Mesenchymal stromal cells-derived extracellular vesicles alleviate systemic sclerosis via miR-29a-3p.

Systemic sclerosis (SSc) is a potentially lethal disease with no curative treatment. Mesenchymal stromal cells (MSCs) have proved efficacy in SSc but no data is available on MSC-derived extracellular vesicles (EVs) in this multi-organ fibrosis disease. Small size (ssEVs) and large size EVs (lsEVs) were isolated from murine MSCs or human adipose tissue-derived MSCs (ASCs). Control antagomiR (Ct) or antagomiR-29a-3p (A29a) were transfected in MSCs and ASCs before EV production. EVs were injected in the HOCl-induced SSc model at day 21 and euthanasized at day 42. We found that both ssEVs and lsEVs were effective to slow-down the course of the disease. All disease parameters improved in skin and lungs. Interestingly, down-regulating miR-29a-3p in MSCs totally abolished therapeutic efficacy. Besides, we demonstrated a similar efficacy of human ASC-EVs and importantly, EVs from A29a-transfected ASCs failed to improve skin fibrosis. We identified Dnmt3a, Pdgfrbb, Bcl2, Bcl-xl as target genes of miR-29a-3p whose regulation was associated with skin fibrosis improvement. Our study highlights the therapeutic role of miR-29a-3p in SSc and the importance of regulating methylation and apoptosis.

A benzaldehyde-indole fused chromophore-based fluorescent probe for double-response to cyanide and hypochlorite in living cells.

With the rapid development of various industries, cyanide (CN-) and hypochlorite (ClO-) have a tremendously adverse effect on the health of humans and animals. In this study, a fluorescent probe HHTB based on a benzaldehyde-indole fused chromophore was designed to detect cyanide and hypochlorite simultaneously. The synthesized probe was found to have strong anti-interference ability. In addition, the designed probe could respond rapidly to ClO- in just 80 s, while the color changed visibly from red to colorless. Moreover, the response time to CN- was longer (about 160 s), with the apparent color change from red to light red. The ratiometric and colorimetric absorbance variation of HHTB was due to the nucleophilic attack of CN- on the indole CN functional group and the strong oxidization of ClO- which destroyed the CC bonds and the conjugation systems. Furthermore, the probe HHTB responding to ClO- and CN- presented high sensitivity, as the calculated detection limits were 1.18 nM and 1.40 nM, respectively. The probe was also found to have low biological toxicity and was used in living cells successfully. Therefore, it has good application prospect in the field of cell imaging and biomedicine. The binding mechanism of HHTB-CN and the reaction mechanism of HHTB and ClO- were further elucidated by a series of experiments.

In Vivo High-resolution Ratiometric Fluorescence Imaging of Inflammation Using NIR-II Nanoprobes with 1550 nm Emission.

Quantitatively imaging the spatiotemporal distribution of biological events in living organisms is essential to understand fundamental biological processes. Self-calibrating ratiometric fluorescent probes enable accurate and reliable imaging and sensing, but conventional probes using wavelength of 400-900 nm suffer from extremely low resolution for in vivo application due to the disastrous photon scattering and tissue autofluorescence background. Here, we develop a NIR-IIb (1500-1700 nm) emissive nanoprobe for high-resolution ratiometric fluorescence imaging in vivo. The obtained nanoprobe shows fast ratiometric response to hypochlorous acid (HOCl) with a detection limit down to 500 nM, through an absorption competition-induced emission (ACIE) bioimaging system between lanthanide-based downconversion nanoparticles and Cy7.5 fluorophores. Additionally, we demonstrate the superior spatial resolution of 1550 nm to a penetration depth of 3.5 mm in a scattering tissue phantom, which is 7.1-fold and 2.1-fold higher than that of 1064 and 1344 nm, respectively. With this nanoprobe, clear anatomical structures of lymphatic inflammation in ratiometric channel are observed with a precise resolution of ∼477 µm. This study will motivate the further research on the development of NIR-II probes for high-resolution biosensing in vivo.

Evaluation of residual toxicity of hypochlorite-treated water using bioluminescent microbes and microalgae: Implications for ballast water management.

