Personalized Management of Fatigue in Individuals With Myalgic Encephalomyelitis/Chronic Fatigue Syndrome and Long COVID Using a Smart Digital mHealth Solution: Protocol for a Participatory Design Approach.

BACKGROUND: Fatigue is the most common symptom in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and long COVID, impacting patients' quality of life; however, there is currently a lack of evidence-based context-aware tools for fatigue self-management in these populations. OBJECTIVE: This study aimed to (1) address fatigue in ME/CFS and long COVID through the development of digital mobile health solutions for self-management, (2) predict perceived fatigue severity using real-time data, and (3) assess the feasibility and potential benefits of personalized digital mobile health solutions. METHODS: The MyFatigue project adopts a patient-centered approach within the participatory health informatics domain. Patient representatives will be actively involved in decision-making processes. This study combines inductive and deductive research approaches, using qualitative studies to generate new knowledge and quantitative methods to test hypotheses regarding the relationship between factors like physical activity, sleep behaviors, and perceived fatigue in ME/CFS and long COVID. Co-design methods will be used to develop a personalized digital solution for fatigue self-management based on the generated knowledge. Finally, a pilot study will evaluate the feasibility, acceptance, and potential benefits of the digital health solution. RESULTS: The MyFatigue project opened to enrollment in November 2023. Initial results are expected to be published by the end of 2024. CONCLUSIONS: This study protocol holds the potential to expand understanding, create personalized self-management approaches, engage stakeholders, and ultimately improve the well-being of individuals with ME/CFS and long COVID. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/50157.

The Conners Continuous Performance Test CPT3™: Is it a reliable marker to predict neurocognitive dysfunction in Myalgic encephalomyelitis/chronic fatigue syndrome?

Introduction: The main objective is to delimit the cognitive dysfunction associated with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) in adult patients by applying the Continuous Performance Test (CPT3™). Additionally, provide empirical evidence on the usefulness of this computerized neuropsychological test to assess ME/CFS. Method: The final sample (n = 225; 158 Patients/67 Healthy controls) were recruited in a Central Sensitization Syndromes (CSS) specialized unit in a tertiary hospital. All participants were administered this neuropsychological test. Results: There were significant differences between ME/CFS and healthy controls in all the main measures of CPT3™. Mainly, patients had a worse indicator of inattentiveness, sustained attention, vigilance, impulsivity, slow reaction time, and more atypical T-scores, which is associated with a likelihood of having a disorder characterized by attention deficits, such as Attention Deficit Hyperactivity Disorder (ADHD). In addition, relevant correlations were obtained between the CPT3™ variables in the patient's group. The most discriminative indicators of ME/CFS patients were Variability and Hit Reaction Time, both measures of response speed. Conclusion: The CPT3™ is a helpful tool to discriminate neurocognitive impairments from attention and response speed in ME/CFS patients, and it could be used as a marker of ME/CFS severity for diagnosing or monitoring this disease.

Encouraging People with Spinal Cord Injury to Take Part in Physical Activity in the COVID-19 Epidemic through the mHealth ParaSportAPP.

BACKGROUND: Although mHealth tools have great potential for health interventions, few experimental studies report on their use by people with spinal cord injuries in physical activity. OBJECTIVE: The main objective of this study was to analyze the effect of the ParaSportAPP on different physical and psychological variables in people with paraplegia. METHODS: Fourteen of these subjects made up the final sample. All the participants performed two pre-tests (control period) and a post-test with 8 months between the evaluations (COVID-19 broke out between pre-test 2 and the post-test). The ParaSportAPP was installed on their smartphones when they performed pre-test 2. The same tests were performed in the same order in all the evaluations: (i) the questionnaires PASIPD, HADS, RS-25; SCIM III and AQoL-8D, (ii) respiratory muscle strength, (iii) spirometry and (iv) cardiopulmonary exercise test. RESULTS: The results showed no differences in any of the variables studied between the measurement times. CONCLUSIONS: Although none of the variables experienced improvements, the ParaSportAPP mobile application was able to lessen the impact of the pandemic on the variables studied.

