OxPhos Dysfunction Causes Hypermetabolism and Reduces Lifespan in Cells and in Patients with Mitochondrial Diseases

Patients with primary mitochondrial diseases present with fatigue and multi-system disease, are often lean, and die prematurely, but the mechanistic basis for this clinical picture remains unclear. Integrating data from 17 cohorts of patients with mitochondrial diseases (n=690), we find that clinical mitochondrial disorders increase resting energy expenditure, a state termed hypermetabolism. In a longitudinal cellular model of primary patient-derived fibroblasts from multiple donors, we show that genetic and pharmacological disruptions of oxidative phosphorylation (OxPhos) similarly trigger increased energy consumption in a cell-autonomous manner, despite near-normal OxPhos coupling efficiency. Hypermetabolism was associated with mtDNA instability, activation of the integrated stress response, increased extracellular secretion of age-related cytokines and metabokines including GDF15, as well as an accelerated rate of telomere erosion and epigenetic aging, and a reduced Hayflick limit. Finally, we generate a longitudinal RNASeq and DNA methylation resource dataset, which reveals conserved, energetically demanding, genome-wide recalibrations to OxPhos dysfunction. Hypermetabolism, or the increased energetic cost of living in mitochondrial diseases, has important biological and clinical implications.

Vaccine genetics of IGHV1-2 VRC01-class broadly neutralizing antibody precursor naïve human B cells

A successful HIV vaccine eliciting broadly neutralizing antibodies (bnAbs) must overcome the hurdle of being able to activate naive precursor B cells encoding features within their germline B cell receptors (BCR) that allow recognition of broadly neutralizing epitopes. Knowledge of whether bnAb precursor B cells are circulating at sufficient frequencies within individuals in communities heavily impacted by HIV may be important. Using a germline-targeting eOD-GT8 immunogen and high-throughput droplet-based single-cell BCR sequencing, we demonstrate that large numbers of paired BCR sequences from multiple donors can be efficiently screened to elucidate precursor frequencies of rare, naive VRC01-class B cells. Further, we analyzed IGHV1-2 allelic usage among three different cohorts; we find that IGHV1-2 alleles traditionally thought to be incompatible with VRC01-class responses are relatively common in various human populations and that germline variation within IGHV1-2 associates with gene usage frequencies in the naive BCR repertoire.

Functional heterogeneity of IFN-γ–licensed mesenchymal stromal cell immunosuppressive capacity on biomaterials

Mesenchymal stromal cells (MSCs) are increasingly combined with biomaterials to enhance their therapeutic properties, including their immunosuppressive function. However, clinical trials utilizing MSCs with or without biomaterials have shown limited success, potentially due to their functional heterogeneity across different donors and among different subpopulations of cells. Here, we evaluated the immunosuppressive capacity, as measured by the ability to reduce T-cell proliferation and activation, of interferon-gamma (IFN-γ)–licensed MSCs from multiple donors on fibrin and collagen hydrogels, the two most commonly utilized biomaterials in combination with MSCs in clinical trials worldwide according to ClinicalTrials.gov. Variations in the immunosuppressive capacity between IFN-γ–licensed MSC donors on the biomaterials correlated with the magnitude of indoleamine-2,3-dioxygenase activity. Immunosuppressive capacity of the IFN-γ–licensed MSCs depended on the αV/α5 integrins when cultured on fibrin and on the α2/β1 integrins when cultured on collagen. While all tested MSCs were nearly 100% positive for these integrins, sorted MSCs that expressed higher levels of αV/α5 integrins demonstrated greater immunosuppressive capacity with IFN-γ licensing than MSCs that expressed lower levels of these integrins on fibrin. These findings were equivalent for MSCs sorted based on the α2/β1 integrins on collagen. These results demonstrate the importance of integrin engagement to IFN-γ licensed MSC immunosuppressive capacity and that IFN-γ–licensed MSC subpopulations of varying immunosuppressive capacity can be identified by the magnitude of integrin expression specific to each biomaterial.

Assessing donor-to-donor variability in human intestinal organoid cultures

Donor-to-donor variability in primary human organoid cultures has not been well characterized. As these cultures contain multiple cell types, there is greater concern that variability could lead to increased noise. In this work we investigated donor-to-donor variability in human gut adult stem cell (ASC) organoids. We examined intestinal developmental pathways during culture differentiation in ileum- and colon-derived cultures established from multiple donors, showing that differentiation patterns were consistent among cultures. This finding indicates that donor-to-donor variability in this system remains at a manageable level. Intestinal metabolic activity was evaluated by targeted analysis of central carbon metabolites and by analyzing hormone production patterns. Both experiments demonstrated similar metabolic functions among donors. Importantly, this activity reflected intestinal biology, indicating that these ASC organoid cultures are appropriate for studying metabolic processes. This work establishes a framework for generating high confidence data using human primary cultures through thorough characterization of variability.