Total residual oxidants (TRO) in treated ballast water can produce various disinfection by-products (DBPs) depending on local conditions, such as salinity and organic matter content in water. Because TRO and DBPs are known to be harmful to aquatic organisms and humans, ecotoxicity tests have been proposed for screening the residual toxicity before discharging treated ballast water. In the present study, we aimed to address the decay rates and toxicity changes of TRO under various conditions in salinity, initial TRO concentrations, and residence time of TRO. In addition, the toxicological sensitivities of bioluminescent bacteria Vibrio fischeri and a commonly-used microalgae Skeletonema costatum relative to the residual toxicity of TRO and six selected DBPs were determined. Decay rate of TRO concentration increased as a function of salinity and was affected by the initial concentrations of TRO. Unexpectedly, significant bioluminescence inhibition was observed for hypochlorite-treated water at < 0.1 mg L-1 TRO (expressed as Cl2), which is a lower concentration than the maximum allowable discharge concentration (MADC) to marine waters established by the International Maritime Organization (IMO). The ecotoxicological thresholds of no observed effective concentration and median effect concentration for all tested DBPs were about 3-10 times lower for V. fischeri than for S. costatum. The results indicate that bioluminescent microbes possess an ecologically-relevant sensitivity to both TRO and DBPs in ballast water. In general, bioassay using V. fischeri was potentially more effective than microalgae for screening the total toxicity of TRO and DBPs in treated ballast water, especially given that ballast water usually contains a highly variable and complex mixture of toxicants.

Disinfection by-Products and Ecotoxic Risk Associated with Hypochlorite Treatment of Tramadol.

In recent years, many studies have highlighted the consistent finding of tramadol (TRA) in the effluents from wastewater treatment plants (WTPs) and also in some rivers and lakes in both Europe and North America, suggesting that TRA is removed by no more than 36% by specific disinfection treatments. The extensive use of this drug has led to environmental pollution of both water and soil, up to its detection in growing plants. In order to expand the knowledge about TRA toxicity as well as the nature of its disinfection by-products (DBPs), a simulation of the waste treatment chlorination step has been reported herein. In particular, we found seven new by-products, that together with TRA, have been assayed on different living organisms (Aliivibrio fischeri, Raphidocelis subcapitata and Daphnia magna), to test their acute and chronic toxicity. The results reported that TRA may be classified as a harmful compound to some aquatic organisms whereas its chlorinated product mixture showed no effects on any of the organisms tested. All data suggest however that TRA chlorination treatment produces a variety of DBPs which can be more harmful than TRA and a risk for the aquatic environment and human health.

Irritant-induced asthma to hypochlorite in mice due to impairment of the airway barrier.

Inhalation of commonly present irritants, such as chlorine and chlorine derivatives, can cause adverse respiratory effects, including irritant-induced asthma (IIA). We hypothesize that due to airway barrier impairment, exposure to hypochlorite (ClO-) can result in airway hypersensitivity. C57Bl/6 mice received an intra-peritoneal (i.p.) injection of the airway damaging agent naphthalene (NA, 200 mg/kg body weight) or vehicle (mineral oil, MO). In vivo micro-computed tomography (CT) images of the lungs were acquired before and at regular time points after the i.p. TREATMENT: After a recovery period of 14 days an intranasal (i.n.) challenge with 0.003% active chlorine (in ClO-) or vehicle (distilled water, H2O) was given, followed by assessment of the breathing frequency. One day later, pulmonary function, along with pulmonary inflammation was determined. Lung permeability was assessed by means of total broncho-alveolar lavage (BAL) protein content and plasma surfactant protein (SP)-D levels. In vivo micro-CT imaging revealed enlargement of the lungs and airways early after NA treatment, with a return to normal at day 14. When challenged i.n. with ClO-, NA-pretreated mice immediately responded with a sensory irritant response. Twenty-four hours later, NA/ClO- mice showed airway hyperreactivity (AHR), accompanied by a neutrophilic and eosinophilic inflammation. NA administration followed by ClO- induced airway barrier impairment, as shown by increased BAL protein and plasma SP-D concentrations; histology revealed epithelial denudation. These data prove that NA-induced lung impairment renders the lungs of mice more sensitive to an airway challenge with ClO-, confirming the hypothesis that incomplete barrier repair, followed by irritant exposure results in airway hypersensitivity.

