Protocol for enrichment and functional analysis of human hematopoietic cells from umbilical cord blood.

Umbilical cord blood (CB) is a donor source for hematopoietic cell therapies. Understanding what drives hematopoietic stem and progenitor cell function is critical to our understanding of the usage of CB in hematopoietic cell therapies. Here, we describe how to isolate and analyze the function of human hematopoietic cells from umbilical CB. This protocol demonstrates assays that measure phenotypic properties and hematopoietic cell potency. For complete details on the use and execution of this protocol, please refer to Broxmeyer et al.1.

DiMANI: diffusion MRI for anatomical nuclei imaging-Application for the direct visualization of thalamic subnuclei.

The thalamus is a centrally located and heterogeneous brain structure that plays a critical role in various sensory, motor, and cognitive processes. However, visualizing the individual subnuclei of the thalamus using conventional MRI techniques is challenging. This difficulty has posed obstacles in targeting specific subnuclei for clinical interventions such as deep brain stimulation (DBS). In this paper, we present DiMANI, a novel method for directly visualizing the thalamic subnuclei using diffusion MRI (dMRI). The DiMANI contrast is computed by averaging, voxelwise, diffusion-weighted volumes enabling the direct distinction of thalamic subnuclei in individuals. We evaluated the reproducibility of DiMANI through multiple approaches. First, we utilized a unique dataset comprising 8 scans of a single participant collected over a 3-year period. Secondly, we quantitatively assessed manual segmentations of thalamic subnuclei for both intra-rater and inter-rater reliability. Thirdly, we qualitatively correlated DiMANI imaging data from several patients with Essential Tremor with the localization of implanted DBS electrodes and clinical observations. Lastly, we demonstrated that DiMANI can provide similar features at 3T and 7T MRI, using varying numbers of diffusion directions. Our results establish that DiMANI is a reproducible and clinically relevant method to directly visualize thalamic subnuclei. This has significant implications for the development of new DBS targets and the optimization of DBS therapy.

Effects of Lung Injury and Abdominal Insufflation on Respiratory Mechanics and Lung Volume During Time-Controlled Adaptive Ventilation.

BACKGROUND: Lung volume measurements are important for monitoring functional aeration and recruitment, and may help guide adjustments in ventilator settings. The expiratory phase of APRV may provide physiologic information about lung volume based on the expiratory flow-time slope, angle, and time to approach a no-flow state (TExp). We hypothesized that expiratory flow rate would correlate with estimated lung volume (ELV), as measured using a modified nitrogen washout/washin technique in a large animal lung injury model. METHODS: Eight pigs (35.2±1.0kg) were mechanically ventilated using an Engström Carescape R860 on the APRV mode. All settings were held constant except the expiratory duration (TLow), which was adjusted based on the expiratory flow curve. Abdominal pressure was increased to 15mmHg in normal and Tween-injured lungs to replicate a combination of pulmonary and extrapulmonary lung injury. ELV was estimated using the Carescape FRC InView Tool. The expiratory flow-time slope and TExp were measured from the expiratory flow profile. RESULTS: Lung elastance increased with Tween-induced lung injury from 29.3±7.3cmH2O/L to 39.9±15.1cmH2O/L and chest wall elastance increased with increasing intra-abdominal pressures from 15.3±4.1cmH2O/L to 25.7±10.0cmH2O/L in the normal lung and 15.8±6.0cmH2O/L to 33.0±6.2cmH2O/L in the Tween-injured lung (p=0.39). ELV decreased from 1.90±0.83L in the Tween-Injured lung to 0.67±0.1L by increasing intra-abdominal pressures to 15mmHg. This had a significant correlation with a TExp decrease from 2.3±0.8s to 1.0±0.1s in the Tween-injured group with increasing insufflation pressures (ρ = 0.95) and with the expiratory flow-time slope, which increased from 0.29±0.06L/s2 to 0.63±0.05L/s2 (ρ = 0.78). CONCLUSIONS: Changes in ELV over time, and the TExp and flow-time slope, can be used to demonstrate evolving lung injury during APRV. Using the slope to infer changes in functional lung volume represents a unique, reproducible, real-time, bedside technique that does not interrupt ventilation and may be used for clinical interpretation.

Coupled social and ecological change drove the historical extinction of the California grizzly bear (Ursus arctos californicus).

