ßPix Guanine Nucleotide Exchange Factor Regulates Regeneration of Injured Peripheral Axons.

Axon regeneration is essential for successful recovery after peripheral nerve injury. Although growth cone reformation and axonal extension are crucial steps in axonal regeneration, the regulatory mechanisms underlying these dynamic processes are poorly understood. Here, we identify ßPix (Arhgef7), the guanine nucleotide exchange factor for Rac1 GTPase, as a regulator of axonal regeneration. After sciatic nerve injury in mice, the expression levels of ßPix increase significantly in nerve segments containing regenerating axons. In regrowing axons, ßPix is localized in the peripheral domain of the growth cone. Using ßPix neuronal isoform knockout (NIKO) mice in which the neuronal isoforms of ßPix are specifically removed, we demonstrate that ßPix promotes neurite outgrowth in cultured dorsal root ganglion neurons and in vivo axon regeneration after sciatic nerve crush injury. Activation of cJun and STAT3 in the cell bodies is not affected in ßPix NIKO mice, supporting the local action of ßPix in regenerating axons. Finally, inhibiting Src, a kinase previously identified as an activator of the ßPix neuronal isoform, causes axon outgrowth defects in vitro, like those found in the ßPix NIKO neurons. Altogether, these data indicate that ßPix plays an important role in axonal regrowth during peripheral nerve regeneration.

ßPix heterozygous mice have defects in neuronal morphology and social interaction.

ßPix activates Rho family small GTPases, Rac1 and Cdc42 as a guanine nucleotide exchange factor. Although overexpression of ßPix in cultured neurons indicates that ßPix is involved in spine morphogenesis and synapse formation in vitro, the in vivo role of ßPix in the neuron is not well understood. Recently, we generated ßPix knockout mice that showed lethality at embryonic day 9.5. Here, we investigate the neuronal role of ßPix using ßPix heterozygous mice that are viable and fertile. ßPix heterozygous mice show decreased expression levels of ßPix proteins in various tissues including the brain. Cultured hippocampal neurons from ßPix heterozygous mice show a decrease in neurite length and complexity as well as synaptic density. Both excitatory and inhibitory synapse densities are decreased in these neurons. Golgi-staining of hippocampal tissues from the brain of these mice show reduced dendritic complexity and spine density in the hippocampal neurons. Expression levels of NMDA- and AMPA-receptor subunits and Git1 protein in hippocampal tissues are also decreased in these mice. Behaviorally, ßPix heterozygous mice exhibit impaired social interaction. Altogether, these results indicate that ßPix is required for neurite morphogenesis and synapse formation, and the reduced expression of ßPix proteins results in a defect in social behavior.

Early and Delayed Meniscal Shrinkage After Fresh-Frozen Lateral Meniscal Allograft Transplantation: Magnetic Resonance Imaging Study With a Midterm Follow-up.