Vascular endothelial growth factor-D modulates oxidant-antioxidant balance of human vascular endothelial cells.

Vascular endothelial growth factor-D (VEGF-D) is an angiogenic and lymphangiogenic glycoprotein that facilitates tumour growth and distant organ metastasis. Our previous studies showed that VEGF-D stimulates the expression of proteins involved in cell-matrix interactions and promoting the migration of endothelial cells. In this study, we focused on the redox homoeostasis of endothelial cells, which is significantly altered in the process of tumour angiogenesis. Our analysis revealed up-regulated expression of proteins that form the antioxidant barrier of the cell in VEGF-D-treated human umbilical endothelial cells and increased production of reactive oxygen and nitrogen species in addition to a transient elevation in the total thiol group content. Despite a lack of changes in the total antioxidant capacity, modification of the antioxidant barrier induced by VEGF-D was sufficient to protect cells against the oxidative stress caused by hypochlorite and paraquat. These results suggest that exogenous stimulation of endothelial cells with VEGF-D induces an antioxidant response of cells that maintains the redox balance. Additionally, VEGF-D-induced changes in serine/threonine kinase mTOR shuttling between the cytosol and nucleus and its increased phosphorylation at Ser-2448, lead us to the conclusion that the observed shift in redox balance is regulated via mTOR kinase signalling.

Contribution of protein isoaspartate methyl transferase (PIMT) in the survival of Salmonella Typhimurium under oxidative stress and virulence.

The enteric pathogen Salmonella Typhimurium (ST) survives inside the oxidative environment of phagocytic cells. Phagocyte generated oxidants primarily target proteins and modify amino acids in them. These modifications render the targeted proteins functionally inactive. Conversion of Asp to iso-Asp is one of the several known oxidant mediated amino acids modifications. By repairing iso-Asp to Asp, protein-isoaspartyl methyltransferase (PIMT) maintains the activities of proteins and thus helps in cellular survival under oxidative stress. To elucidate the role of PIMT in ST survival under oxidative stress, we have constructed a pimt gene deletion strain (Δpimt strain) of ST. The Δpimt strain grows normally in various culture media in vitro. However, in comparison to wild type ST, the Δpimt strain is found significantly (p<0.001) more susceptible to H2O2 and hypochlorite (HOCl). Further, the Δpimt mutant strain shows hypersusceptibility (p<0.001) to INF-γ stimulated macrophages. This susceptibility is reversed by pharmacological inhibition of reactive oxygen species (ROS) but not reactive nitrogen species (RNS) production. Further, plasmid based complementation enhances the survival of Δpimt mutant strain against oxidants in vitro and also inside the macrophages. In mice model, the LD50 for wild type ST and mutant Δpimt has been 1.73×10(4) and 1.38×10(5), respectively. Further, the mutant strain shows reduced dissemination to spleen and liver in mice. Following infection with a mixture of wild type ST and the Δpimt mutant (co-infection experiment), we recover significantly (p<0.001) less numbers of mutant bacteria from the spleen and liver of mice.

Novel Electrokinetic Microfluidic Detector for Evaluating Effectiveness of Microalgae Disinfection in Ship Ballast Water.

Ship ballast water treatment methods face many technical challenges. The effectiveness of every treatment method usually is evaluated by using large scale equipment and a large volume of samples, which involves time-consuming, laborious, and complex operations. This paper reports the development of a novel, simple and fast platform of methodology in evaluating the efficiency and the best parameters for ballast water treatment systems, particularly in chemical disinfection. In this study, a microfluidic chip with six sample wells and a waste well was designed, where sample transportation was controlled by electrokinetic flow. The performance of this microfluidic platform was evaluated by detecting the disinfection of Dunaliella salina (D. salina) algae in ballast water treated by sodium hypochlorite (NaClO) solution. Light-induced chlorophyll fluorescence (LICF) intensity was used to determine the viability of microalgae cells in the system, which can be operated automatically with the dimension of the detector as small as 50 mm × 24 mm × 5 mm. The 40 µL volume of sample solution was used for each treatment condition test and the validity of detection can be accomplished within about five min. The results show that the viability of microalgae cells under different treatment conditions can be determined accurately and further optimal treatment conditions including concentrations of NaClO and treatment time can also be obtained. These results can provide accurate evaluation and optimal parameters for ballast water treatment methods.

