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Some evidence suggests that teeth treated with endodontic surgery and considered to have healed over the short term are seen to relapse when evaluated again after 3 or more years. However, long-term evidence is limited. This study compares healing after endodontic microsurgery over long-term (5–9 years) vs middle-term (1–4 years) follow-up and assesses the influence of different healing predictors over time. A retrospective study was made, comparing the endodontic microsurgery healing rates after 1–4 vs 5–9 years of follow-up. Healing was assessed based on clinical and radiographic parameters. Simple binary logistic regression models were used to analyze the influence of patient age and gender, the type of tooth, previous radiographic lesion size, apical extent of previous root canal filling, the presence of a post, type of restoration, and interproximal bone level upon the endodontic microsurgery healing rate. A sensitivity analysis was used excluding cases of vertical root fracture. Two calibrated observers independently evaluated the periapical radiographs. A total of 332 patients (60% women and 40% men) were included in the study. Of the 332 analyzed teeth, 198 were subjected to middle-term follow-up (1–4 years), with a healing rate of 86.9%, while 134 were subjected to long-term follow-up (5–9 years), with a healing rate of 67.2%. There were no statistically significant differences in terms of gender, age, type of tooth, size of the lesion, apical extent of previous root canal filling, presence of a post, or type of restoration. The regression models identified 2 statistically significant associations: cohort and interproximal bone level (P <.05). A success rate of 86.9% was recorded after 1-4 years of follow-up, vs 67.2% after 5–9 years. Excluding cases of vertical root fractures, in the shortest follow-up cohort (1–4 years), the healing rate was 92.5%, vs 82.6% in the cohort with longer follow-up (5–9 years). The prognosis was influenced by the crestal bone level in relation to the cementoenamel junction of the tooth, being significantly poorer when probing depth was >3 mm mesial or distal to the treated tooth. [ABSTRACT FROM AUTHOR]

Objective: The objective of this study was to answer the question: Do conventional radiographs (periapical/panoramic) afford better diagnostic outcomes than cone beam computed tomography (CBCT) as a complement for clinical diagnosis of apical lesions with persistent apical periodontitis or disease after root canal treatment?Study Design: Five electronic databases were searched and provided information to enable construction of a table to determine primary diagnostic measures and secondary parameters. The evidence was appraised with the Quality Assessment of Diagnostic Accuracy Studies tool and GRADEpro software.Results: Twenty-seven articles (9903 diagnostic images) were included. The pooled sensitivity, specificity, area under the receiver operating characteristic curve (AUCROC), positive predictive value, negative predictive value, negative likelihood ratio, and accuracy were 0.58, 1, 0.77, 1, 0.68, 0.45, and 0.79, respectively.Conclusions: Moderate certainty evidence suggested that conventional radiographs showed poor sensitivity and excellent specificity but good diagnostic performance in terms of AUCROC and accuracy. Sensitivity, AUCROC, and negative likelihood ratio values could be reduced if the time elapsed to diagnosis after root canal treatment exceeded 5 years. The use of CBCT with a reduced field of view or a 2D radiographic technique should be weighed considering patient-specific and indication-oriented criteria as taking precedence over the therapeutic goal. [ABSTRACT FROM AUTHOR]

In recent years, the concern derived from the presence of emerging contaminants in the environment and the possible effects on the One Health trilogy has increased. This study determined the concentration of pharmaceutical contaminants of emerging concern and their relationship with the extracellular enzymatic activity of microbial communities from two rivers in western Cuba. Two sampling stations were analyzed; one in the Almendares River (urban) and the other in the San Juan River (rural), taking into account the pollution sources that arrive at these stations and previous physicochemical characterizations. Extracellular protease, acid phosphatase, alkaline phosphatase, lipase, and catalase activities in water and sediments were determined and correlated with contaminants of emerging concern determined by liquid chromatography with mass spectrometry. This study evidenced the presence of different pharmaceutical contaminants found in the categories of antihypertensives, stimulants, anti-inflammatories, and antibiotics in both rivers. Concentrations of contaminants of emerging concern were greater in the Almendares River compared to the San Juan River. In addition, through the canonical redundancy analysis, the influence of these contaminants on the extracellular enzymatic activities of microbial communities was documented, where in most cases they inhibit protease, phosphatase, and lipase activities and enhance catalase activity in response to oxidative stress. The present investigation constitutes the first report in Cuba of the presence of pharmaceutical contaminants of emerging concern and one of the few works that exist in the Latin American region. [Display omitted] • First determination of pharmaceutical emerging contaminants in Cuban rivers. • Antihypertensives, stimulants, antiinflammatories and antibiotics where detected. • Contaminants of emerging concern inhibit protease, phosphatases and lipase activity. • Contaminants of emerging concern enhance catalase activity. [ABSTRACT FROM AUTHOR]

A study was performed of the healing rate of teeth subjected to endodontic microsurgery after a minimum follow-up of 5 years with an analysis of the influence of different pre- and postoperative factors on the outcome. This was a retrospective study of patients subjected to endodontic microsurgery with the use of mineral trioxide aggregate (MTA) for retrograde filling between January 2011 and December 2015. In patients with multiple treated teeth, only 1 random tooth was selected for the statistical analysis. Clinical and radiographic parameters were used to assess healing. Simple binary logistic regression models were used to analyze the influence of patient age and sex, the type of tooth, prior radiographic lesion size, the presence of a post, the type of restoration, and the apical extent of prior filling of the root canal on the endodontic microsurgery success rate. Two calibrated observers evaluated the periapical radiographs on an independent basis. A total of 111 patients (63.1% women and 36.9% men) were included in the study. Of the 111 teeth analyzed, 90 were classified as healed (mean healing rate = 81.1%). Patient age and sex, the presence of a post, the type of tooth, the type of restoration, and the apical extent of prior filling of the root canal had no significant impact on the outcome. Large lesions (>5 mm) were associated with a lower healing rate than smaller lesions, although the difference was not significant. Anterior teeth had a significantly higher healing rate (93.8% maxillary and 100% mandibular) than molars (70.8% maxillary and 57.1% mandibular) (P <.05). The differences between the anterior teeth and the molars were statistically significant. The mean healing rate of teeth subjected to endodontic microsurgery was 81% after 5–9 years of follow-up. The success rate was lower for upper and lower molars than for teeth in the anterior zone, although the sample was small and further studies are needed to establish whether the type of tooth influences the treatment outcome. [ABSTRACT FROM AUTHOR]

