Evaluation of borinic acids as new,fast hydrogen peroxide–responsive triggers

Hydrogen peroxide (H₂O₂) is responsible for numerous damages when overproduced, and its detection is crucial for a better understanding of H₂O₂-mediated signaling in physiological and pathological processes. For this purpose, various “off–on” small fluorescent probes relying on a boronate trigger have been prepared, and this design has also been involved in the development of H₂O₂-activated prodrugs or theranostic tools. However, this design suffers from slow kinetics, preventing activation by H₂O₂ with a short response time. Therefore, faster H₂O₂-reactive groups are awaited. To address this issue, we have successfully developed and characterized a prototypic borinic-based fluorescent probe containing a coumarin scaffold. We determined its in vitro kinetic constants toward H₂O₂-promoted oxidation. We measured 1.9 × 10⁴ m⁻¹⋅s⁻¹ as a second-order rate constant, which is 10,000-fold faster than its well-established boronic counterpart (1.8 m⁻¹⋅s⁻¹). This improved reactivity was also effective in a cellular context, rendering borinic acids an advantageous trigger for H₂O₂-mediated release of effectors such as fluorescent moieties.

Reactive oxygen species (ROS) are involved in various physiological processes. In particular, hydrogen peroxide (H₂O₂) plays a critical role in the regulation of numerous biological activities as a signaling molecule (1, 2). However, aberrant production or accumulation of H₂O₂ leads to oxidative stress conditions, which can cause lesions associated with aging, cancer (3), and several neurodegenerative diseases such as Alzheimer’s or Parkinson’s (4, 5). Differentiation of physiological or abnormal conditions is closely connected with slight changes in H₂O₂ levels. However, the generation and degradation of H₂O₂ are variable within different cellular compartments, and this small molecule is highly diffusive, rendering the capture of small H₂O₂ fluctuations and the study of its spatial and temporal dynamics difficult. Therefore, the development of selective and sensitive H₂O₂-reactive tools for applications in a biological context represents a challenge for a better understanding of H₂O₂-mediated signaling in physiological and pathological processes or the use of H₂O₂ activation for the release of biological effectors (6).Numerous strategies have been developed to implement H₂O₂-reactive molecular triggers, as exemplified by “off–on” small fluorescent probes. Such probes have attracted particular attention due to their easy implementation, high expected signal-to-noise ratio, and compatibility with standard equipment present in cellular biology research environments (7–9). Activation in such a context is triggered or modified by H₂O₂-mediated transformation of a suitable chemical species. Several approaches have been explored including probes based on arylsulfonyl ester perhydrolysis (10), oxidation of arylboronates (11), Baeyer–Villiger oxidation of diketones (12), Tamao oxidation of silanes (13), a tandem Payne–Dakin reaction (14) or a seleno-Mislow–Evans rearrangement (15). Among them, designs based on the boronate esters oxidation pioneered by Chang are the most explored, due to their remarkable stability, low toxicity profile, ease of preparation, and specificity toward H₂O₂, as illustrated in recent reviews (16–18). Upon reaction with H₂O₂, these compounds undergo an oxidative conversion into aryl borate esters that further hydrolyze into the corresponding phenols along with borate esters or boric acid (Scheme 1A). This conversion turns on probe fluorescence or activates drug release either directly or via the degradation of a self-immolative spacer. This chemospecific and biologically compatible reaction allowed, for instance, developing highly selective fluorescent probes for H₂O₂ imaging in cells (19–23). However, H₂O₂-triggered conversion of boronic acids to phenols is still not completely satisfactory in a biological context (24) since most of these probes have second-order reaction rate constants of 0.1 to 1.0 m⁻¹⋅s⁻¹ (14). In cells, H₂O₂ is present in the 1 to 100 nm concentration range in physiological conditions and could reach up to 100 μm under oxidative stress conditions (25). Therefore, most of the boronate-based systems need an incubation time longer than 30 min for activation at an H₂O₂ concentration of 100 μm. At such a time scale, H₂O₂ typically diffuses over a distance of 2 mm (evaluated as (D_H2O2τ)^0.5 with D_H2O2 = 1.7 × 10⁻⁹ m²⋅s⁻¹ from ref. 26 and τ = 30 min). Hence, to improve spatial resolution for H₂O₂ imaging, alternative H₂O₂ triggers with rapid reaction rates allowing real-time activation by H₂O₂ are still required.Open in a separate windowScheme 1.(A) Current boronic acid (R = H) or boronate (R,R = tetramethylethylene) as H₂O₂-responsive group releasing a hydroxyaryl as effector and a boric acid or a borate ester respectively. (B) This study: a borinic acid as H₂O₂-responsive group releasing a hydroxyaryl as effector and a boronic acid.To address this issue, we envisioned the use of borinic acids, structures in which one of the boron–oxygen bonds of the boronic acid is replaced by a boron–carbon bond. Due to these electronic modifications, borinic acids exhibit more electrophilic properties (27–30) compared to their boronic acid counterparts and could be more prone to rapid oxidation. These structures have been mainly exploited as catalysts in various reactions such as epoxide ring opening (31), hydrosilylation (32), transamidation (33), aldol reaction (34, 35), C–H activation (36, 37), selective monoalkylation, acylation and sulfonation of diols (38, 39), or regioselective glycosylation reactions (40–42). Surprisingly, the reactivity of these borinic species remains underexplored (43–45), probably due to their limited synthetic access (46–49). They were usually obtained through the addition of strong organometallic reagents (RLi/RMgBr) onto boron-based electrophiles such as trialkylborates, boron halides, diborane, or boronate esters. To date, a detailed study of the reactivity of borinic acids toward oxidation including reaction with H₂O₂ has not been reported and their use as triggers for the direct release of a probe or an effector has not been considered.Herein, we report the design, synthesis, and evaluation of a borinic-triggered prototypic probe prone to direct and rapid activation by the H₂O₂ molecule (Scheme 1B). We establish a detailed kinetic analysis of the H₂O₂-promoted oxidation of this borinic acid as well as a comparative study with its corresponding boronic analog. Furthermore, we demonstrate the shorter response time of the borinic trigger compared to the boronic trigger against H₂O₂-mediated oxidation in a cellular environment.     