Amelioration of systemic fibrosis in mice by angiotensin II receptor blockade.

OBJECTIVE: Systemic sclerosis (SSc) is characterized by microvascular damage, fibrosis of skin and visceral organs, and autoimmunity. Previous studies have shown that angiotensin II is involved in the synthesis of type I collagen. We investigated whether the blockade of angiotensin II receptor type I (AT1 ) by irbesartan reduces skin and lung fibrosis in 2 murine models of SSc. METHODS: SSc was induced by daily intradermal injection of HOCl into the backs of BALB/c mice (HOCl-induced SSc). Mice were treated daily with irbesartan by oral gavage. RESULTS: Irbesartan reduced dermal thickness, collagen concentration, Smad2/3, and α-smooth muscle actin expression, as well as fibroblast proliferation and H-Ras expression in the skin of mice with HOCl-induced SSc. Mice treated with irbesartan also displayed less lung fibrosis, less inflammation, and a lower concentration of collagen in the lungs than untreated mice. Exhaled nitric oxide, inducible nitric oxide synthase, and 3-nitrotyrosine expression in the lungs were decreased following irbesartan treatment. Moreover, irbesartan reduced the number and the proliferation of splenic B and T cells and the serum levels of anti-DNA topoisomerase I autoantibodies. CONCLUSION: Irbesartan, an AT1 antagonist, prevents fibrosis and inflammation and inhibits nitric oxide production in HOCl-induced models of systemic fibrosis. Our findings extend the indication of an AT1 antagonist to SSc patients with diffuse fibrosis, especially those with lung involvement.

The HOCl dry fog-is it safe for human cells?

This study aims to investigate if high-concentration HOCl fogging disinfection causes cytotoxicity and genotoxicity to cultured primary human skin fibroblasts. The cells were exposed to a dry fog of HOCl produced from solutions with a concentration of 300 ppm (5.72 mM) or 500 ppm (9.53 mM). After four times when fibroblasts were exposed to aerosolized HOCl at a concentration of 500 ppm for 9 minutes, significant cytotoxicity and genotoxicity effects were observed. Significant changes in the morphology of fibroblasts and cell death due to membrane disruption were observed, independent of the number of exposures. Flow cytometry analyses performed under these experimental conditions indicated a decrease in the number of cells with an intact cell membrane in the exposed samples compared to the sham samples, dropping to 49.1% of the total cells. Additionally, under the same conditions, the neutral comet assay results demonstrated significant DNA damage in the exposed cells. However, no analogous damages were found when the cells were exposed to aerosolized HOCl generated from a 300-ppm solution for 3 minutes, whether once or four times. Therefore, we have concluded that aerosolized HOCl in dry fog, with a concentration exceeding 300 ppm, can cause cytotoxic and genotoxic effects on human skin fibroblasts.

Curcumin from Curcuma longa Linn. (Family: Zingiberaceae) attenuates hypochlorous acid-induced cytotoxicity and oxidative damage to human red blood cells.

Hypochlorous acid (HOCl) is a major oxidant produced by activated neutrophils via the myeloperoxidase catalyzed reaction. The production of HOCl eliminates a wide range of pathogens. However, HOCl can also cause significant oxidative damage in cells and tissues where it is generated. The protective effect of curcumin was studied on HOCl-induced oxidative damage to human red blood cells (RBC). Isolated RBC were incubated with HOCl at 37 °C in absence or presence of different concentrations of curcumin. Hemolysates were prepared and assayed for various biochemical parameters. Treatment of RBC with HOCl alone increased hemolysis, protein carbonyls, heme degradation and chloramines as compared to untreated control cells. This was accompanied by reduction in glutathione level, total sulfhydryls and free amino groups. HOCl also lowered the activities of major antioxidant enzymes and diminished the antioxidant power of RBC. Pre-treatment of RBC with different concentrations of curcumin resulted in concentration-dependent attenuation in all these parameters while curcumin alone had no significant effect. Scanning electron microscopy showed that curcumin prevented HOCl-induced morphological changes in RBC and restored their normal biconcave shape. Thus curcumin can be used as a chemoprotective agent to mitigate HOCl-induced oxidative damage to cells. These results also explain the beneficial effects of curcumin against Helicobacter pylori induced stomach ulcers, caused by excessive production of HOCl at the site of bacterial infection.