Highlights: [•] Univocal evidence for blocking using within-subjects designs have been reported in vertebrates but not in invertebrates. [•] Within-subjects blocking was observed in rats, humans and snails. [•] Blocking seems to be a prevalent phenomenon, and cue competition a characteristic of learning across different animal phyla. [ABSTRACT FROM AUTHOR]

During hematopoiesis, a unique hematopoietic stem cell (HSC) from the bone marrow gives rise to a subset of mature blood cells that directs all the immune responses. Recent studies have shown that this well-defined, hierarchical process is regulated in part by epigenetic mechanisms. Changes in the DNA methylation profile have a critical role in the division of these stem cells into the myeloid and lymphoid lineages and in the establishment of a specific phenotype and functionality in each terminally differentiated cell type. In this review, we describe how the DNA methylation patterns are modified during hematopoietic differentiation and what their role is in cell plasticity and immune function. An in-depth knowledge of these epigenetic mechanisms will help clarify how cell type-specific gene programs are established, and how they can be leveraged in the development of novel strategies for treating immune system-related pathologies. [Copyright &y& Elsevier]

We present the case of a 63-year-old man with a bovine aortic arch variation, who presented episodes of mild hemoptysis secondary to a 4.5-cm (diameter) aneurysm of the innominate artery that compressed the trachea and obliterated the right subclavian artery. Surgery, performed through a median sternotomy, consisted of a bypass from the ascending aorta to both common carotid arteries using a Dacron graft, and exclusion of the aneurysm by ligature and direct thrombin injection. Computed tomography angiography at 30 days showed a patent bypass, successful aneurysm exclusion, and improvement of the tracheal compression. The patient is currently asymptomatic at 12 months following the procedure. [Copyright &y& Elsevier]

Background: Recent evidence regarding carotid revascularization advises against carotid angioplasty and stenting (CAS) in patients aged >70 years with conventional risk for carotid endarterectomy (CEA). The poor outcome of transfemoral CAS in this age group may be explained by the anatomic characteristics of the aortic trunk and supra-aortic vessels in elderly patients, as well as by a high prevalence of aortic arch atheromatosis. Transcervical CAS with flow reversal for cerebral protection avoids these unfavorable characteristics. This study analyzed the short-term and middle-term results of transcervical CAS with flow reversal in patients aged >70 years at high risk for CEA. Methods: Between January 2006 and January 2011, 219 cases of >70% carotid artery stenosis in high-risk patients aged >70 years (55.7% asymptomatic and 44.3% symptomatic) were treated by transcervical CAS. All patients underwent complete neurologic examination by a stroke neurologist before and after the procedure. Primary end points were stroke, death, or myocardial infarction (MI), technical success, and complications at 30 days. During follow-up, we analyzed the rate of restenosis ≥50% and ipsilateral stroke. Data were collected prospectively and outcome was analyzed in all cases, including technical failures. Results: The 30-day combined stroke/death/MI rate was 2.2% (stroke, 1.8%; stroke/death, 2.2%; and MI, 0.45%). In symptomatic patients, stroke/death/MI was 5.1% (stroke, 4.1%; stroke/death, 5.1%). None of the asymptomatic patients suffered stroke, MI, or death postoperatively. Technical success was 96.3% (four inability to cross lesion, two major common carotid dissections, one failed preangioplasty, one stent thrombosis). One cervical hematoma required surgical drainage. At follow-up (18.8 ± 16.9 months), cumulative (standard error) incidence of >70% restenosis was 3% (1%) at 1 year and 8% (3%) at 2 and 3 years. Only one patient experienced ipsilateral stroke during follow-up. Overall survival (standard error) was 94% (2%) at 1 year and 90% (3%) at 2 and 3 years. Conclusions: In our experience, transcervical CAS with flow reversal is a safe technique for treating carotid stenosis in patients aged >70 years. We believe that avoiding the aortic arch and tortuous supra-aortic vessels is responsible for the favorable results in this study. [ABSTRACT FROM AUTHOR]

Abstract: In a 2-D computer based search task, human participants were exposed to a compound stimulus containing both geometric and non-geometric information (a rectangle with colored walls) in such a way that a non-geometric cue, C1, was paired with a geometric cue, G1. Previous reinforcement of either kind of cue (geometric and non-geometric) resulted in second order conditioning (SOC) when the participants were tested with the cue that was never paired with reinforcement (Experiment 1). Similarly, if one of the cues was reinforced following the non-reinforced exposure to the compound, a sensory preconditioning (SPC) effect was observed (Experiment 3). These results show that associations can be formed between geometric and non-geometric cues, a finding that is incompatible with the concept of a geometric module impenetrable to non-geometric information. In Experiments 2 and 4, we found evidence for SOC and SPC using exclusively geometric cues, suggesting that the associative learning principles that apply to other domains also rule spatial geometry learning in humans. This research suggests that spatial representations can be enlarged by successively integrating information bits through the linkage of common elements. [Copyright &y& Elsevier]

The case of a 17-year-old adolescent boy with severe polytrauma is reported. Among other injuries, he presented with aortic rupture distal to the origin of the subclavian artery with no bleeding into the mediastinum. The lesion was repaired by placement of a Cook TX2 endovascular graft (Cook Incorporated, Bloomington, Ind). One year later, he was hospitalized with acute heart failure. Computed tomography angiography showed nearly complete stent graft occlusion and no evidence of altered integrity of the device. A right axillofemoral bypass was performed, allowing conversion to successful definitive repair consisting of an extra-anatomic bypass from the ascending aorta to the supraceliac abdominal aorta. [Copyright &y& Elsevier]