Individual heterogeneity screened umbilical cord-derived mesenchymal stromal cells with high Treg promotion demonstrate improved recovery of mouse liver fibrosis

Background To investigate the heterogeneities of human umbilical cord mesenchymal stromal cells (HUCMSCs) derived from different donors and their therapeutic variations when applied to mouse liver fibrosis model. Methods The characteristics of HUCMSCs derived from multiple donors were comprehensively analyzed including expressions of surface markers, viability, growth curve, karyotype analysis, tumorigenicity, differentiation potentials, and immune regulation capability. Then, the HUCMSCs with distinct immunomodulatory effects were applied to treat mouse liver fibrosis and their therapeutic effects were observed. Results The HUCMSCs derived from multiple donors kept a high consistency in surface marker expressions, viability, growth curve, and tumorigenicity in nude mice but had robust heterogeneities in differentiation potentials and immune regulations. In addition, three HUCMSC lines applied to mice liver fibrosis model had different therapeutic outcomes, in line with individual immune regulation capability. Conclusion The HUCMSCs derived from different donors have individual heterogeneity, which potentially lead to distinct therapeutic outcomes in mouse liver fibrosis, indicating we could make use of the donor-variation of MSCs to screen out guaranteed general indicators of MSCs for specific diseases in further stromal cell therapy.

An Evaluation of the Sagarmatha National Park Forestry Project (Snpfp), Khumbu, Nepal: A Community Stakeholder Approach

This study adopts a community stakeholder approach to analyze the stakeholders' perceptions on foreign aid and NGO-driven reforestation programme in an impacted community. The focus of the study, the Sagarmatha National Park Forestry Project (SNPFP), has operated in Khumbu, Nepal for thirty years. The overall performance and impacts of the SNPFP were assessed by interviewing key informants with regard to their experience and perceptions. Qualitative analysis revealed the gap in the involvement of multiple donors and identified how these gaps impacted on quality of foreign aid and NGO-led project. The implications of this study include the recommendation that the immediate stakeholders in a local area are a reliable source of information to measure the value of foreign aid and NGO performance. Furthermore, the future of natural resource conservation and rural development led by foreign aid and NGOs depends on collaboration between the local people, the NGOs, donors, and the government.

An Evaluation of the Sagarmatha National Park Forestry Project (Snpfp), Khumbu, Nepal: A Community Stakeholder Approach

This study adopts a community stakeholder approach to analyze the stakeholders' perceptions on foreign aid and NGO-driven reforestation programme in an impacted community. The focus of the study, the Sagarmatha National Park Forestry Project (SNPFP), has operated in Khumbu, Nepal for thirty years. The overall performance and impacts of the SNPFP were assessed by interviewing key informants with regard to their experience and perceptions. Qualitative analysis revealed the gap in the involvement of multiple donors and identified how these gaps impacted on quality of foreign aid and NGO-led project. The implications of this study include the recommendation that the immediate stakeholders in a local area are a reliable source of information to measure the value of foreign aid and NGO performance. Furthermore, the future of natural resource conservation and rural development led by foreign aid and NGOs depends on collaboration between the local people, the NGOs, donors, and the government.

Vaccine genetics of IGHV1-2 VRC01-class broadly neutralizing antibody precursor naïve human B cells

ABSTRACTA successful HIV vaccine must overcome the hurdle of being able to activate naïve precursor B cells encoding features within their germline B cell receptors (BCR) that allow recognition of broadly neutralizing epitopes. Knowledge of whether broadly neutralizing antibody (bnAb) precursor B cells are circulating at sufficient frequencies within individuals in communities heavily impacted by HIV may be important. Using a germline-targeting eOD-GT8 immunogen and high-throughput droplet-based single cell BCR sequencing, we demonstrate that large numbers of paired BCR sequences from multiple donors can be efficiently screened to elucidate precursor frequencies of rare, naïve VRC01-class B cells. The results indicate that IGHV1-2 alleles incompatible with VRC01-class responses are relatively common in various human populations, and germline variation within IGHV1-2 associates with gene usage frequencies in the naïve BCR repertoire.