Identification of proteins susceptible to thiol oxidation in endothelial cells exposed to hypochlorous acid and N-chloramines.

Hypochlorous acid (HOCl) is a potent oxidant produced by the enzyme myeloperoxidase, which is released by neutrophils under inflammatory conditions. Although important in the immune system, HOCl can also damage host tissue, which contributes to the development of disease. HOCl reacts readily with free amino groups to form N-chloramines, which also cause damage in vivo, owing to the extracellular release of myeloperoxidase and production of HOCl. HOCl and N-chloramines react readily with cellular thiols, which causes dysfunction via enzyme inactivation and modulation of redox signaling processes. In this study, the ability of HOCl and model N-chloramines produced on histamine and ammonia at inflammatory sites, to oxidize specific thiol-containing proteins in human coronary artery endothelial cells was investigated. Using a proteomics approach with the thiol-specific probe, 5-iodoacetamidofluorescein, we show that several proteins including peptidylprolyl isomerase A (cyclophilin A), protein disulfide isomerase, glyceraldehyde-3-phosphate dehydrogenase and galectin-1 are particularly sensitive to oxidation by HOCl and N-chloramines formed at inflammatory sites. This will contribute to cellular dysfunction and may play a role in inflammatory disease pathogenesis.

Hypochlorite-induced oxidative stress elevates the capability of HDL in promoting breast cancer metastasis.

BACKGROUND: Previous studies suggest that oxidative stress plays an important role in the development of breast cancer. There is a significant inverse relationship between HDL and the risk and mortality of breast cancer. However, it is well known that under conditions of oxidative stress, such as breast cancer, HDL can be oxidatively modifiedand these modifications may have an effect on the functions of HDL. The purpose of this study is to determine the different effects of normal and oxidized (caused by hypochlorite-induced oxidative stress) HDL on breast cancer cell metastasis. METHODS: Human breast cancer cell lines were treated with normal and hypochlorite-oxidized HDL, and then cell metastasis potency in vivo and the abilities of migration, invasion, adhesion to HUVEC and ECM in vitro were examined. Integrin expression and PKC activity were evaluated, and PKC inhibitor and PKC siRNA was applied. RESULTS: We found hypochlorite-oxidized HDL dramatically promotes breast cancer cell pulmonary metastasis (133.4% increase at P < 0.0 l for MDA-MB-231 by mammary fat pad injection; 164.3% increase at P < 0.01 for MCF7 by tail vein injection) and hepatic metastasis (420% increase at P < 0.0 l for MDA-MB-231 by mammary fat pad injection; 1840% fold increase at P < 0.001 for MCF7 by tail vein injection) in nude mice, and stimulates higher cell invasion (85.1% increase at P < 0.00 l for MDA-MB-231; 88.8% increase at P < 0.00 l for MCF7;), TC-HUVEC adhesion (43.4% increase at P < 0.00 l for MDA-MB-231; 35.2% increase at P < 0.00 l for MCF7), and TC-ECM attachment (41.0% increase at P < 0.00 l for MDA-MB-231; 26.7% increase at P < 0.05 for MCF7) in vitro compared with normal HDL. The data also shows that the PKC pathway is involved in the abnormal actions of hypochlorite-oxidized HDL. CONCLUSIONS: Our study demonstrated that HDL under hypochlorite-induced oxidative stress stimulates breast cancer cell migration, invasion, adhesion to HUVEC and ECM, thereby promoting metastasis of breast cancer. These results suggest that HDL-based treatments should be considered for treatment of breast cancer patients.

The antioxidant role of thiocyanate in the pathogenesis of cystic fibrosis and other inflammation-related diseases.