Large carnivores (order Carnivora) are among the world's most threatened mammals due to a confluence of ecological and social forces that have unfolded over centuries. Combining specimens from natural history collections with documents from archival records, we reconstructed the factors surrounding the extinction of the California grizzly bear (Ursus arctos californicus), a once-abundant brown bear subspecies last seen in 1924. Historical documents portrayed California grizzlies as massive hypercarnivores that endangered public safety. Yet, morphological measurements on skulls and teeth generate smaller body size estimates in alignment with extant North American grizzly populations (approx. 200 kg). Stable isotope analysis (δ13C, δ15N) of pelts and bones (n = 57) revealed that grizzlies derived less than 10% of their nutrition from terrestrial animal sources and were therefore largely herbivorous for millennia prior to the first European arrival in this region in 1542. Later colonial land uses, beginning in 1769 with the Mission era, led grizzlies to moderately increase animal protein consumption (up to 26% of diet), but grizzlies still consumed far less livestock than otherwise claimed by contemporary accounts. We show how human activities can provoke short-term behavioural shifts, such as heightened levels of carnivory, that in turn can lead to exaggerated predation narratives and incentivize persecution, triggering rapid loss of an otherwise widespread and ecologically flexible animal.

Paradoxical Modulation of STN ß-Band Activity with Medication Compared to Deep Brain Stimulation.

BACKGROUND: Excessive subthalamic nucleus (STN) ß-band (13-35 Hz) synchronized oscillations has garnered interest as a biomarker for characterizing disease state and developing adaptive stimulation systems for Parkinson's disease (PD). OBJECTIVES: To report on a patient with abnormal treatment-responsive modulation in the ß-band. METHODS: We examined STN local field potentials from an externalized deep brain stimulation (DBS) lead while assessing PD motor signs in four conditions (OFF, MEDS, DBS, and MEDS+DBS). RESULTS: The patient presented here exhibited a paradoxical increase in ß power following administration of levodopa and pramipexole (MEDS), but an attenuation in ß power during DBS and MEDS+DBS despite clinical improvement of 50% or greater under all three therapeutic conditions. CONCLUSIONS: This case highlights the need for further study on the role of ß oscillations in the pathophysiology of PD and the importance of personalized approaches to the development of ß or other biomarker-based DBS closed loop algorithms. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Active contact proximity to the cerebellothalamic tract predicts initial therapeutic current requirement with DBS for ET: an application of 7T MRI.

Objective: To characterize how the proximity of deep brain stimulation (DBS) active contact locations relative to the cerebellothalamic tract (CTT) affect clinical outcomes in patients with essential tremor (ET). Background: DBS is an effective treatment for refractory ET. However, the role of the CTT in mediating the effect of DBS for ET is not well characterized. 7-Tesla (T) MRI-derived tractography provides a means to measure the distance between the active contact and the CTT more precisely. Methods: A retrospective review was conducted of 12 brain hemispheres in 7 patients at a single center who underwent 7T MRI prior to ventral intermediate nucleus (VIM) DBS lead placement for ET following failed medical management. 7T-derived diffusion tractography imaging was used to identify the CTT and was merged with the post-operative CT to calculate the Euclidean distance from the active contact to the CTT. We collected optimized stimulation parameters at initial programing, 1- and 2-year follow up, as well as a baseline and postoperative Fahn-Tolosa-Marin (FTM) scores. Results: The therapeutic DBS current mean (SD) across implants was 1.8 mA (1.8) at initial programming, 2.5 mA (0.6) at 1 year, and 2.9 mA (1.1) at 2-year follow up. Proximity of the clinically-optimized active contact to the CTT was 3.1 mm (1.2), which correlated with lower current requirements at the time of initial programming (R2 = 0.458, p = 0.009), but not at the 1- and 2-year follow up visits. Subjects achieved mean (SD) improvement in tremor control of 77.9% (14.5) at mean follow-up time of 22.2 (18.9) months. Active contact distance to the CTT did not predict post-operative tremor control at the time of the longer term clinical follow up (R2 = -0.073, p = 0.58). Conclusion: Active DBS contact proximity to the CTT was associated with lower therapeutic current requirement following DBS surgery for ET, but therapeutic current was increased over time. Distance to CTT did not predict the need for increased current over time, or longer term post-operative tremor control in this cohort. Further study is needed to characterize the role of the CTT in long-term DBS outcomes.

Insights into highly engraftable hematopoietic cells from 27-year cryopreserved umbilical cord blood.

