Inner-filter effect of nitrogen-doped carbon quantum dots-MnO2 nanotubes for smartphone-integrated dual-mode sensing of glutathione and captopril.

Nitrogen-doped carbon quantum dots (N-CQDs) exhibit unique fluorescence properties and are considered one of the best candidates for the development of fluorescence-based sensors for the detection of many analytes. In this work, a smartphone-assisted fluorescent sensor has been developed using N-CQDs and MnO2 nanotubes (MnO2 NTs) for the detection of glutathione (GSH) and captopril (CAP). N-CQDs were facilely synthesized via the solvothermal method, where o-phenylenediamine (o-PD) and urea were used as nitrogen precursors. Likewise, MnO2 NTs were synthesized using the hydrothermal method. Relying on the excellent fluorescence quenching ability of MnO2 NTs, a nanocomposite of N-CQDs and MnO2 NTs is prepared, wherein the fluorescence intensity of N-CQDs was effectively quenched in the presence of MnO2 NTs via the inner-filter effect (IFE). The addition of thiolated compounds (GSH and CAP) helped in the recovery of the fluorescence of N-CQDs by triggering the redox reaction and decomposing the MnO2 NTs. An investigation of fluorescence along with smartphone-based studies by evaluating the gray measurement using Image J software showed a great response towards GSH and CAP providing LODs of 4.70 µM and 5.22 µM (fluorometrically) and 5.76 µM and 2.81 µM (smartphone-based), respectively. The practical applicability of the sensing system has been verified using human blood plasma samples.

An innate pathogen sensing strategy involving ubiquitination of bacterial surface proteins.

Sensing of pathogens by ubiquitination is a critical arm of cellular immunity. However, universal ubiquitination targets on microbes remain unidentified. Here, using in vitro, ex vivo, and in vivo studies, we identify the first protein-based ubiquitination substrates on phylogenetically diverse bacteria by unveiling a strategy that uses recognition of degron-like motifs. Such motifs form a new class of intra-cytosolic pathogen-associated molecular patterns (PAMPs). Their incorporation enabled recognition of nonubiquitin targets by host ubiquitin ligases. We find that SCFFBW7 E3 ligase, supported by the regulatory kinase, glycogen synthase kinase 3ß, is crucial for effective pathogen detection and clearance. This provides a mechanistic explanation for enhanced risk of infections in patients with chronic lymphocytic leukemia bearing mutations in F-box and WD repeat domain containing 7 protein. We conclude that exploitation of this generic pathogen sensing strategy allows conservation of host resources and boosts antimicrobial immunity.

Differential Ubiquitination as an Effective Strategy Employed by the Blood-Brain Barrier for Prevention of Bacterial Transcytosis.

The protective mechanisms of blood-brain barrier (BBB) prohibiting entry of pathogens into central nervous system (CNS) are critical for maintenance of brain homeostasis. These include various intracellular defense mechanisms that are vital to block transcytosis of neurotropic pathogens into the CNS. However, mechanistic details of coordination between these defense pathways remain unexplored. In this study, we established that BBB-driven ubiquitination acts as a major intracellular defense mechanism for clearance of Streptococcus pneumoniae, a critical neurotropic pathogen, during transit through BBB. Our findings suggest that the BBB employs differential ubiquitination with either K48- or K63-ubiquitin (Ub) chain topologies as an effective strategy to target S. pneumoniae toward diverse killing pathways. While K63-Ub decoration triggers autophagic killing, K48-Ub directs S. pneumoniae exclusively toward proteasomes. Time-lapse fluorescence imaging involving proteasomal marker LMP2 revealed that in the BBB, the majority of the ubiquitinated S. pneumoniae was cleared by proteasome. Fittingly, inhibition of proteasome and autophagy pathway led to accumulation of K48-Ub- and K63-Ub-marked S. pneumoniae, respectively, and triggered significant increases in intracellular S. pneumoniae burden. Moreover, genetic impairment of either K48- or K63-Ub chain formation demonstrated that although both chain types are key in disposal of intracellular S. pneumoniae, K48-Ub chains and subsequent proteasomal degradation have more pronounced contributions to intracellular S. pneumoniae killing in the BBB. Collectively, these observations, for the first time, illustrated a pivotal role of differential ubiquitination deployed by BBB in orchestrating a symphony of intracellular defense mechanisms for interception and degradation of S. pneumoniae, blocking its entry into the brain, which could be exploited to prevent bacterial CNS infections. IMPORTANCE The blood-brain barrier (BBB) represents a unique cellular barrier that provides structural integrity and protection to the CNS from pathogen invasion. Recently, ubiquitination, which is key for cellular homeostasis, was shown to be involved in pathogen clearance. In this study, we deciphered that the BBB deploys differential ubiquitination as an effective strategy to prevent S. pneumoniae trafficking into the brain. The different ubiquitin chain topologies formed on S. pneumoniae dictated the selection of downstream degradative pathways, namely, autophagy and proteasomes, among which the contribution of the proteasomal system in S. pneumoniae killing is more pronounced. Overall our study revealed how the BBB deploys differential ubiquitination as a strategy for synchronization of various intracellular defense pathways, which work in tandem to ensure the brain's identity as an immunologically privileged site.