The activating killer cell lectin-like receptor NKG2D plays a key role in the natural killer (NK) cell-mediated lysis of tumours and infected cells. Unlike other receptors, the ligands recognised by NKG2D are ‘induced-self’ ligands on stressed cells. This system requires precise regulation because inappropriate expression of NKG2D ligands might compromise NK cell activation. For therapeutic purposes it is essential to understand the mechanisms that regulate the expression and function of the NKG2D system. This review focuses on the importance of the signalling pathways involved in the regulation of the NKG2D receptor and its ligand expression in arming the immune response against infected or tumour cells and for the identification of new molecular targets and therapeutic strategies. [Copyright &y& Elsevier]

Background: The use of carotid stenting in octogenarian patients is controversial; some authors consider this population at high risk for the procedure. Anatomic vascular complexity may be an important reason for the high reported rates of periprocedural thromboembolic complications. Transcervical carotid angioplasty and stenting (TCS) with flow reversal avoids aortic arch instrumentation. In this study, we analyzed our experience with TCS in octogenarian patients and compared the results with those of carotid endarterectomy (CEA) in the same age group in terms of safety. Methods: The study included 81 patients, ≥80 years, a retrospective cohort of 45 consecutive patients treated with CEA (January 2002 to January 2005), and a prospective cohort of 36 consecutive patients treated with TCS with protective flow reversal (January 2005 to January 2007). Patients were considered symptomatic according to the North American Symptomatic Carotid Endarterectomy Trial (NASCET) criteria. Stenting indication was established on the SAPPHIRE criteria. General anesthesia was used in patients undergoing CEA, and local anesthesia in those receiving TCS. Primary endpoints were: stroke, death, or acute myocardial infarction within 30 days. Secondary endpoints were peripheral nerve paralysis and cervical hematoma. Statistical significance for between-group differences was assessed by Pearson χ2 or Fisher exact test, and Student t test. A P value of <.05 was considered statistically significant. Follow-up was limited to 30 days. Results: Baseline epidemiological characteristics and revascularization indications were similar between both groups. Mean age was significantly higher in the TCS group (83.5 ± 3.35) than the CEA group (81.7 ± 1.55) (P = .004). Percentage of symptomatic lesions was similar: 30.6% in TCS vs 44.4% in CEA (P = .2). Comorbid conditions (respiratory or cardiac) were more frequent in TCS group (61.6% vs 26.6%; P = .002). There were no significant differences between groups for the primary endpoints: 4.4% (one stroke, one acute myocardial infarction) for CEA vs 0% for TCS (P = .5). Among CEA patients, there were two peripheral nerve palsies (4.4%) and one cervical hematoma (2.2%); there were no such complications with TCS (P = .5 and P = 1, respectively). In one asymptomatic TCS patient, Doppler study at 24 hours following the procedure showed a common carotid artery dissection, which was treated by a common carotid to internal carotid bypass. Conclusions: In this preliminary experience, transcervical carotid angioplasty and stenting with flow reversal for cerebral protection was as safe at short term as carotid endarterectomy in octogenarian patients, who additionally had considerable comorbidity; thus, it may be possible to extend the indications for carotid revascularization in this population. Studies in larger patient series are required to confirm the trends observed in this study. [Copyright &y& Elsevier]

Background: Carotid angioplasty and stenting (CAS) with cerebral embolic protection is a safe alternative to carotid endarterectomy in high-risk patients. Among the various systems proposed for cerebral protection, transcervical CAS avoids crossing the lesion without protection and eliminates the complications associated with transfemoral access. This study analyzes our experience and the results obtained with a transcervical stenting technique for carotid revascularization. Methods: From January 2005 to June 2006, 62 CAS were performed in our center in high-risk patients with >70% stenosis (38.7% had a previous neurologic event and 61.3% were asymptomatic). The indications for CAS were severe heart disease (45.1%), severe pulmonary disease (6.4%), paralysis of the contralateral laryngeal nerve (6.4%), recurrent stenosis (3.2%), and high carotid lesion (1.6%). Twenty-one patients were >80 years old. A complete neurologic examination was performed by a stroke neurologist in all patients before and after stenting. The protection system used was carotid flow reversal by transcervical access. Transcranial Doppler monitoring was done during the procedure in 35 patients. We analyzed technical success, the presence of high-intensity transient signals during the procedure, neurologic morbidity and mortality at 30 days and 6 months, and stent patency at 6 months (range, 1 to 18 months). Technical success was 96.8%. Perioperative high-intensity transient signals were observed in two patients (5.7%). In the immediate postoperative period, one patient had a transient ischemic attack of the anterior cerebral artery and another had a stroke, with contralateral hemiplegia. At 48 hours after discharge, a third patient returned to the hospital with a severe cerebral hemorrhage that required surgical drainage; hence, neurologic morbidity was 4.9%. There were no deaths at 6 months. Among the total, 98.4% of the stents remained patent, two showed restenosis of 50% to 70%, and one restenosis of >70%. No patients presented a neurologic event during the follow-up. Conclusions: Transcervical carotid artery stenting with flow reversal cerebral protection is a relatively simple, safe technique that avoids instrumentation of the aortic arch and crossing the target lesion without protection. It is less expensive than techniques requiring a filter device and provides excellent outcome with an acceptable incidence of complications. [Copyright &y& Elsevier]