Cystic fibrosis (CF) is a pleiotropic disease, originating from mutations in the CF transmembrane conductance regulator (CFTR). Lung injuries inflicted by recurring infection and excessive inflammation cause approximately 90% of the morbidity and mortality of CF patients. It remains unclear how CFTR mutations lead to lung illness. Although commonly known as a Cl(-) channel, CFTR also conducts thiocyanate (SCN(-)) ions, important because, in several ways, they can limit potentially harmful accumulations of hydrogen peroxide (H(2)O(2)) and hypochlorite (OCl(-)). First, lactoperoxidase (LPO) in the airways catalyzes oxidation of SCN(-) to tissue-innocuous hypothiocyanite (OSCN(-)), while consuming H(2)O(2). Second, SCN(-) even at low concentrations competes effectively with Cl(-) for myeloperoxidase (MPO) (which is released by white blood cells), thus limiting OCl(-) production by the enzyme. Third, SCN(-) can rapidly reduce OCl(-) without catalysis. Here, we show that SCN(-) and LPO protect a lung cell line from injuries caused by H(2)O(2); and that SCN(-) protects from OCl(-) made by MPO. Of relevance to inflammation in other diseases, we find that in three other tested cell types (arterial endothelial cells, a neuronal cell line, and a pancreatic beta cell line) SCN(-) at concentrations of > or =100 microM greatly attenuates the cytotoxicity of MPO. Humans naturally derive SCN(-) from edible plants, and plasma SCN(-) levels of the general population vary from 10 to 140 microM. Our findings raise the possibility that insufficient levels of antioxidant SCN(-) provide inadequate protection from OCl(-), thus worsening inflammatory diseases, and predisposing humans to diseases linked to MPO activity, including atherosclerosis, neurodegeneration, and certain cancers.

Airborne fungi and bacteria in child daycare centers and the effectiveness of weak acid hypochlorous water on controlling microbes.

A three-week-long biological sampling scheme was conducted in two child daycare centers (CDCCs) in order to investigate interdiurnal and diurnal variations in indoor airborne microbes as well as the efficiency of weak acid hypochlorous water (WAHW) on disinfecting indoor microbes. During the second week of sampling, WAHW was sprayed using a fogger in the classroom after children had left and before they returned the next morning. An identical cycle of experiments was performed twice in the winter and spring. Without WAHW intervention, the respective mean of the indoor concentrations and I/O ratios were 8732-47581 CFU m(-3) and 0.96-2.53 for fungi, and 6706-28998 CFU m(-3) and 1.10-11.92 for bacteria, showing severe bio-contamination in the CDCCs. Moreover, a relatively high level of bacterial pollution was found at noon, whereas a greater fungal pollution could be detected in the morning and at noon. Among five school days, the fungal and bacterial pollution may be higher on Monday and on Wednesday, Thursday, and Friday, respectively. Furthermore, with WAHW intervention, the indoor microbial concentrations and I/O ratios were decreased significantly. The reduction of I/O ratios caused by WAHW disinfection was accomplished in the morning for bacteria and in the morning, at noon, and in the afternoon for fungi. In conclusion, this study clearly clarified the risky period during which children may be exposed to hazardous environments, and demonstrated the effectiveness of spraying WAHW the night before on decontaminating indoor airborne microbes on the following day, especially in the case of fungi.

Does Hypochlorous Acid Cause Ototoxicity? An Experimental Study.