PURPOSE: To evaluate whether fresh-frozen meniscal allograft shrinkage occurs only during the first year of the early remodeling period or progresses over the delayed period of midterm years and to determine whether these changes were associated with certain clinical and radiologic outcomes. METHODS: We retrospectively reviewed meniscal allograft transplantations (MATs) performed by 1 senior surgeon (S-I.B.) using fresh-frozen allograft from 2008 to 2013. The inclusion criteria were the patients who had midterm follow-up magnetic resonance imaging (MRI) scans between 3 and 6 years after isolated lateral MATs. We excluded the graft tears found on the 1-year or midterm MRI scans. MATs were indicated for the treatment persistent compartmental pain in young to middle-aged, physically active patients who had well-aligned nonarthritic joint without ligament insufficiency. The meniscal width of the transplants at the midbody and posterior horn was measured on day 2 (as a reference), at 1 year (after early remodeling period), and after 3 to 6 years (delayed period) postoperatively. Joint space width changes during each interval were measured on 45° flexion posteroanterior views. The Lysholm score and Tegner activity scale were used to evaluate clinical outcomes. RESULTS: Eighty-four isolated lateral MATs with the midterm MRI scans were identified. Of these, 17 graft tears were found; therefore, we analyzed 67 patients (32 male and 35 female patients) with a mean age of 30.9 years (range, 15-52 years). The mean relative meniscal width at the midbody decreased to 93.7% (95% confidence interval [CI], 91.8%-95.6%; P < .001) at 1 year postoperatively and to 88.0% (95% CI, 85.6%-90.3%; P < .001) at the midterm follow-up of 4.0 ± 1.0 years. The posterior horn shrank less than the midbody during the same period (96.0%; 95% CI, 94.8%-97.1%) at 1 year (P < .001) and 92.5% (95% CI, 91.0%-94.1%) at the last follow-up (P < .001). Although there was no severe shrinkage (>50% of the initial size), the incidence of moderate (25%-50%) changes at the midbody increased from 1 (1.5%) at 1 year to 5 (7.5%) at the last follow-up, respectively. We could not find any significant positive correlations between the relative meniscal width and patient-reported outcomes or joint space width changes after 1 year or at the last follow-up. CONCLUSIONS: Shrinkage of fresh-frozen meniscal transplants occurred during both the early remodeling and delayed midterm periods. Although the changes were greater in the midbody than in the posterior horn, the overall changes were less than those of the previous studies using cryopreserved grafts. We could not find that the meniscal shrinkage over the midterm period were significantly associated with inferior outcomes in this series. LEVEL OF EVIDENCE: Level IV, therapeutic case series.

Total Knee Arthroplasty With Patellar Retention: The Severity of Patellofemoral Osteoarthritis Did Not Affect the Clinical and Radiographic Outcomes.

BACKGROUND: The purpose of this study is to compare the clinical and radiographic outcomes of total knee arthroplasty (TKA) with patellar retention in accordance with the severity of patellofemoral arthritis. METHODS: We retrospectively reviewed patients who underwent TKA with patellar retention using the NexGen LPS or LPS-flex system between September 2010 and May 2015. The radiographic severity of patellofemoral arthritis was categorized according to the Iwano classification system, and subjects were divided into mild (stage 0-I) and moderate to severe (stage II-IV) groups. Clinical outcomes were evaluated using the Hospital for Special Surgery score, Knee Society Score, function score, Western Ontario and McMaster Universities Osteoarthritis Index, and Feller score. Radiographic outcomes were assessed using the congruence angle, patellar tilt angle, and lateral patellar displacement. The minimum follow-up for clinical and radiographic evaluation was 2 years. Clinical and radiographic outcomes were compared between the 2 groups preoperatively and at the time of the last follow-up. RESULTS: Four hundred seventy-four knees were enrolled and assigned to mild (n = 208) or moderate to severe (n = 266) groups. The preoperative Feller score was significantly lower in the moderate to severe group (P = .030), whereas the postoperative clinical and radiographic results did not differ significantly between the 2 groups. CONCLUSION: Clinical and radiographic outcomes did not differ in accordance with the severity of patellofemoral arthritis after a minimum 2 years of follow-up of patients treated with TKA with patellar retention. Good outcomes were obtained with patellar retention in TKA, even in patients with advanced patellofemoral osteoarthritis.

Using a Tibial Short Extension Stem Reduces Tibial Component Loosening After Primary Total Knee Arthroplasty in Severely Varus Knees: Long-term Survival Analysis With Propensity Score Matching.