Human recombinant copper–zinc superoxide dismutase (CuZnSOD) was inactivated by peroxynitrite, the product of the reaction between nitric oxide and superoxide. The concentration of peroxynitrite that decreased the activity by 50% (IC50) was ∼100 μM at 5 μM CuZnSOD and the inactivation was higher at alkaline pH. Stopped-flow determinations showed that the second-order rate constant for the direct reaction of peroxynitrite with CuZnSOD was (9.4 ± 1.0) × 103 M-1 s-1 per monomer at pH 7.5 and 37°C. Addition of peroxynitrite (1 mM) to CuZnSOD (0.5 mM) in the presence of the spin trap 2-methyl-2-nitrosopropane led to the electron paramagnetic resonance detection of an anisotropic signal typical of a protein radical adduct. Treatment with Pronase revealed a nearly isotropic signal consistent with the formation of histidinyl radical. The effects of nitrite, hydrogen peroxide, bicarbonate, and mannitol on the inactivation were assessed. Considering the mechanism accepted for the reaction of CuZnSOD with hydrogen peroxide and the fact that CuZnSOD promotes the nitration of phenolics by peroxynitrite, we herein propose that peroxynitrite reacts with CuZnSOD leading to nitrogen dioxide plus a copper-bound hydroxyl radical species that reacts with histidine residues, forming histidinyl radical. [Copyright &y& Elsevier]

Cells must increase their mass in coordination with cell cycle progression to ensure that their size and macromolecular composition remain constant for any given proliferation rate. To this end, growth factors activate early signaling cascades that simultaneously promote cell mass increase and induce cell cycle entry. Nonetheless, the mechanism that controls the concerted regulation of cell growth and cell cycle entry in mammals remains unknown. The phosphatidylinositol 3-kinase (PI3K)/protein kinase B pathway regulates cell cycle entry by inactivating forkhead transcription factors and promoting cyclin D synthesis. PI3K/protein kinase B-derived signals also affect activation of p70 S6 kinase and the mammalian target of rapamycin, enzymes involved in cell growth control. We previously showed that enhancement of PI3K activation accelerates cell cycle entry, whereas reduction of PI3K activation retarded this process. Here we examined whether expression of different PI3K mutants affects cell growth during cell division. We show that diminishing or enhancing the magnitude of PI3K activation in a transient manner reduces or increases, respectively, the protein synthesis rate. Alteration of cell growth and cell cycle entry by PI3K forms appears to be concerted, because it results in lengthening or shortening of cell division time without altering cell size. In support of a central role for PI3K in growth control, expression of a deregulated, constitutive active PI3K mutant affects p70 S6 kinase and mammalian target of rapamycin activities and increases cell size. Together, the results show that transient PI3K activation regulates cell growth and cell cycle in a coordinated manner, which in turn controls cell division time. [ABSTRACT FROM AUTHOR]

Highlights • This is a case of a WHO Grade I meningioma excised in an uncomplicated operation. • 13 months later the patient presented with subcutaneous metastases. • Proximity was close to her original scar, suggesting intra-operative seeding. • Risk factors and treatment options for this rare occurrence are reviewed. Abstract Meningiomas are the most frequent primary brain tumours and are often managed with surgical excision. We present the case of a young woman with the unusual phenomenon of iatrogenic subcutaneous seeding from an intracranial meningioma. We discuss the risk factors, possible mechanisms and management of this. [ABSTRACT FROM AUTHOR]

Children who receive cranial radiation therapy (RT) as a component of treatment for malignancy are often at risk of long-term central endocrine toxicity secondary to radiation to the hypothalamic-pituitary axis (HPA). A comprehensive analysis was performed of central endocrine late effects in survivors of childhood cancer treated with RT as part of the Pediatric Normal Tissue Effects in the Clinic (PENTEC) consortium. A systematic review of the risk of RT-related central endocrine effects was performed in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). A total of 4629 publications were identified, of which 16 met criteria for inclusion in dose modeling analysis, with a total of 570 patients in 19 cohorts. Eighteen cohorts reported outcomes for growth hormone deficiency (GHD), 7 reported outcomes for central hypothyroidism (HT), and 6 reported outcomes for adrenocorticotropic hormone (ACTH) deficiency. Normal tissue complication probability modeling for GHD (18 cohorts, 545 patients) yielded D 50 = 24.9 Gy (95% CI, 20.9-28.0) and γ 50 = 0.5 (95% CI, 0.27-0.78). The normal tissue complication probability model fit for whole brain irradiation in children with a median age of >5 years indicated a 20% risk of GHD for patients who receive a mean dose of 21 Gy in 2-Gy fractions to the HPA. For HT, among 7 cohorts (250 patients), D 50 = 39 Gy (95% CI, 34.1-53.2) and γ 50 = 0.81 (95% CI, 0.46-1.35), with a 20% risk of HT in children who receive a mean dose of 22 Gy in 2-Gy fractions to the HPA. For ACTH deficiency (6 cohorts, 230 patients), D 50 = 61 Gy (95% CI, 44.7-119.4) and γ 50 = 0.76 (95% CI, 0.5-1.19); there is a 20% risk of ACTH deficiency in children who receive a mean dose of 34 Gy in 2-Gy fractions to the HPA. RT dose to the HPA increases the risk of central endocrine toxicity, including GHD, HT, and ACTH deficiency. In some clinical situations, these toxicities may be difficult to avoid, and counseling of patients and families with respect to anticipated outcomes is important. [ABSTRACT FROM AUTHOR]

In recent years, the macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage CSF (GM-CSF) cytokines have been identified as opposing regulators of the inflammatory program. However, the two cytokines are simultaneously present in the inflammatory milieu, and it is not clear how cells integrate these signals. In order to understand the regulatory networks associated with the GM/M-CSF signaling axis, we analyzed DNA methylation in human monocytes. Our results indicate that GM-CSF induces activation of the inflammatory program and extensive DNA methylation changes, while M-CSF-polarized cells are in a less differentiated state. This inflammatory program is mediated via JAK2 associated with the GM-CSF receptor and the downstream extracellular signal-regulated (ERK) signaling. However, PI3K signaling is associated with a negative regulatory loop of the inflammatory program and M-CSF autocrine signaling in GM-CSF-polarized monocytes. Our findings describe the regulatory networks associated with the GM/M-CSF signaling axis and how they contribute to the establishment of the inflammatory program associated with monocyte activation. • GM-CSF and M-CSF show opposing actions that are reflected at the transcriptional level • GM-CSF and M-CSF induce permanent DNA methylation changes during monocyte polarization • Chromatin acts as an integration node, incorporating multiple inflammatory signals Rodriguez et al. show that GM-CSF and M-CSF give rise to opposing phenotypes in the context of inflammation. Inflammation induced by GM-CSF is mediated by JAK2 and the downstream MAPK signaling pathway. However, PI3K signaling is associated with a negative regulatory loop of this program and the stimulation of autocrine production of M-CSF. [ABSTRACT FROM AUTHOR]