Over-expression of peptide deformylase in chloroplasts confers actinonin resistance, but is not a suitable selective marker system for plastid transformation.

Arabidopsis thaliana peptide deformylase PDF1B was expressed in tobacco chloroplasts using spectinomycin as the selective agent. The foreign protein accumulated in chloroplasts (6% of the total soluble protein) and was enzymatically active. Transplastomic plants were evaluated for resistance to the peptide deformylase inhibitor actinonin. In vitro seed germination in the presence of actinonin and in planta application of the inhibitor demonstrated the resistance of the transformed plants. In addition, transgenic leaf explants were able to develop shoots via organogenesis in the presence of actinonin. However, when the combination of the PDF1B gene and actinonin was used as the primary selective marker system for chloroplast transformation of tobacco, all developed shoots were escapes. Therefore, under the experimental conditions tested, the use of this system for plastid transformation would be limited to function as a secondary selective marker.

Spanish validation of the Neurogenic Bowel Dysfunction score -NBD score- in patients with central neurological injury. / Validación al español de la versión en inglés del Neurogenic Bowel Dysfunction score (NBD score) en pacientes con lesión neurológica de origen central.

OBJECTIVE: to validate into Spanish the Neurogenic Bowel Dysfunction score (NBD score) that quantifies intestinal dysfunction severity in patients with disabilities due to central neurological injury and satisfaction with bowel management. MATERIAL: 59 patients, 30 patients affected by intestinal disability due to spinal cord injury and 29 patients with intestinal disability due to stroke. RESULTS: The result of the reliability of the construction of the Spanish translation of the NBD score for the whole group of patients shows a Cronbach's α for all the variables of 0.970 and the result of the reliability of the NBD score for the whole group in test-retest, using the interclass correlation coefficient, was 0.970 (95% CI 0.954-0.980). CONCLUSIONS: The Spanish version of the NBD score is a valid tool for use in our environment; it will allow a more real approach to the disability situation of each patient in relation to neurogenic intestinal dysfunction and knowledge of the degree and the involvement and effectiveness of management through different therapeutic proposals.

HIV-1 p24-immunoglobulin fusion molecule: a new strategy for plant-based protein production.

We describe the engineering of a human immunodeficiency virus-1 (HIV-1) p24-immunoglobulin A (IgA) antigen-antibody fusion molecule for therapeutic purposes and its enhancing effect on fused antigen expression in tobacco plants. Although many recombinant proteins have been expressed in transgenic plants as vaccine candidates, low levels of expression are a recurring problem. In this paper, using the HIV p24 core antigen as a model vaccine target, we describe a strategy for increasing the yield of a recombinant protein in plants. HIV p24 antigen was expressed as a genetic fusion with the alpha2 and alpha3 constant region sequences from human Ig alpha-chain and targeted to the endomembrane system. The expression of this fusion protein was detected at levels approximately 13-fold higher than HIV p24 expressed alone, and a difference in the behaviour of the two recombinant proteins during trafficking in the plant secretory pathway has been identified. Expressing the antigen within the context of alpha-chain Ig sequences resulted in the formation of homodimers and the antigen was correctly recognized by specific antibodies. Furthermore, the HIV p24 elicited T-cell and antibody responses in immunized mice. The use of Ig fusion partners is proposed as a generic platform technology for up-regulating the expression of antigens in plants, and may represent the first step in a strategy to design new vaccines with enhanced immunological properties.

Transgenic plants expressing antibodies: a model for phytoremediation.

The feasibility of using antibody expressing transgenic plants either to neutralize bioactive molecules in the rhizosphere, or to accumulate and concentrate the molecules in leaves has been demonstrated in a model system consisting of hydroponic Nicotiana plant cultures expressing a murine monoclonal IgG1. Two transgenic plant types were used; in the first, functional antibody was rhizosecreted and shown to bind with antigen in the surrounding medium to form an immune complex. In the second, a transmembrane sequence retained monoclonal antibody in the plants, on the plasma membrane. Antigen added to the nutrient medium around the roots of mIgG plants was transported within 24 h to the topmost leaves of the plant where it was sequestered as an immune complex by binding to antibody on the cell membrane. Concentration of immune complex in the leaf tissue remained constant over a 72 h period after removal of antigen from nutrient medium. Free antigen was not detected in the leaves of wild-type plants. The two strategies of rhizosecretion-mediated binding and sequestration in leaf tissue could potentially be used in the phytoremediation of any pollutant for which it is possible to generate a monoclonal antibody.