BACKGROUND: Patients with severe preoperative varus deformity have been reported to have high rates of loosening after total knee arthroplasty (TKA), primarily on the tibial side. This study investigated whether a short extension stem for the tibial component in severely varus knees would reduce the failure rate due to loosening on the tibial side. METHODS: Patients who underwent TKA, performed by a single surgeon using a single implant between November 1998 and January 2009, were retrospectively evaluated. Patients diagnosed with primary osteoarthritis, having a hip-knee-ankle axis greater than varus 8° on preoperative long-film radiographs, and postoperatively followed up for more than 2 years were included. Patients were divided into "stem" and "nonstem" groups, followed by 1:1 propensity score matching according to age, gender, body mass index, preoperative mechanical axis, and postoperative alignment. Tibial loosening rates in the 2 groups were compared. RESULTS: The study cohort included 602 patients, divided into "stem" and "nonstem" groups. Propensity score matching yielded 88 pairs of patients. Mean follow-up duration was similar in the stem and nonstem groups (109.22 vs 103.81 months, P = .451). None of the patients in the stem group, compared with 5 in the nonstem group, experienced aseptic loosening. The overall implant survival rate was significantly higher in the stem group than in the nonstem group (P = .0201). CONCLUSION: Using a short extension stem for the tibial component in primary TKA in patients with severe varus deformity greater than 8° may reduce the rate of loosening of the tibial side and increase the longevity of the implant. LEVEL OF EVIDENCE: Level III.

Slitrks control excitatory and inhibitory synapse formation with LAR receptor protein tyrosine phosphatases.

The balance between excitatory and inhibitory synaptic inputs, which is governed by multiple synapse organizers, controls neural circuit functions and behaviors. Slit- and Trk-like proteins (Slitrks) are a family of synapse organizers, whose emerging synaptic roles are incompletely understood. Here, we report that Slitrks are enriched in postsynaptic densities in rat brains. Overexpression of Slitrks promoted synapse formation, whereas RNAi-mediated knockdown of Slitrks decreased synapse density. Intriguingly, Slitrks were required for both excitatory and inhibitory synapse formation in an isoform-dependent manner. Moreover, Slitrks required distinct members of the leukocyte antigen-related receptor protein tyrosine phosphatase (LAR-RPTP) family to trigger synapse formation. Protein tyrosine phosphatase σ (PTPσ), in particular, was specifically required for excitatory synaptic differentiation by Slitrks, whereas PTPδ was necessary for inhibitory synapse differentiation. Taken together, these data suggest that combinatorial interactions of Slitrks with LAR-RPTP family members maintain synapse formation to coordinate excitatory-inhibitory balance.

Cyanine-based fluorescent probe for highly selective detection of glutathione in cell cultures and live mouse tissues.

Glutathione (GSH) plays a crucial role in human pathologies. Near-infrared fluorescence-based sensors capable of detecting intracellular GSH in vivo would be useful tools to understand the mechanisms of diseases. In this work, two cyanine-based fluorescent probes, 1 and 2, containing sulfonamide groups were prepared. Evaluation of the fluorescence changes displayed by probe 1, which contains a 2,4-dinitrobenzenesulfonamide group, shows that it is cell-membrane-permeable and can selectively detect thiols such as GSH, cysteine (Cys), and homocysteine (Hcy) in living cells. The response of 1 to thiols can be reversed by treatment with N-methylmaleimide (NMM). Probe 2, which possesses a 5-(dimethylamino)naphthalenesulfonamide group, displays high selectivity for GSH over Cys and Hcy, and its response can be reversed using NMM. The potential biological utility of 2 was shown by its use in fluorescence imaging of GSH in living cells. Furthermore, probe 2 can determine changes in the intracellular levels of GSH modualated by H2O2. The properties of 2 enable its use in monitoring GSH in vivo in a mouse model. The results showed that intravenous injection of 2 into a mouse generates a dramatic image in which strong fluorescence is emitted from various tissues, including the liver, kidney, lung, and spleen. Importantly, 2 can be utilized to monitor the depletion of GSH in mouse tissue cells promoted by excessive administration of the painkiller acetaminophen. The combined results coming from this effort suggest that the new probe will serve as an efficient tool for detecting cellular GSH in animals.

Analysis of stored and transplanted cord blood units from KoreaCORD: reappraisal of banking guidelines and selection strategy.

