Determination of IgG1 and IgG3 SARS-CoV-2 Spike Protein and Nucleocapsid Binding-Who Is Binding Who and Why?

The involvement of immunoglobulin (Ig) G3 in the humoral immune response to SARS-CoV-2 infection has been implicated in the pathogenesis of acute respiratory distress syndrome (ARDS) in COVID-19. The exact molecular mechanism is unknown, but it is thought to involve this IgG subtype's differential ability to fix, complement and stimulate cytokine release. We examined the binding of convalescent patient antibodies to immobilized nucleocapsids and spike proteins by matrix-assisted laser desorption/ionization-time of flight (MALDI-ToF) mass spectrometry. IgG3 was a major immunoglobulin found in all samples. Differential analysis of the spectral signatures found for the nucleocapsid versus the spike protein demonstrated that the predominant humoral immune response to the nucleocapsid was IgG3, whilst for the spike protein it was IgG1. However, the spike protein displayed a strong affinity for IgG3 itself, as it would bind from control plasma samples, as well as from those previously infected with SARS-CoV-2, similar to the way protein G binds IgG1. Furthermore, detailed spectral analysis indicated that a mass shift consistent with hyper-glycosylation or glycation was a characteristic of the IgG3 captured by the spike protein.

Abnormal secretion of melatonin and cortisol in relation to sleep disturbances in children with Williams syndrome.

OBJECTIVE: A high rate of sleep disturbances has been reported in individuals with Williams syndrome (WS) but the underlying aetiology has yet to be identified. Melatonin and cortisol levels display circadian rhythmicity and are known to affect and regulate sleep/wake patterns. The current study examined the levels of these two endocrine markers and explored a possible relationship with sleep patterns in children with WS. METHODS: Twenty-five children with WS and 27 typically developing age- and gender-matched comparison children were recruited. Saliva was collected from each child at three time points: 4-6 pm, before natural bedtime, and after awakening. The levels of salivary melatonin and cortisol were analysed by specific enzyme-linked immunoassays. Sleep patterns were examined using actigraphy and the Children's Sleep Habit Questionnaire. RESULTS: The WS group had shallower drops in cortisol and less pronounced increase in melatonin at bedtime compared to the controls. Furthermore, they also had significantly higher levels of cortisol before bedtime. CONCLUSIONS: Increased bedtime cortisol and less pronounced rise in melatonin levels before sleep may play a role in the occurrence of sleep disturbances, such as delayed sleep onset, observed in children with WS. As both markers play a significant role in our circadian rhythm and sleep/wake cycle, it is necessary to examine sleep using multi-system analysis.

Determination of urinary cortisol, cortisone and 6-sulfatoxymelatonin using dilute and shoot ultra-high pressure liquid chromatography-tandem mass spectrometry.

Human sleep is a natural part of every individual's life. Clear relationship between sleep and endocrine system has been already established. In particular, melatonin and cortisol are known to affect and regulate sleep/wake patterns. Here we report the development of an ultra-high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous measurement of 6-sulfatoxymelatonin (MT6s), cortisol and cortisone in urine. A separate method was developed for measurement of creatinine in urine. These levels were used to normalise the levels of analytes. First void morning urine samples were collected from 24 healthy volunteers. Samples were diluted 1:1 in water prior to injection onto reversed-phase C18 column and analysed using UHPLC-MS/MS method. Linear calibrations were obtained for all analytes with correlation coefficient in the range 0.998-0.999. The observed concentration was found to be in the range 92-105% for cortisol, 92-107% for cortisone and between 93 and 120% for MT6s of the reference levels. The total run time of 6 min with all peaks of interest eluting within 3 min was obtained. This demonstrates the feasibility of utilising the method for large multi-scale studies, where high throughput is required for studying the circadian rhythm of melatonin and cortisol secretion. These hormones play significant role in circadian rhythm and sleep/wake cycle; therefore it is important to monitor the levels of these endocrine markers in individuals suffering from sleep disorders. It is also beneficial with clinical applications to analyse melatonin and cortisol simultaneously in order to assess their interrelationships of these substances, such as their effect on diurnal rhythm and sleep.