The extradomain A of fibronectin (EDA) combined with poly(I:C) enhances the immune response to HIV-1 p24 protein and the protection against recombinant Listeria monocytogenes-Gag infection in the mouse model.

The development of effective vaccines against HIV-1 infection constitutes one of the major challenges in viral immunology. One of the protein candidates in vaccination against this virus is p24, since it is a conserved HIV antigen that has cytotoxic and helper T cell epitopes as well as B cell epitopes that may jointly confer enhanced protection against infection when used in immunization-challenge approaches. In this context, the adjuvant effect of EDA (used as EDAp24 fusion protein) and poly(I:C), as agonists of TLR4 and TLR3, respectively, was assessed in p24 immunizations using a recombinant Listeria monocytogenes HIV-1 Gag proteins (Lm-Gag, where p24 is the major antigen) for challenge in mice. Immunization with EDAp24 fusion protein together with poly(I:C) adjuvant induced a specific p24 IFN-γ production (Th1 profile) as well as protection against a Lm-Gag challenge, suggesting an additive or synergistic effect between both adjuvants. The combination of EDA (as a fusion protein with the antigen, which may favor antigen targeting to dendritic cells through TLR4) and poly(I:C) could thus be a good adjuvant candidate to enhance the immune response against HIV-1 proteins and its use may open new ways in vaccine investigations on this virus.

Intrinsic order and disorder in the bcl-2 member harakiri: insights into its proapoptotic activity.

Harakiri is a BH3-only member of the Bcl-2 family that localizes in membranes and induces cell death by binding to prosurvival Bcl-x(L) and Bcl-2. The cytosolic domain of Harakiri is largely disorder with residual α-helical conformation according to previous structural studies. As these helical structures could play an important role in Harakiri's function, we have used NMR and circular dichroism to fully characterize them at the residue-atomic level. In addition, we report structural studies on a peptide fragment spanning Harakiri's C-terminal hydrophobic sequence, which potentially operates as a transmembrane domain. We initially checked by enzyme immunoassays and NMR that peptides encompassing different lengths of the cytosolic domain are functional as they bind Bcl-x(L) and Bcl-2. The structural data in water indicate that the α-helical conformation is restricted to a 25-residue segment comprising the BH3 domain. However, structure calculation was precluded because of insufficient NMR restraints. To bypass this problem we used alcohol-water mixture to increase structure population and confirmed by NMR that the conformation in both milieus is equivalent. The resulting three-dimensional structure closely resembles that of peptides encompassing the BH3 domain of BH3-only members in complex with their prosurvival partners, suggesting that preformed structural elements in the disordered protein are central to binding. In contrast, the transmembrane domain forms in micelles a monomeric α-helix with a population close to 100%. Its three-dimensional structure here reported reveals features that explain its function as membrane anchor. Altogether these results are used to propose a tentative structural model of how Harakiri works.

Characterization of a novel interaction between Bcl-2 members Diva and Harakiri.

