Small RNA Sequencing across Diverse Biofluids Identifies Optimal Methods for exRNA Isolation.

Poor reproducibility within and across studies arising from lack of knowledge regarding the performance of extracellular RNA (exRNA) isolation methods has hindered progress in the exRNA field. A systematic comparison of 10 exRNA isolation methods across 5 biofluids revealed marked differences in the complexity and reproducibility of the resulting small RNA-seq profiles. The relative efficiency with which each method accessed different exRNA carrier subclasses was determined by estimating the proportions of extracellular vesicle (EV)-, ribonucleoprotein (RNP)-, and high-density lipoprotein (HDL)-specific miRNA signatures in each profile. An interactive web-based application (miRDaR) was developed to help investigators select the optimal exRNA isolation method for their studies. miRDar provides comparative statistics for all expressed miRNAs or a selected subset of miRNAs in the desired biofluid for each exRNA isolation method and returns a ranked list of exRNA isolation methods prioritized by complexity, expression level, and reproducibility. These results will improve reproducibility and stimulate further progress in exRNA biomarker development.

exRNA Atlas Analysis Reveals Distinct Extracellular RNA Cargo Types and Their Carriers Present across Human Biofluids.

To develop a map of cell-cell communication mediated by extracellular RNA (exRNA), the NIH Extracellular RNA Communication Consortium created the exRNA Atlas resource (https://exrna-atlas.org). The Atlas version 4P1 hosts 5,309 exRNA-seq and exRNA qPCR profiles from 19 studies and a suite of analysis and visualization tools. To analyze variation between profiles, we apply computational deconvolution. The analysis leads to a model with six exRNA cargo types (CT1, CT2, CT3A, CT3B, CT3C, CT4), each detectable in multiple biofluids (serum, plasma, CSF, saliva, urine). Five of the cargo types associate with known vesicular and non-vesicular (lipoprotein and ribonucleoprotein) exRNA carriers. To validate utility of this model, we re-analyze an exercise response study by deconvolution to identify physiologically relevant response pathways that were not detected previously. To enable wide application of this model, as part of the exRNA Atlas resource, we provide tools for deconvolution and analysis of user-provided case-control studies.

Adipose triglyceride lipase promotes prostaglandin-dependent actin remodeling by regulating substrate release from lipid droplets.

Lipid droplets (LDs), crucial regulators of lipid metabolism, accumulate during oocyte development. However, their roles in fertility remain largely unknown. During Drosophila oogenesis, LD accumulation coincides with the actin remodeling necessary for follicle development. Loss of the LD-associated Adipose Triglyceride Lipase (ATGL) disrupts both actin bundle formation and cortical actin integrity, an unusual phenotype also seen when the prostaglandin (PG) synthase Pxt is missing. Dominant genetic interactions and PG treatment of follicles indicate that ATGL acts upstream of Pxt to regulate actin remodeling. Our data suggest that ATGL releases arachidonic acid (AA) from LDs to serve as the substrate for PG synthesis. Lipidomic analysis detects AA-containing triglycerides in ovaries, and these are increased when ATGL is lost. High levels of exogenous AA block follicle development; this is enhanced by impairing LD formation and suppressed by reducing ATGL. Together, these data support the model that AA stored in LD triglycerides is released by ATGL to drive the production of PGs, which promote the actin remodeling necessary for follicle development. We speculate that this pathway is conserved across organisms to regulate oocyte development and promote fertility.

Lentiviral gene therapy for X-linked chronic granulomatous disease recapitulates endogenous CYBB regulation and expression.

X-linked chronic granulomatous disease (X-CGD) is a primary immunodeficiency caused by mutations in the CYBB gene, resulting in the inability of phagocytic cells to eliminate infections. To design a lentiviral vector (LV) capable of recapitulating the endogenous regulation and expression of CYBB, a bioinformatics-guided approach was used to elucidate the cognate enhancer elements regulating the native CYBB gene. Using this approach, we analyzed a 600-kilobase topologically associated domain of the CYBB gene and identified endogenous enhancer elements to supplement the CYBB promoter to develop MyeloVec, a physiologically regulated LV for the treatment of X-CGD. When compared with an LV currently in clinical trials for X-CGD, MyeloVec showed improved expression, superior gene transfer to hematopoietic stem and progenitor cells (HSPCs), corrected an X-CGD mouse model leading to complete protection against Burkholderia cepacia infection, and restored healthy donor levels of antimicrobial oxidase activity in neutrophils derived from HSPCs from patients with X-CGD. Our findings validate the bioinformatics-guided design approach and have yielded a novel LV with clinical promise for the treatment of X-CGD.

Role of Cholesterol Metabolic Enzyme CYP46A1 and Its Metabolite 24S-Hydroxycholesterol in Ischemic Stroke.