Persulfides (RSSH/RSS−) participate in sulfur metabolism and are proposed to transduce hydrogen sulfide (H2S) signaling. Their biochemical properties are poorly understood. Herein, we studied the acidity and nucleophilicity of several low molecular weight persulfides using the alkylating agent, monobromobimane. The different persulfides presented similar pKa values (4.6–6.3) and pH-independent rate constants (3.2–9.0 × 10³ M−1 s−1), indicating that the substituents in persulfides affect properties to a lesser extent than in thiols because of the larger distance to the outer sulfur. The persulfides had higher reactivity with monobromobimane than analogous thiols and putative thiols with the same pKa, providing evidence for the alpha effect (enhanced nucleophilicity by the presence of a contiguous atom with high electron density). Additionally, we investigated two enzymes from the human mitochondrial H2S oxidation pathway that form catalytic persulfide intermediates, sulfide quinone oxidoreductase and thiosulfate sulfurtransferase (TST, rhodanese). The pH dependence of the activities of both enzymes was measured using sulfite and/or cyanide as sulfur acceptors. The TST half-reactions were also studied by stopped-flow fluorescence spectroscopy. Both persulfidated enzymes relied on protonated groups for reaction with the acceptors. Persulfidated sulfide quinone oxidoreductase appeared to have a pKa of 7.8 ± 0.2. Persulfidated TST presented a pKa of 9.38 ± 0.04, probably due to a critical active site residue rather than the persulfide itself. The TST thiol reacted in the anionic state with thiosulfate, with an apparent pKa of 6.5 ± 0.1. Overall, our study contributes to a fundamental understanding of persulfide properties and their modulation by protein environments. [ABSTRACT FROM AUTHOR]

It is well established that proteins and peptides can release sulfur under alkaline treatment, mainly through the β-elimination of disulfides with the concomitant formation of persulfides and dehydroalanine derivatives. In this study, we evaluated the formation of glutathione persulfide (GSSH/GSS-) by exposure of glutathione disulfide (GSSG) to alkaline conditions. The kinetics of the reaction between GSSG and HO- was investigated by UV–Vis absorbance, reaction with 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB), and cold cyanolysis, obtaining an apparent second-order rate constant of ∼10-3 M-1 s-1 at 25 °C. The formation of GSSH and the dehydroalanine derivative was confirmed by HPLC and/or mass spectrometry. However, the mixtures did not equilibrate in a timescale of hours, and additional species, including thiol and diverse sulfane sulfur compounds were also formed, probably through further reactions of the persulfide. Cold cyanolysis is frequently used to quantify persulfides, since it measures sulfane sulfur. This method involves a step in which the sample to be analyzed is incubated with cyanide at alkaline pH. When cold cyanolysis was applied to samples containing GSSG, sulfane sulfur products that were not present in the original sample were measured. Thus, our results reveal the risk of overestimating the amount of sulfane sulfur compounds in samples that contain disulfides due to their decay to persulfides and other sulfane sulfur compounds at alkaline pH. Overall, our study highlights that the β-elimination of disulfides is a potential source of persulfides, although we do not recommend the preparation of GSSH from incubation of GSSG in alkali. Our study also highlights the importance of being cautious when doing and interpreting cold cyanolysis experiments. [Display omitted] • Glutathione disulfide and HO- form persulfide and dehydroalanine derivatives. • They react by a β-elimination mechanism with a rate constant of ∼10-3 M-1 s-1. • Additional species formed include thiol and diverse sulfane sulfur compounds. • We do not recommend preparing persulfides by incubating disulfides in alkali. • Overestimations in cold cyanolysis can occur when the samples contain disulfides. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H2S) is produced endogenously by several enzymatic pathways and modulates physiological functions in mammals. Quantification of H2S in biochemical systems remains challenging because of the presence of interferents with similar reactivity, particularly thiols. Herein, we present a new quantification method based on the formation of pyrene excimers in solution. We synthesized the probe 2-(maleimido)ethyl 4-pyrenylbutanoate (MEPB) and determined that MEPB reacted with H2S in a two-step reaction to yield the thioether-linked dimer (MEPB) 2S, which formed excimers upon excitation, with a broad peak of fluorescence emission centered at 480 nm. In contrast, we found that the products formed with thiols showed peaks at 378 and 398 nm. The difference in emission between the products prevented the interference. Furthermore, we showed that the excimer fluorescence signal yielded a linear response to H2S, with a limit of detection of 54 nM in a fluorometer. Our quantification method with MEPB was successfully applied to follow the reaction of H2S with glutathione disulfide and to quantify the production of H2S from cysteine by Escherichia coli. In conclusion, this method represents an addition to the toolkit of biochemists to quantify H2S specifically and sensitively in biochemical systems. [ABSTRACT FROM AUTHOR]

T cell lymphopoiesis is a complex, stepwise process in which the transcriptional program of the progenitor cells is progressively adapted in order to generate mature phenotypes. This transcriptional program in differentiated cells is also very flexible, allowing the silencing or activation of critical genes in response to extrinsic or intrinsic stimuli, or, in the case of progenitors, to developmental signals. Thus, progenitor and mature cells must maintain a balance between stability, to preserve their phenotypic identity, and plasticity, to respond and adapt to stimuli. A long-standing question is, therefore, how the transcriptional program is regulated to allow both controlled differentiation and a flexible response. Here we review the contribution of epigenetic mechanisms to transcriptional control during CD4 + T cell differentiation and the ways in which these mechanisms interact with key transcription factors to ensure proper maturation and maintenance of cell identity. This article is part of a Directed Issue entitled: Epigenetics dynamics in development and disease. [ABSTRACT FROM AUTHOR]

