Human mesenchymal stem cells/multipotent stromal cells consume accumulated autophagosomes early in differentiation

Introduction: Bone marrow mesenchymal stem cells/multipotent stromal cells (MSCs) are recruited to sites of injury and subsequently support regeneration through differentiation or paracrine activity. During periods of stress such as wound site implant or differentiation, MSCs are subjected to a variety of stressors that might activate pathways to improve cell survival and generate energy. In this study, we monitored MSC autophagy in response to the process of differentiation. Methods: MSC autophagosome structures were observed by using transmission electron microscopy and a tandem green fluorescent protein-red fluorescent protein autophagic flux reporter to monitor the mammalian microtubule-associated protein-1 light chain 3 (LC3) turnover in real time. MSCs were differentiated by using standard osteogenic and adipogenic media, and autophagy was examined during short-term and long-term differentiation via immunoblots for LC3I and II. Autophagy was modulated during differentiation by using rapamycin and bafilomycin treatments to disrupt the autophagosome balance during the early stages of the differentiation process, and differentiation was monitored in the long term by using Von Kossa and Oil Red O staining as well as quantitative polymerase chain reaction analysis of typical differentiation markers. Results: We found that undifferentiated MSCs showed an accumulation of a large number of undegraded autophagic vacuoles, with little autophagic turnover. Stimulation of autophagy with rapamycin led to rapid degradation of these autophagosomes and greatly increased rough endoplasmic reticulum size. Upon induction of osteogenic differentiation, MSC expression of LC3II, a common autophagosome marker, was lost within 12 hours, consistent with increased turnover. However, during adipogenic differentiation, drug treatment to alter the autophagosome balance during early differentiation led to changes in differentiation efficiency, with inhibited adipocyte formation following rapamycin treatment and accelerated fat accumulation following autophagosome blockade by bafilomycin. Conclusions: Our findings suggest that MSCs exist in a state of arrested autophagy with high autophagosome accumulation and are poised to rapidly undergo autophagic degradation. This phenotype is highly sensitive, and a balance of autophagy appears to be key in efficient MSC differentiation and function, as evidenced by our results implicating autophagic flux in early osteogenesis and adipogenesis

Patient Frailty: Key Considerations, Definitions and Practical Implications

By 2020, the elderly (≥65-year-old) world population is projected to exceed one billion individuals. This demographic megatrend has brought topics such as physiological age and frailty to the forefront of medical research efforts around the globe. The concept of frailty has evolved significantly since the mid-twentieth century. The outdated stereotype of a “thin, stooped, slow octogenarian” has transitioned to a more scientific and objective understanding of the problem. Still, a comprehensive and concise definition of “frailty” remains elusive. Until such a definition is firmly established and universally agreed upon, clinicians continue to rely on the somewhat subjective conceptual framework of today. In this chapter, the authors review key issues pertaining to clinical management of frail patients, including diagnosis/identification, preventive strategies, therapeutic approaches, and common pitfalls. The relationship between frailty, various domains of life, and functional status is also discussed. Finally, we will touch upon the concepts of end-of-life and goals of care, focusing on their relationship to frailty

Longitudinal In Vivo Imaging of Retinal Ganglion Cells and Retinal Thickness Changes Following Optic Nerve Injury in Mice

Retinal ganglion cells (RGCs) die in sight-threatening eye diseases. Imaging RGCs in humans is not currently possible and proof of principle in experimental models is fundamental for future development. Our objective was to quantify RGC density and retinal thickness following optic nerve transection in transgenic mice expressing cyan fluorescent protein (CFP) under control of the Thy1 promoter, expressed by RGCs and other neurons.A modified confocal scanning laser ophthalmoscopy (CSLO)/spectral-domain optical coherence tomography (SD-OCT) camera was used to image and quantify CFP+ cells in mice from the B6.Cg-Tg(Thy1-CFP)23Jrs/J line. SD-OCT circle (1 B-scan), raster (37 B-scans) and radial (24 B-scans) scans of the retina were also obtained. CSLO was performed at baseline (n = 11) and 3 (n = 11), 5 (n = 4), 7 (n = 10), 10 (n = 6), 14 (n = 7) and 21 (n = 5) days post-transection, while SD-OCT was performed at baseline and 7, 14 and 35 days (n = 9) post-transection. Longitudinal change in CFP+ cell density and retinal thickness were computed. Compared to baseline, the mean (SD) percentage CFP+ cells remaining at 3, 5, 7, 10, 14 and 21 days post-transection was 86 (9)%, 63 (11)%, 45 (11)%, 31 (9)%, 20 (9)% and 8 (4)%, respectively. Compared to baseline, the mean (SD) retinal thickness at 7 days post-transection was 97 (3)%, 98 (2)% and 97 (4)% for the circle, raster and radial scans, respectively. The corresponding figures at 14 and 35 days post-transection were 96 (3)%, 97 (2)% and 95 (3)%; and 93 (3)%, 94 (3)% and 92 (3)%.Longitudinal imaging showed an exponential decline in CFP+ cell density and a small (≤8%) reduction in SD-OCT measured retinal thickness post-transection. SD-OCT is a promising tool for detecting structural changes in experimental optic neuropathy. These results represent an important step towards translation for clinical use

Prognostication of traumatic brain injury outcomes in older trauma patients: A novel risk assessment tool based on initial cranial CT findings.