BACKGROUND: We analyzed the characteristics of stored and transplanted cord blood (CB) units from the Korean network for public CB donation (KoreaCORD) to reassess the banking guidelines and optimize CB selection based on cell dose and human leukocyte antigen (HLA) mismatching. STUDY DESIGN AND METHODS: We retrospectively reviewed data, with regard to total nucleated cell (TNC) count and HLA match in the KoreaCORD registry from August 2001 to December 2010. RESULTS: A total of 21,914 CB units have been registered, of which 904 units (4.1%) contained less than 5 × 10(8) TNCs, which did not meet the present storage criteria for public CB banking in Korea. Although the proportion of stored CBs providing TNC of 5 × 10(8) to 7.9 × 10(8) was 45.7%, only 22.0% of all transplanted CBs were derived from these stored CBs. In the single CB transplantation setting, 79% (85/108) of CB units provided 4 × 10(7) TNCs/kg or more in the transplanted one-mismatch (1-MM) CB units and 51% (19/37) of CBs provided 6 × 10(7) TNCs/kg or more in the transplanted 2-MM CB units. CONCLUSIONS: The minimal requirement of TNCs for banking of CB units for public banking should be evaluated and increased to support the selection of CB units with higher cell doses, especially for use in the 1- and 2-MM transplant settings.

Correction: ßPix-d promotes tubulin acetylation and neurite outgrowth through a PAK/Stathmin1 signaling pathway.

[This corrects the article DOI: 10.1371/journal.pone.0230814.].

ßPix-d promotes tubulin acetylation and neurite outgrowth through a PAK/Stathmin1 signaling pathway.

Microtubules are a major cytoskeletal component of neurites, and the regulation of microtubule stability is essential for neurite morphogenesis. ßPix (ARHGEF7) is a guanine nucleotide exchange factor for the small GTPases Rac1 and Cdc42, which modulate the organization of actin filaments and microtubules. ßPix is expressed as alternatively spliced variants, including the ubiquitous isoform ßPix-a and the neuronal isoforms ßPix-b and ßPix-d, but the function of the neuronal isoforms remains unclear. Here, we reveal the novel role of ßPix neuronal isoforms in regulating tubulin acetylation and neurite outgrowth. At DIV4, hippocampal neurons cultured from ßPix neuronal isoform knockout (ßPix-NIKO) mice exhibit defects in neurite morphology and tubulin acetylation, a type of tubulin modification which often labels stable microtubules. Treating ßPix-NIKO neurons with paclitaxel, which stabilizes the microtubules, or reintroducing either neuronal ßPix isoform to the KO neurons overcomes the impairment in neurite morphology and tubulin acetylation, suggesting that neuronal ßPix isoforms may promote microtubule stabilization during neurite development. ßPix-NIKO neurons also exhibit lower phosphorylation levels for Stathmin1, a microtubule-destabilizing protein, at Ser16. Expressing either ßPix neuronal isoform in the ßPix-NIKO neurons restores Stathmin1 phosphorylation levels, with ßPix-d having a greater effect than ßPix-b. Furthermore, we find that the recovery of neurite length and Stathmin1 phosphorylation via ßPix-d expression requires PAK kinase activity. Taken together, our study demonstrates that ßPix-d regulates the phosphorylation of Stathmin1 in a PAK-dependent manner and that neuronal ßPix isoforms promote tubulin acetylation and neurite morphogenesis during neuronal development.

Loss of ϐPix Causes Defects in Early Embryonic Development, and Cell Spreading and PlateletDerived Growth Factor-Induced Chemotaxis in Mouse Embryonic Fibroblasts.

ßPix is a guanine nucleotide exchange factor for the Rho family small GTPases, Rac1 and Cdc42. It is known to regulate focal adhesion dynamics and cell migration. However, the in vivo role of ßPix is currently not well understood. Here, we report the production and characterization of ßPix-KO mice. Loss of ßPix results in embryonic lethality accompanied by abnormal developmental features, such as incomplete neural tube closure, impaired axial rotation, and failure of allantoischorion fusion. We also generated ßPix-KO mouse embryonic fibroblasts (MEFs) to examine ßPix function in mouse fibroblasts. ßPix-KO MEFs exhibit decreased Rac1 activity, and defects in cell spreading and platelet-derived growth factor (PDGF)-induced ruffle formation and chemotaxis. The average size of focal adhesions is increased in ßPix-KO MEFs. Interestingly, ßPix-KO MEFs showed increased motility in random migration and rapid wound healing with elevated levels of MLC2 phosphorylation. Taken together, our data demonstrate that ßPix plays essential roles in early embryonic development, cell spreading, and cell migration in fibroblasts.