Umbilical cord blood transplantation is a life-saving treatment for malignant and non-malignant hematologic disorders. It remains unclear how long cryopreserved units remain functional, and the length of cryopreservation is often used as a criterion to exclude older units. We demonstrate that long-term cryopreserved cord blood retains similar numbers of hematopoietic stem and progenitor cells compared with fresh and recently cryopreserved cord blood units. Long-term cryopreserved units contain highly functional cells, yielding robust engraftment in mouse transplantation models. We also leverage differences between units to examine gene programs associated with better engraftment. Transcriptomic analyses reveal that gene programs associated with lineage determination and oxidative stress are enriched in high engrafting cord blood, revealing potential molecular markers to be used as potency markers for cord blood unit selection regardless of length of cryopreservation. In summary, cord blood units cryopreserved for extended periods retain engrafting potential and can potentially be used for patient treatment.

Low-frequency deep brain stimulation reveals resonant beta-band evoked oscillations in the pallidum of Parkinson's Disease patients.

Introduction: Evidence suggests that spontaneous beta band (11-35 Hz) oscillations in the basal ganglia thalamocortical (BGTC) circuit are linked to Parkinson's disease (PD) pathophysiology. Previous studies on neural responses in the motor cortex evoked by electrical stimulation in the subthalamic nucleus have suggested that circuit resonance may underlie the generation of spontaneous and stimulation-evoked beta oscillations in PD. Whether these stimulation-evoked, resonant oscillations are present across PD patients in the internal segment of the globus pallidus (GPi), a primary output nucleus in the BGTC circuit, is yet to be determined. Methods: We characterized spontaneous and stimulation-evoked local field potentials (LFPs) in the GPi of four PD patients (five hemispheres) using deep brain stimulation (DBS) leads externalized after DBS implantation surgery. Results: Our analyses show that low-frequency (2-4 Hz) stimulation in the GPi evoked long-latency (>50 ms) beta-band neural responses in the GPi in 4/5 hemispheres. We demonstrated that neural sources generating both stimulation-evoked and spontaneous beta oscillations were correlated in their frequency content and spatial localization. Discussion: Our results support the hypothesis that the same neuronal population and resonance phenomenon in the BGTC circuit generates both spontaneous and evoked pallidal beta oscillations. These data also support the development of closed-loop control systems that modulate the GPi spontaneous oscillations across PD patients using beta band stimulation-evoked responses.

Surgical management of primary gastric Ewing-like sarcoma at the lesser curvature.

We report the case of a 65-year-old female who presented with a 6-month history of epigastric pain and dyspepsia. Computed tomography of the abdomen and pelvis showed an enhancing nodular lesion and an indeterminate 4 mm lymph node on the lesser curvature of the stomach raising concerns for gastric malignancy. Upper gastrointestinal endoscopy revealed a 10 cm malignant appearing lesion along the gastric lesser curvature. Histopathology demonstrated spindled and small round blue cell tumor with immunohistochemistry staining consistent with Ewing-like sarcoma. After multidisciplinary team discussion the patient was arranged for neoadjuvant chemotherapy with early re-imaging, followed by consideration of gastrectomy. This case highlights the unusual diagnosis of primary gastric Ewing-like sarcoma and the management of this rare condition.

Hibernation and hemostasis.

Hibernating mammals have developed many physiological adaptations to accommodate their decreased metabolism, body temperature, heart rate and prolonged immobility without suffering organ injury. During hibernation, the animals must suppress blood clotting to survive prolonged periods of immobility and decreased blood flow that could otherwise lead to the formation of potentially lethal clots. Conversely, upon arousal hibernators must be able to quickly restore normal clotting activity to avoid bleeding. Studies in multiple species of hibernating mammals have shown reversible decreases in circulating platelets, cells involved in hemostasis, as well as in protein coagulation factors during torpor. Hibernator platelets themselves also have adaptations that allow them to survive in the cold, while those from non-hibernating mammals undergo lesions during cold exposure that lead to their rapid clearance from circulation when re-transfused. While platelets lack a nucleus with DNA, they contain RNA and other organelles including mitochondria, in which metabolic adaptations may play a role in hibernator's platelet resistance to cold induced lesions. Finally, the breakdown of clots, fibrinolysis, is accelerated during torpor. Collectively, these reversible physiological and metabolic adaptations allow hibernating mammals to survive low blood flow, low body temperature, and immobility without the formation of clots during torpor, yet have normal hemostasis when not hibernating. In this review we summarize blood clotting changes and the underlying mechanisms in multiple species of hibernating mammals. We also discuss possible medical applications to improve cold preservation of platelets and antithrombotic therapy.