Rhesus CMV: an emerging animal model for human CMV

Human CMV is the predominant infectious cause of congenital birth defects and an opportunistic pathogen in immunosuppressed individuals, including AIDS patients. Most individuals are infected early during their life followed by life-long latent infection. During this latent phase, frequent reactivation and antigen production continue to stimulate the immune system. While the immune response is able to control the virus, it is unable to eradicate it. Moreover, super-infection by different CMV strains has been observed despite a strong immune response. Long-term immune stimulation by CMV has also been implicated in immune senescence and chronic conditions such as atherosclerosis. CMVs are highly species-specific and the relatedness of CMV genomes exactly mirrors the relatedness of their hosts. Thus, each CMV species is highly adapted to its respective host species, but is unable to infect other, even closely related hosts. While fascinating from an evolutionary perspective, this host restriction prevents studying HCMV in experimental animals. Exceptions are severely immunocompromised mice, e.g. SCID mice, or SCID/NOD mice, which might allow partial reconstitution of CMV infection in rodents. More practical however, is to study CMVs in their natural host, e.g. murine, rat or guinea pig CMVs. However, while these small animal models have many advantages, such as the availability of inbred animals as well as lower cost, the limited homology of the viral genomes with HCMV limits the functional analysis of homologous gene products. The closest relative to HCMV is chimpanzee CMV (CCMV), but this is not a practical animal model since chimps are a protected species, extremely expensive and of very limited availability. In contrast, rhesus macaques are a more widely used experimental animal species and, while more distant than CCMV, rhesus CMV (RhCMV) contains most of the HCMV gene families thus allowing the study of their role in acute and latent CMV infection. In this review we will discuss the current state of developing RhCMV as a model for HCMV.     

Role of the cAMP-binding protein Epac in cardiovascular physiology and pathophysiology

Exchange proteins directly activated by cyclic AMP (Epac) were discovered 10 years ago as new sensors for the second messenger cyclic AMP (cAMP). Epac family, including Epac1 and Epac2, are guanine nucleotide exchange factors for the Ras-like small GTPases Rap1 and Rap2 and function independently of protein kinase A. Given the importance of cAMP in the cardiovascular system, numerous molecular and cellular studies using specific Epac agonists have analyzed the role and the regulation of Epac proteins in cardiovascular physiology and pathophysiology. The specific functions of Epac proteins may depend upon their microcellular environments as well as their expression and localization. This review discusses recent data showing the involvement of Epac in vascular cell migration, endothelial permeability, and inflammation through specific signaling pathways. In addition, we present evidence that Epac regulates the activity of various cellular compartments of the cardiac myocyte and influences calcium handling and excitation–contraction coupling. The potential role of Epac in cardiovascular disorders such as cardiac hypertrophy and remodeling is also discussed.     

Ethical challenges in connection with the use of coercion: a focus group study of health care personnel in mental health care

Background

In recent years, the attention on the use of coercion in mental health care has increased. The use of coercion is common and controversial, and involves many complex ethical challenges. The research question in this study was: What kind of ethical challenges related to the use of coercion do health care practitioners face in their daily clinical work?

Methods

We conducted seven focus group interviews in three mental health care institutions involving 65 multidisciplinary participants from different clinical fields. The interviews were recorded and transcribed verbatim. We analysed the material applying a ‘bricolage’ approach. Basic ethical principles for research ethics were followed. We received permission from the hospitals’ administrations and all health care professionals who participated in the focus group interviews.