Morphologic MRI changes of the anterior cruciate ligament are associated with an increase in the medial tibial plateau bony slope after medial opening wedge high tibial osteotomy in a non-injured ACL population.

BACKGROUND: Medial opening wedge high tibial osteotomy (OWHTO) is a useful treatment for medial osteoarthritis. However, OWHTO sometimes causes a change in tibial slope in the sagittal plane. Although several studies have described the effects of the tibial slope on the biomechanics of the knee, including the anterior cruciate ligament (ACL), there has been little study of the magnetic resonance imaging (MRI) visible changes occurring to the native ACL and the factors affecting them after OWHTO. HYPOTHESIS: We hypothesized that morphologic MRI changes to an uninjured ACL after OWHTO would be associated with increased medial tibial plateau bony slope. PATIENTS AND METHODS: Thirty-three patients who underwent OWHTO and pre/postoperative MRI were included in this retrospective study. The mean period of follow-up MRI was 22.35 (±14.78) months. The patients were divided into two groups according to the occurrence of postoperative ACL morphologic MRI changes defined as mucoid degeneration, ganglion cyst occurrence, or change in the ACL fiber shape (stationary group n=21, altered group n=12). The medial tibial plateau bony slope (MTS) and anterior tibial translation (ATT) were evaluated on MRI. Logistic regression analysis was used to determine factors affecting the occurrence of postoperative ACL morphologic changes. RESULTS: Postoperative MTS and the difference between pre- and post values (ΔMTS), postoperative ATT and the difference between pre- and post values (ΔATT) were significantly different between stationary and altered groups. ΔMTS was associated with postoperative morphologic changes to the ACL (odds ratio: 0.30, 95% confidence interval=0.11-0.82, p=0.019). CONCLUSION: The occurrence of morphologic ACL change after OWHTO is associated with the amount of MTS change. LEVEL OF EVIDENCE: III, Retrospective comparative study.

Hinge Fractures Are Underestimated on Plain Radiographs After Open Wedge Proximal Tibial Osteotomy: Evaluation by Computed Tomography.

BACKGROUND: Lateral hinge fracture (LHF) after medial open wedge high tibial osteotomy (MOWHTO) may not be recognized on perioperative plain radiographs. Such cases may be identified at follow-up and misdiagnosed as delayed LHF. PURPOSE: This study aimed to investigate the extent of LHF misdiagnosis and to determine whether patients with LHFs have inferior clinical outcomes after MOWHTO. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Fifty-one knees in 50 patients (36 women, 14 men; mean age, 51.8 years; range, 24-64 years) who had undergone MOWHTO with locking plate fixation between October 2013 and April 2016 were retrospectively reviewed. LHFs identified on intraoperative fluoroscopy and immediate postoperative radiographs were compared with the actual incidence based on computed tomography (CT) scans performed within 2 days of surgery. Delayed LHFs, not visible on the CT scans but found on later follow-up radiographs, were also assessed. More frequent radiographic check-ups were recommended in patients with LHFs, and weightbearing was delayed until evident callus formation was seen on follow-up radiographs for type 2 or 3 fractures. The loss of correction, the time of union, and complication rate were compared between the knees with LHF and those without LHF. Clinical outcome was measured according to the Knee Society (KS) scores. RESULTS: Overall, 14 early LHFs (27.5%) were identified on CT scans. Of these, 7 LHFs (13.7%) were observed on perioperative radiographs, and the remaining 7 LHFs were identified on later radiographs. Delayed LHFs occurred in 2 cases (3.9%). In the 16 knees with LHF, minimal loss of correction was observed 1 month to 1 year postoperatively without statistical significance. No alignment changes were seen in the 35 knees without LHFs. In the LHF versus no LHF groups, no significant differences were seen regarding time of union (5.3 ± 1.7 months vs 5.4 ± 1.8 months, respectively; P = .898) and postoperative KS scores (knee score, 96.6 ± 2.5 vs 95.3 ± 6.4, P = .435; functional score, 94.4 ± 9.6 vs 89.1 ± 10.9, P = .107). No other complications occurred in either group. CONCLUSION: Most LHFs after MOWHTO occurred intraoperatively, but half (7/14) were not identified on postoperative radiographs. CT scans would enable detection of early LHFs that would otherwise have been mistaken for delayed LHF. However, clinical outcomes did not differ between patients with and without LHF.