Interactions within proteins of the Bcl-2 family are key in the regulation of apoptosis. The death-inducing members control apoptotic mechanisms partly by antagonizing the prosurvival proteins through heterodimer formation. Structural and biophysical studies on these complexes are providing important clues to understand their function. To help improve our knowledge on protein-protein interactions within the Bcl-2 family we have studied the binding between two of its members: mouse Diva and human Harakiri. Diva has been shown to perform both prosurvival and killing activity. In contrast, Harakiri induces cell death by interacting with antiapoptotic Bcl-2 members. Here we show using ELISA and NMR that Diva and Harakiri can interact in vitro. Combining the NMR data with the previously reported three-dimensional structure of Diva we find that Harakiri binds to a specific region in Diva. This interacting surface is equivalent to the known binding area of prosurvival Bcl-2 members from the reported structures of the complexes, suggesting that Diva could function at the structural level similarly to the antiapoptotic proteins of the Bcl-2 family. We illustrate this result by building a structural model of the heterodimer using molecular docking and the NMR data as restraints. Moreover, combining circular dichroism and NMR we also show that Harakiri is largely unstructured with residual (13%) α-helical conformation. This result agrees with intrinsic disorder previously observed in other Bcl-2 members. In addition, Harakiri constructs of different length were studied to identify the region critical for the interaction. Differential affinity for Diva of these constructs suggests that the amino acid sequence flanking the interacting region could play an important role in binding.

Antibody processing and engineering in plants, and new strategies for vaccine production.

The use of transgenic plants for the production of recombinant proteins is not a universal solution for all proteins. The choice of this expression system depends very much on the type of protein and its applications. Many proteins will best be made by conventional microbial fermentation, similarly, we are already identifying proteins where plants represent the only practical option for one reason or another. It will be important to understand better the cellular mechanisms of protein folding, assembly and processing in plants, in order to maximise the potential of transgenic plants as a protein production system. One of the main advantages that plants offer is that they are higher eukaryotic organisms with an endomembrane system. Therefore, they fold and assemble recombinant proteins using protein chaperones that are homologous to those in mammalian cells, and they perform post-translational modifications. This allows, for example, the expression of monoclonal antibodies, first described in 1989, as well as a range of other types of immunoglobulin molecules and multimeric complexes.

Highly immunogenic and protective recombinant vaccine candidate expressed in transgenic plants.

Vaccine development has been hampered by difficulties in developing new and safe adjuvants, so alternative technologies that offer new avenues forward are urgently needed. The goal of this study was to express a monoclonal recombinant immune complex in a transgenic plant. A recombinant protein consisting of a tetanus toxin C fragment-specific monoclonal antibody fused with the tetanus toxin C fragment was designed and expressed. Immune complex formation occurred between individual fusion proteins to form immune complex-like aggregates that bound C1q and FcgammaRIIa receptor and could be targeted to antigen-presenting cells. Unlike antigen alone, the recombinant immune fusion complexes were highly immunogenic in mice and did not require coadministration of an adjuvant (when injected subcutaneously). Indeed, these complexes elicited antibody titers that were more than 10,000 times higher than those observed in animals immunized with the antigen alone. Furthermore, animals immunized with only 1 mug of recombinant immune complex without adjuvant were fully protected against lethal challenge. This the first report on the use of a genetic fusion between antigen and antibody to ensure an optimal expression ratio between the two moieties and to obtain fully functional recombinant immune complexes as a new vaccine model.

Rhizosecretion of a monoclonal antibody protein complex from transgenic tobacco roots.

The secretion of a functional, full-length monoclonal antibody complex from transgenic Nicotiana tabacum roots has been demonstrated. Initially, seeds were germinated on nitrocellulose membranes and antibody secretion detected from the developing roots. Plants were then established in hydroponic culture and secretion into the growth medium measured over 25 days. Western blotting indicated that full-length antibody was present in the medium along with other fragments. Secreted antibody was shown to be functional by binding to antigen in ELISA studies. In contrast, no antibody could be detected from transgenic Nicotiana in which the same antibody was expressed as a membrane protein in the plasmalemma. These results indicate that antibody accumulation in the growth medium is genuinely caused by rhizosecretion and not cell damage. Addition of gelatin to plant growth medium markedly increased levels of antibody accumulation. The mean antibody yield per plant was calculated to be 11.7 microg per gram root dry weight per day. Rhizosecretion may be a viable alternative to agricultural production or cell culture for the generation of monoclonal antibodies in transgenic plants. It may also give rise to novel applications for antibodies expressed in plants such as removal or neutralisation of environmental pollutants and attenuation of pathogens which infect the plant via the rhizosphere.