INTRODUCTION: Advanced age has been traditionally associated with worse traumatic brain injury (TBI) outcomes. Although prompt neurosurgical intervention (NSI, craniotomy or craniectomy) may be life-saving in the older trauma patient, it does not guarantee survival and/or return to preinjury functional status. The aim of this study was to determine whether a simple score, based entirely on the initial cranial computed tomography (CCT) is predictive of the need for NSI and key outcome measures (e.g., morbidity and mortality) in the older (age 45+ years) TBI patient subset. We hypothesized that increasing number of categorical CCT findings is independently associated with NSI, morbidity, and mortality in older patients with severe TBI. METHODS: After IRB approval, a retrospective study of patients 45 years and older was performed using our Regional Level 1 Trauma Center registry data between June 2003 and December 2013. Collected variables included patient demographics, Injury Severity Score (ISS), Abbreviated Injury Scale Head (AISh), brain injury characteristics on CCT, Glasgow Coma Scale (GCS), Intensive Care Unit (ICU) and hospital length of stay (LOS), all-cause morbidity and mortality, functional independence scores, as well as discharge disposition. A novel CCT scoring tool (CCTST, scored from 1 to 8+) was devised, with one point given for each of the following findings: subdural hematoma, epidural hematoma, subarachnoid blood, intraventricular blood, cerebral contusion/intraparenchymal blood, skull fracture, pneumocephalus, brain edema/herniation, midline shift, and external (skin/face) trauma. Descriptive statistics and univariate analyses were conducted with 30-day mortality, in-hospital morbidity, and need for NSI as primary end-points. Secondary end-points included the length of stay in the ICU (ICULOS), step-down unit (SDLOS), and the hospital (HLOS) as well as patient functional outcomes, and postdischarge destination. Factors associated with the need for NSI were determined using matched NSI ( RESULTS: A total of 620 patients were analyzed, including 310 patients who underwent NSI and 310 age- and ISS-matched non-NSI controls. Average patient age was 72.8 ± 13.4 years (64.1% male, 99% blunt trauma, mean ISS 25.1 ± 8.68, and mean AISh/GCS of 4.63/10.9). CCTST was the only variable independently associated with NSI (AOR 1.23, 95% CI 1.06-1.42) and was inversely proportional to initial GCS and functional outcome scores on discharge. Increasing CCTST was associated with greater mortality, morbidity, HLOS, SDLOS, ICULOS, and ventilator days. On multivariate analysis, factors independently associated with mortality included AISh (AOR 2.70, 95% CI 1.21-6.00), initial GCS (AOR 1.14, 1.07-1.22), and CCTST (AOR 1.31, 1.09-1.58). Variables independently associated with in-hospital morbidity included CCTST (AOR 1.16, 1.02-1.34), GCS (AOR 1.05, 1.01-1.09), and NSI (AOR 2.62, 1.69-4.06). Multivariate models incorporating factors independently associated with each respective outcome displayed good overall predictive characteristics for mortality (AUC 0.787) and in-hospital morbidity (AUC 0.651). Finally, modified CCTST demonstrated good overall predictive ability for NSI (AUC 0.755). CONCLUSION: This study found that the number of discrete findings on CCT is independently associated with major TBI outcome measures, including 30-day mortality, in-hospital morbidity, and NSI. Of note, multivariate models with best predictive characteristics incorporate both CCTST and GCS. CCTST is easy to calculate, and this preliminary investigation of its predictive utility in older patients with TBI warrants further validation, focusing on exploring prognostic synergies between CCTST, GCS, and AISh. If independently confirmed to be predictive of clinical outcomes and the need for NSI, the approach described herein could lead to a shift in both operative and nonoperative management of patients with TBI

A high-Q superconducting toroidal medium frequency detection system with a capacitively adjustable frequency range >180 kHz

We describe a newly developed polytetrafluoroethylene/copper capacitor driven by a cryogenic piezoelectric slip-stick stage and demonstrate with the chosen layout cryogenic capacitance tuning of ≈60 pF at ≈10 pF background capacitance. Connected to a highly sensitive superconducting toroidal LC circuit, we demonstrate tuning of the resonant frequency between 345 and 685 kHz, at quality factors Q > 100 000. Connected to a cryogenic ultra low noise amplifier, a frequency tuning range between 520 and 710 kHz is reached, while quality factors Q > 86 000 are achieved. This new device can be used as a versatile image current detector in high-precision Penning-trap experiments or as an LC-circuit-based haloscope detector to search for the conversion of axion-like dark matter to radio-frequency photons. This new development increases the sensitive detection bandwidth of our axion haloscope by a factor of ≈1000