RESUMO
Photosynthetic organisms frequently experience abiotic stress that restricts their growth and development. Under such circumstances, most absorbed solar energy cannot be used for CO2 fixation and can cause the photoproduction of reactive oxygen species (ROS) that can damage the photosynthetic reaction centers of PSI and PSII, resulting in a decline in primary productivity. This work describes a biological "switch" in the green alga Chlamydomonas reinhardtii that reversibly restricts photosynthetic electron transport (PET) at the cytochrome b6f (Cyt b6f) complex when the capacity for accepting electrons downstream of PSI is severely limited. We specifically show this restriction in STARCHLESS6 (sta6) mutant cells, which cannot synthesize starch when they are limited for nitrogen (growth inhibition) and subjected to a dark-to-light transition. This restriction represents a form of photosynthetic control that causes diminished electron flow to PSI and thereby prevents PSI photodamage but does not appear to rely on a ΔpH. Furthermore, when electron flow is restricted, the plastid alternative oxidase (PTOX) becomes active, functioning as an electron valve that dissipates some excitation energy absorbed by PSII and allows the formation of a proton motive force (PMF) that would drive some ATP production (potentially sustaining PSII repair and nonphotochemical quenching [NPQ]). The restriction at the Cyt b6f complex can be gradually relieved with continued illumination. This study provides insights into how PET responds to a marked reduction in availability of downstream electron acceptors and the protective mechanisms involved.
Assuntos
Complexo Citocromos b6f , Elétrons , Complexo Citocromos b6f/metabolismo , Transporte de Elétrons , Fotossíntese/fisiologia , Oxirredução , Oxidantes , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , LuzRESUMO
Environmental stresses dramatically impact the balance between the production of photosynthetically derived energetic electrons and Calvin-Benson-Bassham cycle (CBBC) activity; an imbalance promotes accumulation of reactive oxygen species and causes cell damage. Hence, photosynthetic organisms have developed several strategies to route electrons toward alternative outlets that allow for storage or harmless dissipation of their energy. In this work, we explore the activities of three essential outlets associated with Chlamydomonas reinhardtii photosynthetic electron transport: (i) reduction of O2 to H2O through flavodiiron proteins (FLVs) and (ii) plastid terminal oxidases (PTOX) and (iii) the synthesis of starch. Real-time measurements of O2 exchange have demonstrated that FLVs immediately engage during dark-to-light transitions, allowing electron transport when the CBBC is not fully activated. Under these conditions, we quantified maximal FLV activity and its overall capacity to direct photosynthetic electrons toward O2 reduction. However, when starch synthesis is compromised, a greater proportion of the electrons is directed toward O2 reduction through both the FLVs and PTOX, suggesting an important role for starch synthesis in priming/regulating CBBC and electron transport. Moreover, partitioning energized electrons between sustainable (starch; energetic electrons are recaptured) and nonsustainable (H2O; energetic electrons are not recaptured) outlets is part of the energy management strategy of photosynthetic organisms that allows them to cope with the fluctuating conditions encountered in nature. Finally, unmasking the repertoire and control of such energetic reactions offers new directions for rational redesign and optimization of photosynthesis to satisfy global demands for food and other resources.
Assuntos
Transporte de Elétrons/fisiologia , Fotossíntese/fisiologia , Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/fisiologia , Escuridão , Luz , Oxirredutases/metabolismo , Oxigênio/metabolismo , Proteínas de Plantas/metabolismo , Plastídeos/metabolismo , Plastídeos/fisiologiaRESUMO
PURPOSE: The spine, hip, and knee are anatomically and biomechanically connected. Total hip arthroplasty (THA) and total knee arthroplasty (TKA) are commonly employed to treat degenerative changes in the hip and knee, while fusion is used for spine degeneration. Spine deformity correction results in changes in sagittal alignment and pelvic parameters, and patients with stiff spines have higher rates of THA dislocation and revision due to instability. The goal of this study was to determine the prevalence of total joint arthroplasty (TJA) in adult spinal deformity (ASD) patients at our institution. METHODS: Following Institutional Review Board approval, we retrospectively reviewed a list of cases performed by the senior author from 4/2017 to 5/2021. Patients > 18 years old undergoing preoperative evaluation for symptomatic lumbar degeneration or ASD were included. Patients < 18 years old, those diagnosed with adolescent idiopathic scoliosis, and non-fusion cases were excluded. Perioperative full-length standing EOS images were examined for the presence or absence of THA, TKA, or both. Demographic data was collected from patient electronic medical records, and statistical analyses were completed. RESULTS: 572 consecutive cases were reviewed, and 322 were excluded. 250 cases (97M:153F) were included in the final analysis, with a mean age of 61.8 ± 11.2 years. A total of 74 patients had a TJA (29.4%). THA was present in 41 patients (16.4%), and TKA was present in 49 patients (19.6%). Males had a higher prevalence of TJA, THA, and TKA (29.9%, 16.5%, and 21.6%) than females (29.4%, 16.3%, and 18.3%). CONCLUSIONS: This study revealed a high prevalence TJA rate of 29.4% in ASD at our institution. This rate surpasses the prevalence rate reported among the general population in previous studies. High prevalence of patients with ASD and TJA may merit special surgical consideration.