BACKGROUND: For several decades, it has been recognized that overactivation of the glutamate-gated N-methyl-D-aspartate receptors (NMDARs) and subsequent Ca2+ toxicity play a critical role in ischemic brain injury. 24S-hydroxycholesterol (24S-HC) is a major cholesterol metabolite in the brain, which has been identified as a potent positive allosteric modulator of NMDAR in rat hippocampal neurons. We hypothesize that 24S-HC worsens ischemic brain injury via its potentiation of the NMDAR, and reducing the production of 24S-HC by targeting its synthetic enzyme CYP46A1 provides neuroprotection. METHODS: We tested this hypothesis using electrophysiological, pharmacological, and transgenic approaches and in vitro and in vivo cerebral ischemia models. RESULTS: Our data show that 24S-HC potentiates NMDAR activation in primary cultured mouse cortical neurons in a concentration-dependent manner. At 10 µmol/L, it dramatically increases the steady-state currents by 51% and slightly increases the peak currents by 20%. Furthermore, 24S-HC increases NMDA and oxygen-glucose deprivation-induced cortical neuronal injury. The increased neuronal injury is largely abolished by NMDAR channel blocker MK-801, suggesting an NMDAR-dependent mechanism. Pharmacological inhibition of CYP46A1 by voriconazole or gene knockout of Cyp46a1 dramatically reduces ischemic brain injury. CONCLUSIONS: These results identify a new mechanism and signaling cascade that critically impacts stroke outcome: CYP46A1 → 24S-HC → NMDAR → ischemic brain injury. They offer proof of principle for further development of new strategies for stroke intervention by targeting CYP46A1 or its metabolite 24S-HC.

Autologous Ex Vivo Lentiviral Gene Therapy for Adenosine Deaminase Deficiency.

BACKGROUND: Severe combined immunodeficiency due to adenosine deaminase (ADA) deficiency (ADA-SCID) is a rare and life-threatening primary immunodeficiency. METHODS: We treated 50 patients with ADA-SCID (30 in the United States and 20 in the United Kingdom) with an investigational gene therapy composed of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with a self-inactivating lentiviral vector encoding human ADA. Data from the two U.S. studies (in which fresh and cryopreserved formulations were used) at 24 months of follow-up were analyzed alongside data from the U.K. study (in which a fresh formulation was used) at 36 months of follow-up. RESULTS: Overall survival was 100% in all studies up to 24 and 36 months. Event-free survival (in the absence of reinitiation of enzyme-replacement therapy or rescue allogeneic hematopoietic stem-cell transplantation) was 97% (U.S. studies) and 100% (U.K. study) at 12 months; 97% and 95%, respectively, at 24 months; and 95% (U.K. study) at 36 months. Engraftment of genetically modified HSPCs persisted in 29 of 30 patients in the U.S. studies and in 19 of 20 patients in the U.K. study. Patients had sustained metabolic detoxification and normalization of ADA activity levels. Immune reconstitution was robust, with 90% of the patients in the U.S. studies and 100% of those in the U.K. study discontinuing immunoglobulin-replacement therapy by 24 months and 36 months, respectively. No evidence of monoclonal expansion, leukoproliferative complications, or emergence of replication-competent lentivirus was noted, and no events of autoimmunity or graft-versus-host disease occurred. Most adverse events were of low grade. CONCLUSIONS: Treatment of ADA-SCID with ex vivo lentiviral HSPC gene therapy resulted in high overall and event-free survival with sustained ADA expression, metabolic correction, and functional immune reconstitution. (Funded by the National Institutes of Health and others; ClinicalTrials.gov numbers, NCT01852071, NCT02999984, and NCT01380990.).

Sequestration to lipid droplets promotes histone availability by preventing turnover of excess histones.

Because both dearth and overabundance of histones result in cellular defects, histone synthesis and demand are typically tightly coupled. In Drosophila embryos, histones H2B, H2A and H2Av accumulate on lipid droplets (LDs), which are cytoplasmic fat storage organelles. Without LD binding, maternally provided H2B, H2A and H2Av are absent; however, how LDs ensure histone storage is unclear. Using quantitative imaging, we uncover when during oogenesis these histones accumulate, and which step of accumulation is LD dependent. LDs originate in nurse cells (NCs) and are transported to the oocyte. Although H2Av accumulates on LDs in NCs, the majority of the final H2Av pool is synthesized in oocytes. LDs promote intercellular transport of the histone anchor Jabba and thus its presence in the ooplasm. Ooplasmic Jabba then prevents H2Av degradation, safeguarding the H2Av stockpile. Our findings provide insight into the mechanism for establishing histone stores during Drosophila oogenesis and shed light on the function of LDs as protein-sequestration sites.

Discovery of a conserved translationally repressive upstream open reading frame within the iron-deficiency response regulator IDEF2.

BACKGROUND: Iron (Fe) deficiency affects 30-50% of the world's population. Genetic biofortification of staple crops is a promising strategy for improving human nutrition, but the number of effective precision breeding targets for Fe biofortification is small. Upstream open reading frames (uORFs) are cis-regulatory elements within the 5' leader sequence (LS) of genes that generally repress translation of the main open reading frame (mORF). RESULTS: We aligned publicly available rice (Oryza sativa L.) ribo-seq datasets and transcriptomes to identify putative uORFs within important Fe homeostasis genes. A dual luciferase assay (DLA) was used to determine whether these uORFs cause repression of mORF translation and pinpoint LS regions that can be mutated for mORF derepression. A translationally repressive uORF region was identified in two positive regulators of the Fe-deficiency response: IDEF1 and IDEF2. The IDEF2-uORF peptide was highly conserved among monocots and a mutation series in the 5' LS of the wheat (Triticum aestivum L.) TaIDEF2-A1 gene demonstrated variable mORF derepression. CONCLUSIONS: Together these results reveal a possible regulatory mechanism by which IDEF2 transcription factors modulate the Fe deficiency response in monocots, and highlight novel precision breeding targets to improve crop nutrition and abiotic stress tolerance.