Impaired quality of life in growth hormone-deficient adults is independent of the altered skeletal muscle oxidative metabolism found in conditions with peripheral fatigue.

CONTEXT: Growth hormone-deficient (GHD) adults often report impaired quality of life (QoL) - with fatigue, a key element. This deficit can improve following GH replacement. The basis of this response is unclear. Perturbations in skeletal muscle metabolism have been demonstrated in several conditions in which fatigue is a prominent symptom. We wished to define the role of skeletal muscle metabolism in the impaired QoL observed in patients with GHD. OBJECTIVE: To compare in vivo skeletal muscle mitochondrial oxidative phosphorylation using phosphorus-31 magnetic resonance spectroscopy in matched untreated GHD adults, treated GHD adults and healthy volunteers. DESIGN: Twenty-two untreated GHD adults, 23 treated GHD adults and 20 healthy volunteers were recruited at a regional centre. All patients underwent assessment of muscle mitochondrial function (τ1/2 PCr) and proton handling using spectroscopy. Fasting biochemical analyses and anthropometric measurement were obtained. All patients completed the QoL-AGHDA and physical activity assessment (IPAQ) questionnaires. RESULTS: Untreated and treated GHD adults complained of significantly increased fatigue and an impaired QoL (P = 0·002) when compared to healthy controls. There was no difference in maximal mitochondrial function (P = 0·53) nor pH recovery (P = 0·38) of skeletal muscle between the three groups. Untreated GHD patients had significantly lower IGF-1 than both treated GHD and healthy volunteers (P < 0·001), but there was no association between τ1/2 PCr and serum IGF-1 (r = -0·13, P = 0·32). CONCLUSIONS: The impaired QoL seen in GHD adults is not associated with the skeletal muscle spectroscopic 'footprint' of altered mitochondrial oxidative function, anaerobic glycolysis or proton clearance that are a feature of several conditions in which fatigue is a prominent feature. These data suggest that the pathophysiology of fatigue and impaired QoL in GHD may have a significant central rather than peripheral (skeletal muscle) component.

Graves' immune reconstitution inflammatory syndrome in childhood.

BACKGROUND: The use of hematopoietic stem cell transplantations (HSCTs) as a curative therapy for life-threatening immunodeficiencies has had a profound impact on clinical outcomes. A subset of patients may experience immune reconstitution inflammatory syndrome (IRIS) post-transplant affecting the thyroid gland, but this has received little attention in the pediatric literature. We present the clinical, biochemical, and cytological course of patients with Graves' disease after HSCT in the pediatric population. PATIENTS AND METHODS: Four children (median age 1.5 years, range 2 months-9 years) underwent HSCT. The conditioning regimen included chemotherapy but not radiotherapy. None of the children or their donors had evidence of thyroid disease pre-HSCT or during the follow-up period. Engraftment was uneventful in all, with stable donor T-cell chimerism, and none had evidence of graft-versus-host disease. RESULTS: Patients developed Graves' disease soon after undergoing HSCT, with a median time interval between HSCT and Graves' disease of 22 months (range 16-28 months). Graves' disease was diagnosed on the basis of clinical and biochemical parameters, including a suppressed thyrotropin, raised free thyroxine, and raised thyrotropin receptor antibodies. Three patients were hypothyroid initially (suggestive of a Th1 profile) before Graves' disease (suggestive of a Th2 profile). In three patients, the clinical picture changed rapidly with hypothyroidism abruptly followed by profound thyroid hormone excess. The onset of Graves' IRIS coincided with a rapid expansion in naïve and total CD4. CONCLUSIONS: Immunological dysregulation during T-cell engraftment is the most likely mechanism for developing Graves' IRIS after allogenic HSTC. Clinicians need to be aware that HSCT-engendered immune recovery may result in a particularly aggressive form of autoimmune thyroid disease in children with implications for the developing central nervous system. Careful surveillance of thyroid function post-HSCT is essential.