LIN28B and Let-7 in Diffuse Midline Glioma: A Review.

Diffuse midline glioma (DMG) is the most lethal of all childhood cancers. DMGs are driven by histone-tail-mutation-mediated epigenetic dysregulation and partner mutations in genes controlling proliferation and migration. One result of this epigenetic and genetic landscape is the overexpression of LIN28B RNA binding protein. In other systems, LIN28B has been shown to prevent let-7 microRNA biogenesis; however, let-7, when available, faithfully suppresses tumorigenic pathways and induces cellular maturation by preventing the translation of numerous oncogenes. Here, we review the current literature on LIN28A/B and the let-7 family and describe their role in gliomagenesis. Future research is then recommended, with a focus on the mechanisms of LIN28B overexpression and localization in DMG.

BATF sustains homeostasis and functionality of bone marrow Treg cells to preserve homeostatic regulation of hematopoiesis and development of B cells.

Bone marrow Treg cells (BM Tregs) orchestrate stem cell niches crucial for hematopoiesis. Yet little is known about the molecular mechanisms governing BM Treg homeostasis and function. Here we report that the transcription factor BATF maintains homeostasis and functionality of BM Tregs to facilitate homeostatic regulation of hematopoiesis and B cell development. Treg-specific ablation of BATF profoundly compromised proportions of BM Tregs associated with reduced expression of Treg effector molecules, including CD44, ICOS, KLRG1, and TIGIT. Moreover, BATF deficiency in Tregs led to increased numbers of hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), and granulocyte-macrophage progenitors (GMPs), while reducing the functionality of myeloid progenitors and the generation of common lymphoid progenitors. Furthermore, Tregs lacking BATF failed to support the development of B cells in the BM. Mechanistically, BATF mediated IL-7 signaling to promote expression of effector molecules on BM Tregs and their homeostasis. Our studies reveal a previously unappreciated role for BATF in sustaining BM Treg homeostasis and function to ensure hematopoiesis.

Lateral cerebellothalamic tract activation underlies DBS therapy for Essential Tremor.

BACKGROUND: While deep brain stimulation (DBS) therapy can be effective at suppressing tremor in individuals with medication-refractory Essential Tremor, patient outcome variability remains a significant challenge across centers. Proximity of active electrodes to the cerebellothalamic tract (CTT) is likely important in suppressing tremor, but how tremor control and side effects relate to targeting parcellations within the CTT and other pathways in and around the ventral intermediate (VIM) nucleus of thalamus remain unclear. METHODS: Using ultra-high field (7T) MRI, we developed high-dimensional, subject-specific pathway activation models for 23 directional DBS leads. Modeled pathway activations were compared with post-hoc analysis of clinician-optimized DBS settings, paresthesia thresholds, and dysarthria thresholds. Mixed-effect models were utilized to determine how the six parcellated regions of the CTT and how six other pathways in and around the VIM contributed to tremor suppression and induction of side effects. RESULTS: The lateral portion of the CTT had the highest activation at clinical settings (p < 0.05) and a significant effect on tremor suppression (p < 0.001). Activation of the medial lemniscus and posterior-medial CTT was significantly associated with severity of paresthesias (p < 0.001). Activation of the anterior-medial CTT had a significant association with dysarthria (p < 0.05). CONCLUSIONS: This study provides a detailed understanding of the fiber pathways responsible for therapy and side effects of DBS for Essential Tremor, and suggests a model-based programming approach will enable more selective activation of lateral fibers within the CTT.

Dopamine replacement therapy normalizes reactive step length to postural perturbations in Parkinson's disease.

BACKGROUND: Postural instability is one of the most disabling motor symptoms of Parkinson's disease (PD) given its association with falls and loss of independence. Previous studies have assessed biomechanical measures of reactive stepping in response to perturbations, showing that individuals with PD exhibit inadequate postural responses to regain balance. RESEARCH QUESTION: Does dopamine replacement therapy normalize step length in response to balance perturbations? METHODS: In this study, we estimated reactive step length, to a postural perturbation, retrospectively from a dataset of frontal plane video using 2D motion tracking and direct linear transform methods. We compared two perturbation methods: support surface translation and shoulder pull (the clinical standard) in 14 individuals with PD and 13 without PD (on and off medication), with and without partial body weight support (BWS). The primary outcome was the length of the first step taken to regain balance after the perturbation analyzed with mixed effects ANOVA, with post hoc analysis of anteroposterior (AP) and mediolateral (ML) components. RESULTS: PD OFF medication exhibited shorter reactive step length compared to PD ON and compared to control groups for the surface translation perturbations, but no significant difference was observed for the shoulder pull perturbations. SIGNIFICANCE: Dopamine replacement therapy affects step length in response to perturbation more robustly for surface translations than for a pull by the shoulders.