Naphthalene-based fluorescent probes for glutathione and their applications in living cells and patients with sepsis.

Rationale: Among the biothiols-related diseases, sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection and can result in severe oxidative stress and damage to multiple organs. In this study, we aimed to develop a fluorescence chemosensor that can both detect GSH and further predict sepsis. Methods: In this study, two new naphthalene dialdehyde compounds containing different functional groups were synthesized, and the sensing abilities of these compounds towards biothiols and its applications for prediction of sepsis were investigated. Results: Our study revealed that the newly developed probe 6-methoxynaphthalene-2, 3-dicarbaldehyde (MNDA) has two-photon is capable of detecting GSH in live cells with two-photon microscopy (TPM) under the excitation at a wavelength of 900 nm. Furthermore, two GSH detection probes naphthalene-2,3-dicarboxaldehyde (NDA) and 6-fluoronaphthalene-2,3-dicarbaldehyde (FNDA) not only can detect GSH in living cells, but also showed clinical significance for the diagnosis and prediction of mortality in patients with sepsis. Conclusions: These results open up a promising direction for further medical diagnostic techniques.

SESN2/sestrin2 suppresses sepsis by inducing mitophagy and inhibiting NLRP3 activation in macrophages.

Proper regulation of mitophagy for mitochondrial homeostasis is important in various inflammatory diseases. However, the precise mechanisms by which mitophagy is activated to regulate inflammatory responses remain largely unknown. The NLRP3 (NLR family, pyrin domain containing 3) inflammasome serves as a platform that triggers the activation of CASP1 (caspase 1) and secretion of proinflammatory cytokines. Here, we demonstrate that SESN2 (sestrin 2), known as stress-inducible protein, suppresses prolonged NLRP3 inflammasome activation by clearance of damaged mitochondria through inducing mitophagy in macrophages. SESN2 plays a dual role in inducing mitophagy in response to inflammasome activation. First, SESN2 induces "mitochondrial priming" by marking mitochondria for recognition by the autophagic machinery. For mitochondrial preparing, SESN2 facilitates the perinuclear-clustering of mitochondria by mediating aggregation of SQSTM1 (sequestosome 1) and its binding to lysine 63 (Lys63)-linked ubiquitins on the mitochondrial surface. Second, SESN2 activates the specific autophagic machinery for degradation of primed mitochondria via an increase of ULK1 (unc-51 like kinase 1) protein levels. Moreover, increased SESN2 expression by extended LPS (lipopolysaccharide) stimulation is mediated by NOS2 (nitric oxide synthase 2, inducible)-mediated NO (nitric oxide) in macrophages. Thus, Sesn2-deficient mice displayed defective mitophagy, which resulted in hyperactivation of inflammasomes and increased mortality in 2 different sepsis models. Our findings define a unique regulatory mechanism of mitophagy activation for immunological homeostasis that protects the host from sepsis.

A ratiometric fluorescent probe based on a coumarin-hemicyanine scaffold for sensitive and selective detection of endogenous peroxynitrite.