Assuntos
Artroplastia de Quadril , Artroplastia do Joelho , Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Estudos Retrospectivos , Artroplastia de Quadril/estatística & dados numéricos , Prevalência , Artroplastia do Joelho/estatística & dados numéricos , Artroplastia do Joelho/métodos , Idoso , Curvaturas da Coluna Vertebral/cirurgia , Curvaturas da Coluna Vertebral/epidemiologia , Adulto , Escoliose/cirurgia , Escoliose/epidemiologiaRESUMO
Alveolar epithelial type II cells (AEC2s) are the facultative progenitors of lung alveoli and serve as the surfactant-producing cells of air-breathing organisms. Although primary human AEC2s are difficult to maintain stably in cell cultures, recent advances have facilitated the derivation of AEC2-like cells from human pluripotent stem cells (hPSCs) in vitro. Here, we provide a detailed protocol for the directed differentiation of hPSCs into self-renewing AEC2-like cells that can be maintained for up to 1 year in culture as epithelial-only spheres without the need for supporting mesenchymal feeder cells. The month-long protocol requires recapitulation of the sequence of milestones associated with in vivo development of the distal lung, beginning with differentiation of cells into anterior foregut endoderm, which is followed by their lineage specification into NKX2-1+ lung progenitors and then distal/alveolar differentiation to produce progeny that express transcripts and possess functional properties associated with AEC2s.
Assuntos
Células Epiteliais Alveolares/citologia , Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes/citologia , Células Epiteliais Alveolares/fisiologia , Animais , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Proliferação de Células , Autorrenovação Celular/genética , Autorrenovação Celular/fisiologia , Células Epiteliais/citologia , Células Alimentadoras , Humanos , Pulmão/citologia , Células-Tronco Pluripotentes/fisiologiaRESUMO
Effective derivation of functional airway organoids from induced pluripotent stem cells (iPSCs) would provide valuable models of lung disease and facilitate precision therapies for airway disorders such as cystic fibrosis. However, limited understanding of human airway patterning has made this goal challenging. Here, we show that cyclical modulation of the canonical Wnt signaling pathway enables rapid directed differentiation of human iPSCs via an NKX2-1+ progenitor intermediate into functional proximal airway organoids. We find that human NKX2-1+ progenitors have high levels of Wnt activation but respond intrinsically to decreases in Wnt signaling by rapidly patterning into proximal airway lineages at the expense of distal fates. Using this directed approach, we were able to generate cystic fibrosis patient-specific iPSC-derived airway organoids with a defect in forskolin-induced swelling that is rescued by gene editing to correct the disease mutation. Our approach has many potential applications in modeling and drug screening for airway diseases.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Organoides/metabolismo , Mucosa Respiratória/metabolismo , Fator Nuclear 1 de Tireoide/metabolismo , Via de Sinalização Wnt , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Organoides/citologia , Mucosa Respiratória/citologiaRESUMO
It has been postulated that during human fetal development, all cells of the lung epithelium derive from embryonic, endodermal, NK2 homeobox 1-expressing (NKX2-1+) precursor cells. However, this hypothesis has not been formally tested owing to an inability to purify or track these progenitors for detailed characterization. Here we have engineered and developmentally differentiated NKX2-1GFP reporter pluripotent stem cells (PSCs) in vitro to generate and isolate human primordial lung progenitors that express NKX2-1 but are initially devoid of differentiated lung lineage markers. After sorting to purity, these primordial lung progenitors exhibited lung epithelial maturation. In the absence of mesenchymal coculture support, this NKX2-1+ population was able to generate epithelial-only spheroids in defined 3D cultures. Alternatively, when recombined with fetal mouse lung mesenchyme, the cells recapitulated epithelial-mesenchymal developing lung interactions. We imaged these progenitors in real time and performed time-series global transcriptomic profiling and single-cell RNA sequencing as they moved through the earliest moments of lung lineage specification. The profiles indicated that evolutionarily conserved, stage-dependent gene signatures of early lung development are expressed in primordial human lung progenitors and revealed a CD47hiCD26lo cell surface phenotype that allows their prospective isolation from untargeted, patient-specific PSCs for further in vitro differentiation and future applications in regenerative medicine.