NKG2D is an activating receptor expressed by NK and T cells. NKG2D ligands show a restricted expression in normal tissues, but they are frequently overexpressed in cancer and infected cells. The binding of NKG2D to its ligands activates NK and T cells and promotes cytotoxic lysis of the cells expressing these molecules. The mechanisms involved in the expression of the ligands of NKG2D play a key role in the recognition of stressed cells by the immune system and represent a promising therapeutic target for improving the immune response against cancer or autoimmune disease. In this review, we analyse the recent advances in understanding the regulation of NKG2D ligand expression and their therapeutic implications. [Copyright &y& Elsevier]

Human recombinant MnSOD and CuZnSOD were both inactivated when exposed to simultaneous fluxes of superoxide (JO2・−) and nitric oxide (J・NO). The inactivation was also observed with varying J・NO/JO2・− ratios. Protein-derived radicals were detected in both CuZn and MnSOD by immuno-spin trapping. The formation of protein radicals was followed by tyrosine nitration in the case of MnSOD. When MnSOD was exposed to J・NO and JO2・− in the presence of uric acid, a scavenger of peroxynitrite-derived free radicals, nitration was decreased but inactivation was not prevented. On the other hand, glutathione, known to react with both peroxynitrite and nitrogen dioxide, totally protected MnSOD from inactivation and nitration on addition of authentic peroxynitrite but, notably, it was only partially inhibitory in the presence of the more biologically relevant J・NO and JO2・−. The data are consistent with the direct reaction of peroxynitrite with the Mn center and a metal-catalyzed nitration of Tyr-34 in MnSOD. In this context, we propose that inactivation is also occurring through a ・NO-dependent nitration mechanism. Our results help to rationalize MnSOD tyrosine nitration observed in inflammatory conditions in vivo in the presence of low molecular weight scavengers such as glutathione that otherwise would completely consume nitrogen dioxide and prevent nitration reactions. [Copyright &y& Elsevier]

Targeting epigenetic mechanisms has emerged as a potential therapeutic approach for the treatment of kidney diseases. Specifically, inhibiting the bromodomain and extra-terminal (BET) domain proteins using the small molecule inhibitor JQ1 has shown promise in preclinical models of acute kidney injury (AKI) and chronic kidney disease (CKD). However, its clinical translation faces challenges due to issues with poor pharmacokinetics and side effects. Here, we developed engineered liposomes loaded with JQ1 with the aim of enhancing kidney drug delivery and reducing the required minimum effective dose by leveraging cargo protection. These liposomes efficiently encapsulated JQ1 in both the membrane and core, demonstrating superior therapeutic efficacy compared to freely delivered JQ1 in a mouse model of kidney ischemia-reperfusion injury. JQ1-loaded liposomes (JQ1-NPs) effectively targeted the kidneys and only one administration, one-hour after injury, was enough to decrease the immune cell (neutrophils and monocytes) infiltration to the kidney—an early and pivotal step to prevent damage progression. By inhibiting BRD4, JQ1-NPs suppress the transcription of pro-inflammatory genes, such as cytokines (il-6) and chemokines (ccl2, ccl5). This success not only improved early the kidney function, as evidenced by decreased serum levels of BUN and creatinine in JQ1-NPs-treated mice, along with reduced tissue expression of the damage marker, NGAL, but also halted the production of extracellular matrix proteins (Fsp-1, Fn-1, α-SMA and Col1a1) and the fibrosis development. In summary, this work presents a promising nanotherapeutic strategy for AKI treatment and its progression and provides new insights into renal drug delivery. [Display omitted] • JQ1-NPs circumvent its poor pharmacokinetics and severe side effects of this inhibitor. • JQ1-NPs reach efficiently the kidney of mice with acute kidney injury (AKI). • Administration of JQ1-NPs avoid the AKI induced by bilateral kidney IRI. • JQ1-NPs significantly reduce the recruitment of neutrophils and monocytes to the damaged kidney. • The renal fibrosis development and CKD progression is halted by JQ1-NPs treatment. [ABSTRACT FROM AUTHOR]

Human serum albumin (HSA) contains 17 disulfides and only one reduced cysteine, Cys34, which can be oxidized to a relatively stable sulfenic acid (HSA-SOH). This derivative has been previously detected and quantified. However, its properties are poorly understood. Herein, HSA-SOH formation from the exposure of HSA to hydrogen peroxide was confirmed using the sulfenic acid probe bicyclo [6.1.0]nonyne-biotin (BCN-Bio1), and by direct detection by whole protein mass spectrometry. The decay pathways of HSA-SOH were studied. HSA-SOH reacted with a thiol leading to the formation of a mixed disulfide. The reaction occurred through a concerted or direct displacement mechanism (S N 2) with the thiolate (RS−) as nucleophile towards HSA-SOH. The net charge of the thiolate affected the value of the rate constant. In the presence of hydrogen peroxide, HSA-SOH was further oxidized to sulfinic acid (HSA-SO 2 H) and sulfonic acid (HSA-SO 3 H). The rate constants of these reactions were estimated. Lastly, HSA-SOH spontaneously decayed in solution. Mass spectrometry experiments suggested that the decay product is a sulfenylamide (HSA-SN(R′)R″). Chromatofocusing analysis showed that the overoxidation with hydrogen peroxide predominates at alkaline pH whereas the spontaneous decay predominates at acidic pH. The present findings provide insights into the reactivity and fate of the sulfenic acid in albumin, which are also of relevance to numerous sulfenic acid-mediated processes in redox biology and catalysis. [Display omitted] • A sulfenic acid is formed in human serum albumin upon exposure to hydrogen peroxide. • Once formed, sulfenic acid can follow one of three possible routes. • Sulfenic acid reacts with thiols leading to the formation of mixed disulfides. • Sulfenic acid is further oxidized by hydrogen peroxide to sulfinic or sulfonic acid. • Sulfenic acid decays spontaneously, likely forming a sulfenylamide. [ABSTRACT FROM AUTHOR]