AIM: Hypochlorous acid (HOCl) is a weak acid that ionizes in water. It is an effective antiseptic exhibiting low toxicity on living tissues. We aimed to investigate the ototoxic effects of HOCl on an animal model by using electrophysiological and histological methods. MATERIALS AND METHODS: The study comprised 32 Sprague-Dawley rats, which were separated into four groups: control group (A), saline solution group (B), 70% isopropyl alcohol + 2% chlorhexidine group (C), and HOCl group (D). After recording the auditory brainstem response (ABR) for basal hearing thresholds (8, 16, 24, and 32 kHz), 0.03 ml of the aforementioned materials was injected intratympanically three times every 2 days in groups B, C, and D. ABR measurements were repeated on the 7th and 21st days. All animals were sacrificed, and temporal bones were prepared for examinations of cochlear histology and vascular endothelial growth factor immunohistochemistry. RESULTS: Basal hearing levels were normal across all frequencies and groups, with no statistical differentiation. On the 7th and 21st days after the ABR test, all other groups demonstrated a significant deterioration in hearing levels compared with group A. When the results from 7th and 21st days were compared within group D, a partial recovery was observed. In histopathology, groups C and D demonstrated moderate and severe cochlear degeneration, along with decreased immunoreactivity in the organ of Corti, stria vascularis, and spiral ligament. CONCLUSION: This is the first study to evaluate the safety of using HOCl in otology. Although HOCI is less ototoxic than the disinfectant used, it may have a toxic effect on cochlea.Level of Evidence: Animal Research.

Dosing metric in cellular experiments: The mol/cell metric has its limitations.

It has been argued that the mol/cell metric is more universal than concentration of the toxic agent since in many cases the effect of dose expressed as mol/cell is independent of ex-perimental setup. We confirmed it for hemolysis of erythrocytes in phosphate-buffered saline induced by hypochlorite where the amount of femtomoles/cell of hypochlorite needed for 50% hemolysis was independent of erythrocyte concentration. However, in the presence of blood plasma this metric became dependent on cell concentration. Similarly, the effect of 3-bromopyruvic acid (3-BP) on PEO1 cells as a function of mol/cell ratio depended on the volume of the 3-BP containing medium, due to the reaction of 3-BP with components of the medium. Hemolytic amounts of sodium dodecyl sulfate and Triton X-100 expressed as mol/cell decreased with increasing cell concentration while the effect of DMSO on the viability of a constant number of fibroblasts was independent of the volume of DMSO-containing medium. These results demonstrate that the mol/cell metric is still dependent on experimental conditions when the toxic agent interacts with components of the medium or when its physical state is modified by the target cells, and the effect is independent of the mol/per cell ratio for high excess of a cell damaging agent.

New insights on chemically induced animal models of systemic sclerosis.

PURPOSE OF REVIEW: To discuss the most recent published studies on chemical-induced animal models of systemic sclerosis (SSc) and to precise the important signalling pathways that lead to the initiation and progression of the disease in these models. RECENT FINDINGS: Environmental factors undoubtedly contribute to the initiation and the development of SSc. Among those factors, bleomycin has been identified as a possible SSc-inducing substance in mice. The bleomycin model mimics the inflammatory changes observed in the early phase of the disease. This model has been extensively studied and has allowed identification of several key pathways activated in the human disease. More recently, a new chemical-induced model of scleroderma has been developed in mice using daily intradermal injections of a solution generating hypochlorous acid (HOCl)-model. This HOCl-model recapitulates the whole spectrum of the human disease, as fibrosis, inflammation, autoimmunity and vasculopathy can be observed in mice and brought new arguments for a major role of reactive oxygen species in the induction of local and systemic fibrosis. SUMMARY: Chemically induced models truly develop a SSc-like disease and argue for a crucial role of ROS in SSc.

Maturation of released spores is necessary for acquisition of full spore heat resistance during Bacillus subtilis sporulation.

The first ∼10% of spores released from sporangia (early spores) during Bacillus subtilis sporulation were isolated, and their properties were compared to those of the total spores produced from the same culture. The early spores had significantly lower resistance to wet heat and hypochlorite than the total spores but identical resistance to dry heat and UV radiation. Early and total spores also had the same levels of core water, dipicolinic acid, and Ca and germinated similarly with several nutrient germinants. The wet heat resistance of the early spores could be increased to that of total spores if early spores were incubated in conditioned sporulation medium for ∼24 h at 37°C (maturation), and some hypochlorite resistance was also restored. The maturation of early spores took place in pH 8 buffer with Ca(2+) but was blocked by EDTA; maturation was also seen with early spores of strains lacking the CotE protein or the coat-associated transglutaminase, both of which are needed for normal coat structure. Nonetheless, it appears to be most likely that it is changes in coat structure that are responsible for the increased resistance to wet heat and hypochlorite upon early spore maturation.