Transient regulation of RNA methylation in human hematopoietic stem cells promotes their homing and engraftment.

Enhancing the efficiency of hematopoietic stem cell (HSC) homing and engraftment is critical for cord blood (CB) hematopoietic cell transplantation (HCT). Recent studies indicate that N6-methyladenosine (m6A) modulates the expression of mRNAs that are critical for stem cell function by influencing their stability. Here, we demonstrate that inhibition of RNA decay by regulation of RNA methylation, enhances the expression of the homing receptor chemokine C-X-C receptor-4 (CXCR4) in HSCs. We show that YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), a m6A reader and FTO α-ketoglutarate dependent dioxygenase (FTO), a m6A eraser play an opposite role in this process. Through screening, we identified several FDA-approved compounds that regulate the expression of YTHDF2 and FTO in CB CD34+ cells. We show that transient downregulation of YTHDF2 or activation of FTO by using these compounds inhibits CXCR4 decay in CB HSCs and promotes their homing and engraftment. Our results demonstrate a novel regulation strategy to enhance the function of CB HSCs and provide a translational approach to enhance the clinical efficacy of HCT.

Experimental Models of Mouse and Human Hematopoietic Stem Cell Transplantation.

Experimental hematopoietic stem cell transplantation (HSCT) is an invaluable tool in determining the function and characteristics of hematopoietic stem cells (HSC) from experimental mouse and human donor groups. These groups could include, but are not limited to, genetically altered populations (gene knockout/knockin models), ex vivo manipulated cell populations, or in vivo modulated cell populations. The basic fundamentals of this process involve taking cells from a mouse/human donor source and putting them into another mouse (recipient) after preconditioning of the recipient with either total body irradiation (TBI) for mouse donor cells or into sublethally irradiated immune-deficient mice for human donor cells. Then, at pre-determined time points post-transplant, sampling a small amount of peripheral blood (PB) and at the termination of the evalaution, bone marrow (BM) to determine donor contribution and function by phenotypic analysis. Exploiting the congenic mouse strains of C57BL/6 (CD45.1- CD45.2+), BoyJ (CD45.1+ CD45.2-), and their F1-crossed hybrid C57BL/6 × BoyJ (CD45.1+ CD45.2+), we are able to quantify donor, competitor, and recipient mouse cell contributions to the engraftment state. Human donor cell engraftment (e.g., from the cord blood [CB], mobilized PB, or BM) is assessed by human cell phenotyping in sublethally irradiated immune-deficient mouse recipients (e.g., NOD scid gamma mice that are deficient in B cells, T cells, and natural killer cells and have defective dendritic cells and macrophages). Engraftment of cells from primary mouse recipients into secondary mice allows for an estimation of the self-renewal capacity of the original donor HSC. This chapter outlines concepts, methods, and techniques for mouse and human cell models of HSCT and for assessment of donor cells collected and processed in hypoxia versus ambient air.

Postural instability in Parkinson's disease assessed with clinical "pull test" and standardized postural perturbations: effect of medication and body weight support.

OBJECTIVE: This experiment tested if balance performance differed between a standardized treadmill surface perturbation task and a clinical pull test and was affected by medication or the presence of body weight support in people with Parkinson's disease (PD). METHODS: Twenty-seven individuals were tested (14 PD in both ON- and OFF-medication states). Clinical pull test and rapid forward (backward fall) translations of the support surface were applied to induce postural reactions requiring at least 1 step to restore balance. The effects of pull type (clinical vs. treadmill), partial bodyweight support (0 vs 20% body weight) and group (control, PD ON-meds and PD OFF-meds) on reactive stepping as well as practice/learning effect were examined. The number of steps taken and the first step duration were entered in linear repeated-measures mixed-effect models separately. RESULTS: The effects of pull type, group, and bodyweight support were all significant in both metrics, as was ON- vs. OFF-medication. A significant interaction term (group x pull type) was found in the first step duration, showing that the group difference was greater in treadmill compared to the clinical pull test. A significant practice effect was also observed within and across testing sessions. CONCLUSIONS: A standardized treadmill perturbation performed slightly better than the classical pull test in distinguishing between groups, and partial weight support did not substantially degrade the test's performance to detect the balance deficits in people with PD.