Persulfides (RSSH/RSS2) participate in sulfur trafficking and metabolic processes, and are proposed to mediate the signaling effects of hydrogen sulfide (H2S). Despite their growing relevance, their chemical properties are poorly understood. Herein, we studied experimentally and computationally the formation, acidity, and nucleophilicity of glutathione persulfide (GSSH/GSS2), the derivative of the abundant cellular thiol glutathione (GSH). We characterized the kinetics and equilibrium of GSSH formation from glutathione disulfide and H2S. A pKa of 5.45 for GSSH was determined, which is 3.49 units below that of GSH. The reactions of GSSH with the physiologically relevant electrophiles peroxynitrite and hydrogen peroxide, and with the probe monobromobimane, were studied and compared with those of thiols. These reactions occurred through SN2 mechanisms. At neutral pH, GSSH reacted faster than GSH because of increased availability of the anion and, depending on the electrophile, increased reactivity. In addition, GSS2 presented higher nucleophilicity with respect to a thiolate with similar basicity. This can be interpreted in terms of the so-called a effect, i.e. the increased reactivity of a nucleophile when the atom adjacent to the nucleophilic atom has high electron density. The magnitude of the a effect correlated with the Brønsted nucleophilic factor, bnuc, for the reactions with thiolates and with the ability of the leaving group. Our study constitutes the first determination of the pKa of a biological persulfide and the first examination of thea effect in sulfur nucleophiles, and sheds light on the chemical basis of the biological properties of persulfides. [ABSTRACT FROM AUTHOR]

The free radical nitric oxide (NO) exerts biological effects through the direct and reversible interaction with specific targets (e.g. soluble guanylate cyclase) or through the generation of secondary species, many of which can oxidize, nitrosate or nitrate biomolecules. The NO-derived reactive species are typically short-lived, and their preferential fates depend on kinetic and compartmentalization aspects. Their detection and quantification are technically challenging. In general, the strategies employed are based either on the detection of relatively stable end products or on the use of synthetic probes, and they are not always selective for a particular species. In this study, we describe the biologically relevant characteristics of the reactive species formed downstream from NO, and we discuss the approaches currently available for the analysis of NO, nitrogen dioxide (NO2), dinitrogen trioxide (N2O3), nitroxyl (HNO), and peroxynitrite (ONOO/ONOOH), as well as peroxynitrite-derived hydroxyl (HO) and carbonate anion (CO3) radicals. We also discuss the biological origins of and analytical tools for detecting nitrite (NO2), nitrate (NO3), nitrosyl-metal complexes, S-nitrosothiols, and 3-nitrotyrosine. Moreover, we highlight state- of-the-art methods, alert readers to caveats of widely used techniques, and encourage retirement of approaches that have been supplanted by more reliable and selective tools for detecting and measuring NO-derived oxidants. We emphasize that the use of appropriate analytical methods needs to be strongly grounded in a chemical and biochemical understanding of the species and mechanistic pathways involved. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H2S) participates in prokaryotic metabolism and is associated with several physiological functions in mammals. H2S reacts with oxidized thiol derivatives (i.e. disulfides and sulfenic acids) and thereby forms persulfides, which are plausible transducers of the H2S-mediated signaling effects. The one-cysteine peroxiredoxin alkyl hydroperoxide reductase E from Mycobacterium tuberculosis (MtAhpE-SH) reacts fast with hydroperoxides, forming a stable sulfenic acid (MtAhpE-SOH), which we chose here as a model to study the interactions between H2S and peroxiredoxins (Prx). MtAhpE-SOH reacted with H2S, forming a persulfide (MtAhpE-SSH) detectable by mass spectrometry. The rate constant for this reaction was (1.4 © 0.2) × 103 M -1 s-1 (pH 7.4, 25 °C), six times higher than that reported for the reaction with the main low-molecularweight thiol in M. tuberculosis, mycothiol.H2S was able to complete the catalytic cycle of MtAhpE and, according to kinetic Hydrogen sulfide (H2S) participates in prokaryotic metabolism and is associated with several physiological functions in mammals. H2S reacts with oxidized thiol derivatives (i.e. disulfides and sulfenic acids) and thereby forms persulfides, which are plausible transducers of the H2S-mediated signaling effects. The one-cysteine peroxiredoxin alkyl hydroperoxide reductase E from Mycobacterium tuberculosis (MtAhpE-SH) reacts fast with hydroperoxides, forming a stable sulfenic acid (MtAhpE-SOH), which we chose here as a model to study the interactions between H2S and peroxiredoxins (Prx). MtAhpE-SOH reacted with H2S, forming a persulfide (MtAhpE-SSH) detectable by mass spectrometry. The rate constant for this reaction was (1.4 © 0.2) × 103 M -1 s-1 (pH 7.4, 25 °C), six times higher than that reported for the reaction with the main low-molecularweight thiol in M. tuberculosis, mycothiol.H2S was able to complete the catalytic cycle of MtAhpE and, according to kinetic considerations, it could represent an alternative substrate in M. tuberculosis. MtAhpE-SSH reacted 43 times faster than did MtAhpE-SH with the unspecific electrophile 4,4'-dithiodipyridine, a disulfide that exhibits no preferential reactivity with peroxidatic cysteines, but MtAhpE-SSH was less reactive toward specific Prx substrates such as hydrogen peroxide and peroxynitrite. According to molecular dynamics simulations, this loss of specific reactivity could be explained by alterations in the MtAhpE active site. MtAhpE-SSH could transfer its sulfane sulfur to a low-molecular-weight thiol, a process likely facilitated by the low pKa of the leaving thiolMtAhpE-SH, highlighting the possibility that Prx participates in transpersulfidation. The findings of our study contribute to the understanding of persulfide formation and reactivity. [ABSTRACT FROM AUTHOR]