In the study described herein, the red emitting probe CHCN, which possesses a linked coumarin-hemicyanine scaffold, was developed for detection of peroxynitrite (ONOO(-)) under physiological conditions. The studies show that CHCN displays a dual ratiometric and colorimetric response to ONOO(-) that is caused by an oxidation process. A possible mechanism of this oxidation process was proposed and confirmed by ESI-MS spectra for the first time. CHCN shown highly selective and sensitive towards ONOO(-) with a low limit of detection LOD (49.7 nM). Moreover, CHCN has appreciable cell permeability and, as a result, it is applicable to ratiometric detection of exogenous and endogenous ONOO(-) in living cells during phagocytic immune response. We anticipate that, owing to their ideal properties, probes of this type will find great use in explorations of the role played by ONOO(-) in biology.

Efficient Learning of Image Super-Resolution and Compression Artifact Removal with Semi-Local Gaussian Processes.

Improving the quality of degraded images is a key problem in image processing, but the breadth of the problem leads to domain-specific approaches for tasks such as super-resolution and compression artifact removal. Recent approaches have shown that a general approach is possible by learning application-specific models from examples; however, learning models sophisticated enough to generate high-quality images is computationally expensive, and so specific per-application or per-dataset models are impractical. To solve this problem, we present an efficient semi-local approximation scheme to large-scale Gaussian processes. This allows efficient learning of task-specific image enhancements from example images without reducing quality. As such, our algorithm can be easily customized to specific applications and datasets, and we show the efficiency and effectiveness of our approach across five domains: single-image super-resolution for scene, human face, and text images, and artifact removal in JPEG- and JPEG 2000-encoded images.

Preparation of a cyanine-based fluorescent probe for highly selective detection of glutathione and its use in living cells and tissues of mice.

Glutathione (GSH) is a major endogenous antioxidant that has a central role in cellular defense against toxins and free radicals. This protocol describes the preparation of CPDSA, a cyanine-based near-infrared (NIR) fluorescent probe for the detection of GSH in cells and in vivo. CPDSA is prepared with high yield through a simple two-step process. The first step is to react commercially available IR-780 iodide with excess anhydrous piperazine in anhydrous N,N-dimethyl formamide at 85 °C to form cyanine-piperazine (CP). The second step is the sulfonylation of CP with dansyl chloride in anhydrous dichloromethane. CPDSA selectively detects GSH in cells, and it has been shown to not react with other biothiols such as cysteine (Cys) and homocysteine (Hcy). This probe can also be used to monitor the GSH level of mouse bone marrow-derived neutrophils (BMDNs). The preparation of probe CPDSA takes 2 d, and experiments in cells and mice take 12-13 d.

Dual oxidase 2 in lung epithelia is essential for hyperoxia-induced acute lung injury in mice.

AIMS: Acute lung injury (ALI) induced by excessive hyperoxia has been employed as a model of oxidative stress imitating acute respiratory distress syndrome. Under hyperoxic conditions, overloading quantities of reactive oxygen species (ROS) are generated in both lung epithelial and endothelial cells, leading to ALI. Some NADPH oxidase (NOX) family enzymes are responsible for hyperoxia-induced ROS generation in lung epithelial and endothelial cells. However, the molecular mechanisms of ROS production in type II alveolar epithelial cells (AECs) and ALI induced by hyperoxia are poorly understood. RESULTS: In this study, we show that dual oxidase 2 (DUOX2) is a key NOX enzyme that affects hyperoxia-induced ROS production, particularly in type II AECs, leading to lung injury. In DUOX2 mutant mice (DUOX2(thyd/thyd)) or mice in which DUOX2 expression is knocked down in the lungs, hyperoxia-induced ALI was significantly lower than in wild-type (WT) mice. DUOX2 was mainly expressed in type II AECs, but not endothelial cells, and hyperoxia-induced ROS production was markedly reduced in primary type II AECs isolated from DUOX2(thyd/thyd) mice. Furthermore, DUOX2-generated ROS are responsible for caspase-mediated cell death, inducing ERK and JNK phophorylation in type II AECs. INNOVATION: To date, no role for DUOX2 has been defined in hyperoxia-mediated ALI despite it being a NOX homologue and major ROS source in lung epithelium. CONCLUSION: Here, we present the novel finding that DUOX2-generated ROS induce AEC death, leading to hyperoxia-induced lung injury.