for enterocyte effacement (OI-148). However, many of these islands harbor only genes of unknown function. Here, we demonstrate that OI-29 encodes a newly discovered transcriptional activator, Z0639 (named GmrA), that is required for motility and flagellar synthesis in O157:H7. GmrA directly binds to the promoter of fliA, an RNA polymerase sigma factor, and thereby regulates flagellar genes controlled by FliA. Expression of gmrA is maximal under host conditions (37°C, neutral pH, and physiological osmolarity), and in the presence of host epithelial cells, indicative of a role of this gene in infection by promoting motility. Finally, GmrA was found to be a widespread regulator of bacterial motility and flagellar synthesis in different pathotypes of E. coli. Our work largely enriches our understanding of bacterial motility control, and provides another example of regulators acquired laterally that mediate flagellar synthesis.

Role of sph2 Gene Regulation in Hemolytic and Sphingomyelinase Activities Produced by Leptospira interrogans.

Pathogenic members of the genus Leptospira are the causative agents of leptospirosis, a neglected disease of public and veterinary health concern. Leptospirosis is a systemic disease that in its severest forms leads to renal insufficiency, hepatic dysfunction, and pulmonary failure. Many strains of Leptospira produce hemolytic and sphingomyelinase activities, and a number of candidate leptospiral hemolysins have been identified based on sequence similarity to well-characterized bacterial hemolysins. Five of the putative hemolysins are sphingomyelinase paralogs. Although recombinant forms of the sphingomyelinase Sph2 and other hemolysins lyse erythrocytes, none have been demonstrated to contribute to the hemolytic activity secreted by leptospiral cells. In this study, we examined the regulation of sph2 and its relationship to hemolytic and sphingomyelinase activities produced by several L. interrogans strains cultivated under the osmotic conditions found in the mammalian host. The sph2 gene was poorly expressed when the Fiocruz L1-130 (serovar Copenhageni), 56601 (sv. Lai), and L495 (sv. Manilae) strains were cultivated in the standard culture medium EMJH. Raising EMJH osmolarity to physiological levels with sodium chloride enhanced Sph2 production in all three strains. In addition, the Pomona subtype kennewicki strain LC82-25 produced substantially greater amounts of Sph2 during standard EMJH growth than the other strains, and sph2 expression increased further by addition of salt. When 10% rat serum was present in EMJH along with the sodium chloride supplement, Sph2 production increased further in all strains. Osmotic regulation and differences in basal Sph2 production in the Manilae L495 and Pomona strains correlated with the levels of secreted hemolysin and sphingomyelinase activities. Finally, a transposon insertion in sph2 dramatically reduced hemolytic and sphingomyelinase activities during incubation of L. interrogans at physiologic osmolarity. Complementation of the mutation with the sph2 gene partially restored production of hemolytic and sphingomyelinase activities. These results indicate that the sph2 gene product contributes to the hemolytic and sphingomyelinase activities secreted by L. interrogans and most likely dominates those functions under the culture condition tested.