Human serum albumin (HSA) has a single reduced cysteine residue, Cys34, whose acidity has been controversial. Three experimental approaches (pH-dependence of reactivity towards hydrogen peroxide, ultraviolet titration and infrared spectroscopy) are used to determine that the p K a value in delipidated HSA is 8.1±0.2 at 37 °C and 0.1 M ionic strength. Molecular dynamics simulations of HSA in the sub-microsecond timescale show that while sulfur exposure to solvent is limited and fluctuating in the thiol form, it increases in the thiolate, stabilized by a persistent hydrogen-bond (HB) network involving Tyr84 and bridging waters to Asp38 and Gln33 backbone. Insight into the mechanism of Cys34 oxidation by H 2 O 2 is provided by ONIOM(QM:MM) modeling including quantum water molecules. The reaction proceeds through a slightly asynchronous S N 2 transition state (TS) with calculated Δ ‡ G and Δ ‡ H barriers at 298 K of respectively 59 and 54 kJ mol −1 (the latter within chemical accuracy from the experimental value). A post-TS proton transfer leads to HSA–SO − and water as products. The structured reaction site cages H 2 O 2 , which donates a strong HB to the thiolate. Loss of this HB before reaching the TS modulates Cys34 nucleophilicity and contributes to destabilize H 2 O 2 . The lack of reaction-site features required for differential stabilization of the TS (positive charges, H 2 O 2 HB strengthening) explains the striking difference in kinetic efficiency for the same reaction in other proteins (e.g. peroxiredoxins). The structured HB network surrounding HSA–SH with sequestered waters carries an entropic penalty on the barrier height. These studies contribute to deepen the understanding of the reactivity of HSA–SH, the most abundant thiol in human plasma, and in a wider perspective, provide clues on the key aspects that modulate thiol reactivity against H 2 O 2 . [ABSTRACT FROM AUTHOR]

Nitroalkene fatty acids are formed in vivo and exert protective and anti-inflammatory effects via reversible Michael addition to thiol-containing proteins in key signaling pathways. Nitro-conjugated linoleic acid (NO2-CLA) is preferentially formed, constitutes the most abundant nitrated fatty acid in humans, and contains two carbons that could potentially react with thiols, modulating signaling actions and levels. In this work, we examined the reactions of NO2-CLA with low molecular weight thiols (glutathione, cysteine, homocysteine, cysteinylglycine, and β-mercaptoethanol) and human serum albumin. Reactions followed reversible biphasic kinetics, consistent with the presence of two electrophilic centers in NO2-CLA located on the β- and δ-carbons with respect to the nitro group. The differential reactivity was confirmed by computational modeling of the electronic structure. The rates (kon and koff) and equilibrium constants for both reactions were determined for different thiols. LC-UV-Visible and LC-MS analyses showed that the fast reaction corresponds to β-adduct formation (the kinetic product), while the slow reaction corresponds to the formation of the δ-adduct (the thermodynamic product). The pH dependence of the rate constants, the correlation between intrinsic reactivity and thiol pKa, and the absence of deuterium solvent kinetic isotope effects suggested stepwise mechanisms with thiolate attack on NO2-CLA as rate-controlling step. Computational modeling supported the mechanism and revealed additional features of the transition states, anionic intermediates, and final neutral products. Importantly, the detection of cysteine-δ-adducts in human urine provided evidence for the biological relevance of this reaction. Finally, human serum albumin was found to bind NO2-CLA both non-covalently and to form covalent adducts at Cys-34, suggesting potential modes for systemic distribution. These results provide new insights into the chemical basis of NO2-CLA signaling actions. [ABSTRACT FROM AUTHOR]

Cystathionine β-synthase (CBS) is a pyridoxal phosphate-dependent enzyme that catalyzes the condensation of homocysteine with serine or with cysteine to form cystathionine and either water or hydrogen sulfide, respectively. Human CBS possesses a noncatalytic heme cofactor with cysteine and histidine as ligands, which in its oxidized state is relatively unreactive. Ferric CBS (Fe(III)-CBS) can be reduced by strong chemical and biochemical reductants to Fe(II)-CBS, which can bind carbon monoxide (CO) or nitric oxide (NO.), leading to inactive enzyme. Alternatively, Fe(II)-CBS can be reoxidized by O2 to Fe(III)-CBS, forming superoxide radical anion (O2.¯). In this study, we describe the kinetics of nitrite (NO2-) reduction by Fe(II)-CBS to form Fe(II)NO.-CBS. The second order rate constant for the reaction of Fe(II)-CBS with nitrite was obtained at low dithionite concentrations. Reoxidation of Fe(II)NO.-CBS by O2 showed complex kinetic behavior and led to peroxynitrite (ONOO-) formation, which was detected using the fluorescent probe, coumarin boronic acid. Thus, in addition to being a potential source of superoxide radical, CBS constitutes a previously unrecognized source of NO. and peroxynitrite. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H2S) is increasingly recognized to modulate physiological processes in mammals through mechanisms that are currently under scrutiny. H2S is not able to react with reduced thiols (RSH). However,H2S, more preciselyHS-, is able to react with oxidized thiol derivatives. We performed a systematic study of the reactivity of HS- toward symmetric low molecular weight disulfides (RSSR) and mixed albumin (HSA) disulfides. Correlations with thiol acidity and computational modeling showed that the reaction occurs through a concerted mechanism. Comparison with analogous reactions of thiolates indicated that the intrinsic reactivity of HS- is 1 order of magnitude lower than that of thiolates. In addition, H2S is able to react with sulfenic acids (RSOH). The rate constant of the reaction of H2S with the sulfenic acid formed in HSA was determined. Both reactions of H2S with disulfides and sulfenic acids yield persulfides (RSSH), recently identified post-translational modifications. The formation of this derivative in HSA was determined, and the rate constants of its reactions with a reporter disulfide and with peroxynitrite revealed that persulfides are better nucleophiles than thiols, which is consistent with the α effect. Experiments with cells in culture showed that treatment with hydrogen peroxide enhanced the formation of persulfides. Biological implications are discussed. Our results give light on the mechanisms of persulfide formation and provide quantitative evidence for the high nucleophilicity of these novel derivatives, setting the stage for understanding the contribution contribution of the reactions of H2S with oxidized thiol derivatives to H2S effector processes. [ABSTRACT FROM AUTHOR]