Results

Health care practitioners describe ethical dilemmas they face concerning formal, informal and perceived coercion. They provide a complex picture. They have to handle various ethical challenges, not seldom concerning questions of life and death. In every situation, the dignity of the patient is at stake when coercion is considered as morally right, as well as when coercion is not the preferred intervention. The work of the mental health professional is a complicated “moral enterprise”.The ethical challenges deserve to be identified and handled in a systematic way. This is important for developing the quality of health care, and it is relevant to the current focus on reducing the use of coercion and increasing patient participation. Precise knowledge about ethical challenges is necessary for those who want to develop ethics support in mental health care. Better communication skills among health care professionals and improved therapeutic relationships seem to be vital.

Conclusions

A systematic focus on ethical challenges when dealing with coercion is an important step forward in order to improve health care in the mental health field.

     

Classification of anti-FcepsilonRI and anti-IgE autoantibodies in chronic idiopathic urticaria and correlation with disease severity

BACKGROUND: Circulating autoantibodies against FcepsilonRI, IgE, or both occur in approximately one third of patients with chronic idiopathic urticaria (CIU), but not all autoantibodies initiate histamine release. OBJECTIVE: We sought to classify patients with CIU into subsets on the basis of serum bioactivity and immunoreactivity and to examine the relationship between newly defined subtype and disease severity. METHODS: Sera from patients with CIU (n = 78), dermog-raphism (n = 15), and cholinergic urticaria (n = 10) and sera from healthy subjects (n = 39) were analyzed by means of Western blot analysis for anti-FcepsilonRI autoantibodies and for histamine release from basophils and dermal mast cells. In vivo reactivity of autologous serum was tested by means of intradermal injection, and CIU severity was determined on the basis of clinical interview. RESULTS: We classified sera from patients with CIU into 5 subsets: immunoreactive histamine-releasing anti-FcepsilonRI autoantibodies (n = 20 [26%]); immunoreactive anti-FcepsilonRI autoantibodies without histamine-releasing activity (n = 12 [15%]); anti-IgE-like autoantibodies (n = 7 [9%]); serum containing a mast cell-specific histamine-releasing factor (n = 7 [9%]); and sera with no identifiable factor (n = 32 [41%]). Patients with serum histamine-releasing activity had more severe urticaria than patients without such activity. Positive skin test responses to autologous sera were associated with histamine-releasing anti-FcepsilonRI autoantibodies but not with non-histamine-releasing anti-FcepsilonRI autoantibodies. Neither healthy subjects nor patients with dermographism or cholinergic urticaria had his-tamine-releasing anti-FcepsilonRI autoantibodies. CONCLUSION: These data support the specificity of functional anti-FcepsilonRI autoantibodies to CIU. The identification of distinctive subsets of patients suggests that other pathogenic mechanisms occur in CIU in addition to direct ligation of FcepsilonRI by autoantibodies causing dermal mast cell degranulation. Elucidating these mechanisms might lead to new treatments for CIU.     

Outcome of surgical treatment for early adenocarcinoma of the esophagus or gastro-esophageal junction

Adenocarcinoma of the esophagus, or GEJ, has a poor prognosis. Early lesions [i.e. high grade dysplasia (HGD) or T1-carcinoma] are potentially curable. Local endoscopic therapies are promising treatment options for superficial lesions; however, for deeper lesions, surgical resection is considered to be the treatment of choice. To contribute to therapeutic decision-making, we retrospectively analysed the outcome of transhiatal esophagectomy in 120 patients with pathologically proven HGD (n=13) or T1-adenocarcinoma (n=107) of the distal esophagus or gastro-esophageal junction (GEJ). Tumors were subdivided into six different depths of invasion (T1-mucosal m1-m3, T1-submucosal sm1-sm3), and the frequency of lymphatic dissemination and time to locoregional and/or distant recurrence were analysed. Only one of the 79 T1m1-3/sm1 tumors (1%) showed lymph node metastases as compared with 18 out of 41 T1sm2-3 tumors (44%). There was a significant difference in recurrence-free period between T1m1-m3/sm1 versus T1sm2-sm3 tumor patients (P log rank <0.0001), with 5-year recurrence-free percentages of 97% and 57%, respectively. In multivariate analysis including age, gender, tumor differentiation grade, N-stage and depth of invasion, only N-stage was an independent prognostic factor for recurrence-free period (hazard rate=5.9, 95% CI 1.7–20.7). However, if N-stage was excluded from analysis, only depth of invasion (T1sm2-3 versus T1m1-m3/sm1) was an independent prognostic factor for recurrence-free period (hazard rate=7.5, 95% CI 2.0–27.7). These data indicate that T1m1-m3/sm1 adenocarcinomas of esophagus or GEJ show a very low risk of lymphatic dissemination and are therefore eligible for local endoscopic therapy. After transhiatal surgical resection, almost half of the patients with T1sm2-sm3 lesions develop recurrent disease within 5 years, and therefore need additional therapy to improve survival.