Improving the vitamin D status of vitamin D deficient adults is associated with improved mitochondrial oxidative function in skeletal muscle.

OBJECTIVE: Suboptimal mitochondrial function has been implicated in several disorders in which fatigue is a prominent feature. Vitamin D deficiency is a well-recognized cause of fatigue and myopathy. The aim of this study was to examine the effects of cholecalciferol therapy on skeletal mitochondrial oxidative function in symptomatic, vitamin D-deficient individuals. DESIGN: This longitudinal study assessed mitochondrial oxidative phosphorylation in the gastrosoleus compartment using phosphorus-31 magnetic resonance spectroscopy measurements of phosphocreatine recovery kinetics in 12 symptomatic, severely vitamin D-deficient subjects before and after treatment with cholecalciferol. All subjects had serum assays before and after cholecalciferol therapy to document serum 25-hydroxyvitamin D (25OHD) and bone profiles. Fifteen healthy controls also underwent (31)P-magnetic resonance spectroscopy and serum 25OHD assessment. RESULTS: The phosphocreatine recovery half-time (τ1/2PCr) was significantly reduced after cholecalciferol therapy in the subjects indicating an improvement in maximal oxidative phosphorylation (34.44 ± 8.18 sec to 27.84 ± 9.54 sec, P < .001). This was associated with an improvement in mean serum 25OHD levels (8.8 ± 4.2 nmol/L to 113.8 ± 51.5 nmol/L, P < .001). There was no difference in phosphate metabolites at rest. A linear regression model showed that decreasing serum 25OHD levels was associated with increasing τ1/2PCr (r = -0.41, P = .009). All patients reported an improvement in fatigue after cholecalciferol therapy. CONCLUSIONS: Cholecalciferol therapy augments muscle mitochondrial maximal oxidative phosphorylation after exercise in symptomatic, vitamin D-deficient individuals. This finding suggests that changes in mitochondrial oxidative phosphorylation in skeletal muscle could at least be partly responsible for the fatigue experienced by these patients. For the first time, we demonstrate a link between vitamin D and the mitochondria in human skeletal muscle.

Prevention and treatment of vitamin D deficiency.

Vitamin D insufficiency and deficiency are widespread in many countries. We review the evidence pertaining to its prevention and treatment. Deficiency may be adequately treated with many different therapeutic regimens of either cholecalciferol or ergocalciferol, owing to the high therapeutic index of both compounds. Nevertheless, the current evidence suggests that regular dosing with oral cholecalciferol (e.g., 60,000 IU weekly) may have slight advantages over other regimens when replenishing vitamin D stores following deficiency. For long-term supplementation, smaller regular doses, such as cholecalciferol 1,000 IU daily, or 10,000 IU weekly, are suitable. Giving reliable and specific advice about appropriate sunlight exposure remains difficult because of differing interindividual skin pigmentation and variable sunlight UVB content at different latitudes, at different times of year, and in different terrestrial environments.

Vitamin D status in paediatric patients with cancer.

BACKGROUND: Children with malignant disease are at increased risk of bone disorders and cardiovascular disease. Vitamin D status may influence this risk and so we assessed vitamin D levels in children with malignant disease undergoing active treatment or surveillance post-therapy. PROCEDURE: This was an outpatient-based cross-sectional study of 61 children with a history of malignancy (median age 11.1 years; range 1.5-24.4 years) and 60 control subjects (median age 8.4 years; range 0.2-18.0 years) attending hospital for the management of non-malignant disorders. Serum vitamin D (25-OH-D), parathormone levels and bone biochemistry were determined. Vitamin D status and its relationship to age, sex, ethnicity, time of sampling and presence of malignant disease was determined. RESULTS: Vitamin D status was suboptimal in 62% of cases (25-OH-D < 50 nmol/L [20 ng/ml]). Vitamin D deficiency (25-OH-D < 25 nmol/L [10 ng/ml]) was more common in children with malignant disease than controls (21.3% vs. 3.3%; P = 0.013). Month of sampling (P < 0.001), ethnicity (P < 0.001), older age (P = 0.011), and history of malignancy (P = 0.012) were associated with a poorer vitamin D status. CONCLUSIONS: Vitamin D levels [25-OH-D] are lower in survivors of childhood cancer in comparison to control children with the majority either insufficient or deficient. Assessment and adequate replacement of vitamin D status may be of particular value in this group of children.