The study of circadian typology differences has increased in the last few years. As a result, new instruments have been developed to estimate the individual circadian phase of temporal human behavior, also referred as chronotype. The current review was conducted to evaluate the differences among the questionnaires most frequently used to assess chronotype: the Morningness-Eveningness Questionnaire (MEQ), the Composite Scale of Morningness (CSM), and the Munich Chronotype Questionnaire (MCTQ). Each instrument evaluates a different aspect of chronotype. MEQ is considered to evaluate the phase preferences of individual behavior over a 24-hour day, while MCTQ measures the phase of sleep positions for both free and work days. CSM is similar to MEQ, but is more sensitive to measure shift work. The concept of chronotype has been used to refer to phase positions or phase preferences in the literature reviewed. Most of the time this is a consequence of different interpretations: it is not clear whether phase preferences are a direct manifestation of the individual's internal clock or a result of external cues, e.g., social interaction (including the alarm clock). Also, phase preferences are not uniform throughout life. Therefore, a single assessment, not taking age into consideration, will not accurately describe the sample. We suggest that MCTQ is the best instrument for investigators dealing with desynchronization and as an instrument for sleep phase. Conversely, if the goal is to assess characteristics that change under specific situations - chronotype -, the MEQ should be used.

Background Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a severe multisystemic condition associated with post-infectious onset, impaired natural killer (NK) cell cytotoxicity and impaired ion channel function, namely Transient Receptor Potential Melastatin 3 (TRPM3). Long-term effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has resulted in neurocognitive, immunological, gastrointestinal, and cardiovascular manifestations recently recognised as post coronavirus disease 2019 (COVID-19) condition. The symptomatology of ME/CFS overlaps significantly with post COVID-19; therefore, this research aimed to investigate TRPM3 ion channel function in post COVID-19 condition patients. Methods Whole-cell patch-clamp technique was used to measure TRPM3 ion channel activity in isolated NK cells of N = 5 ME/CFS patients, N = 5 post COVID-19 patients, and N = 5 healthy controls (HC). The TRPM3 agonist, pregnenolone sulfate (PregS) was used to activate TRPM3 function, while ononetin was used as a TRPM3 antagonist. Results As reported in previous research, PregS-induced TRPM3 currents were significantly reduced in ME/CFS patients compared with HC (p = 0.0048). PregS-induced TRPM3 amplitude was significantly reduced in post COVID-19 condition compared with HC (p = 0.0039). Importantly, no significant difference was reported in ME/CFS patients compared with post COVID-19 condition as PregS-induced TRPM3 currents of post COVID-19 condition patients were similar of ME/CFS patients currents (p > 0.9999). Isolated NK cells from post COVID-19 condition and ME/CFS patients were resistant to ononetin and differed significantly with HC (p < 0.0001). Conclusion The results of this investigation suggest that post COVID-19 condition patients may have impaired TRPM3 ion channel function and provide further evidence regarding the similarities between post COVID-19 condition and ME/CFS. Impaired TRPM3 channel activity in post COVID-19 condition patients suggest impaired ion mobilisation which may consequently impede cell function resulting in chronic post-infectious symptoms. Further investigation into TRPM3 function may elucidate the pathomechanism, provide a diagnostic and therapeutic target for post COVID-19 condition patients and commonalities with ME/CFS patients.

Introduction: The behavior of nocturnal rodents is associated with circadian variation, whereby higher levels of activity are concentrated during the nocturnal period, highlighting the need to control circadian rhythms. Objective: The objective of this study is to evaluate whether different times of intervention in a depression model affects performance on animal behavioral tests. Methodology: The stress model used was the inescapable foot shock in 35 male 60-day-old Wistar rats. The animals received intervention in the light phase and in the dark phase, after that they were tested in the light or in the dark phase. Results: The light-dark box test showed that the Control L (tested in the light) was not significantly different from other groups across any of the parameters. However, when comparing the Control D (tested in the dark) to the intervention groups, we observed a difference in the mean length of time spent in the light and in the dark (t=2.56; p=0.045). Comparing the Control D with the experimental inescapable foot shock in the light and tested in the light group, we observed that the intervention group had made more crossings into the light (t=-2.608; p=0.028) and into the dark (t=-2.488; p=0.035). Furthermore, comparing to inescapable foot shock in the light and tested in the dark group, we observed that the treated group had made more crossings into the light side (t =-2.571; p=0.030) and dark side (t=-2.398; p =0.040). Conclusion: These results show that behavioral testing during the animal's period of higher activity revealed differences caused by the intervention, while no differences were apparent when the control group was observed during the day.

Introduction: Mutations and misfolding of membrane proteins are associated with various disorders, hence they make suitable targets in proteomic studies. However, extraction of membrane proteins is challenging due to their low abundance, stability, and susceptibility to protease degradation. Given the limitations in existing protocols for membrane protein extraction, the aim of this investigation was to develop a protocol for a high yield of membrane proteins for isolated Natural Killer (NK) cells. This will facilitate genetic analysis of membrane proteins known as transient receptor potential melastatin 3 (TRPM3) ion channels in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) research.Methods: Two protocols, internally identified as Protocol 1 and 2, were adapted and optimized for high yield protein extraction. Protocol 1 utilized ultrasonic and salt precipitation, while Protocol 2 implemented a detergent and chloroform/methanol approach. Protein concentrations were determined by the Pierce Bicinchoninic Acid (BCA) and the Bio-Rad DC (detergent compatible) protein assays according to manufacturer’s recommendation. Using Protocol 2, protein samples were extracted from NK cells of n = 6 healthy controls (HC) and n = 4 ME/CFS patients. In silico tryptic digest and enhanced signature peptide (ESP) predictor were used to predict high-responding TRPM3 tryptic peptides. Trypsin in-gel digestion was performed on protein samples loaded on SDS-PAGE gels (excised at 150–200 kDa). A liquid chromatography-multiple reaction monitoring (LC-MRM) method was optimized and used to evaluate the detectability of TRPM3 n = 5 proteotypic peptides in extracted protein samples.Results: The detergent-based protocol protein yield was significantly higher (p &lt; 0.05) compared with the ultrasonic-based protocol. The Pierce BCA protein assay showed more reproducibility and compatibility compared to the Bio-Rad DC protein assay. Two high-responding tryptic peptides (GANASAPDQLSLALAWNR and QAILFPNEEPSWK) for TRPM3 were detectable in n = 10 extracted protein samples from NK cells isolated from HC and ME/CFS patients.Conclusion: A method was optimized for high yield protein extraction from human NK cells and for the first time TRPM3 proteotypic peptides were detected using LC-MRM. This new method provides for future research to assess membrane protein structural and functional relationships, particularly to facilitate proteomic investigation of TRPM3 ion channel isoforms in NK cells in both health and disease states, such as ME/CFS.