A Delphi Method for Development of a Barrett's Esophagus Question Prompt List as a Communication Tool for Optimal Patient-physician Communication.

BACKGROUND METHODS: The question prompt list content was derived through a modified Delphi process consisting of 3 rounds. In round 1, experts provided 5 answers to the prompts "What general questions should patients ask when given a new diagnosis of Barrett's esophagus" and "What questions do I not hear patients asking, but given my expertise, I believe they should be asking?" Questions were reviewed and categorized into themes. In round 2, experts rated questions on a 5-point Likert scale. In round 3, experts rerated questions modified or reduced after the previous rounds. Only questions rated as "essential" or "important" were included in Barrett's esophagus question prompt list (BE-QPL). To improve usability, questions were reduced to minimize redundancy and simplified to use language at an eighth-grade level (Fig. 1). RESULTS: Twenty-one esophageal medical and surgical experts participated in both rounds (91% males; median age 52 years). The expert panel comprised of 33% esophagologists, 24% foregut surgeons, and 24% advanced endoscopists, with a median of 15 years in clinical practice. Most (81%), worked in an academic tertiary referral hospital. In this 3-round Delphi technique, 220 questions were proposed in round 1, 122 (55.5%) were accepted into the BE-QPL and reduced down to 76 questions (round 2), and 67 questions (round 3). These 67 questions reached a Flesch Reading Ease of 68.8, interpreted as easily understood by 13 to 15 years olds. CONCLUSIONS: With multidisciplinary input, we have developed a physician-derived BE-QPL to optimize patient-physician communication. Future directions will seek patient feedback to distill the questions further to a smaller number and then assess their usability.

Mental health during the COVID-19 pandemic: the importance of social support in midlife women.

OBJECTIVE: This study aimed to examine sex differences in factors associated with mood and anxiety in midlife men and women during the COVID-19 pandemic. METHODS: During a remote visit, 312 adults aged 40-60 years (167 female; 23.6% perimenopausal) from the Human Connectome Project in Aging completed PROMIS measures of depression, anxiety and anger/irritability; perceived stress; and questions about social support, financial stress and menopause stage. Multivariate linear regression models assessed sex differences in mental health and the association of social support, financial stress and menopause stage with mental health. RESULTS: Anxiety was higher in women than in men (b = 2.39, p = 0.02). For women only, decreased social support was associated with increased anxiety (b = -2.26, p = 0.002), anger/irritability (b = -1.89, p = 0.02) and stress (b = -1.67, p = 0.002). For women only, not having close family was associated with increased depressive symptoms (b = -6.60, p = 0.01) and stress (b = -7.03, p < 0.001). For both sexes, having children was associated with lower depressive symptoms (b = -3.08, p = 0.002), anxiety (b = -1.93, p = 0.07), anger/irritability (b = -2.73, p = 0.02) and stress (b = -1.44, p = 0.07). Menopause stage was unrelated to mental health. CONCLUSION: Social support, but not financial stress, influenced mental health during the COVID-19 pandemic at midlife, particularly for women.

Postoperative complications and unanticipated healthcare encounters following mini-laparotomy vs. laparoscopic/robotic-assisted sacrocolpopexy: a comparative retrospective study.

BACKGROUND: Pelvic organ prolapse is a debilitating condition impacting lives of millions of women worldwide. Sacrocolpopexy (SCP) is considered an effective and durable surgical technique for treatment of apical prolapse. The aim of this study was to compare short-term outcomes including postoperative complications and unanticipated healthcare encounters between patients who underwent SCP with a mini-laparotomy approach compared to patients treated with laparoscopic and robotic-assisted laparoscopic SCP. METHODS: This was a retrospective cohort study including patients treated for apical prolapse at a university affiliated urogynecology practice. Patients over the age of 18 who underwent abdominal SCP between 2019 and 2023 were included. The cohort was formed into two groups: (1) Patients who underwent SCP through a mini-laparotomy incision (Mini-lap group); (2) Patients who underwent laparoscopic or robotic-assisted laparoscopic SCP (Lap/Robot group). RESULTS: A total of 116 patients were included in the final analysis. Ninety patients underwent either laparoscopic or robotic-assisted SCP, whereas 26 patients underwent SCP with a mini-laparotomy approach. Study participants exhibited a mean age of 63.1 ± 10.3 years, mean body mass index (BMI) of 25.8 ± 4.9 Kg/m2, and 77.6% of them identified as Caucasian. Upon comparison of demographic and past medical history between groups there were no statistically significant differences in age, BMI, menopausal status, race, parity or comorbid conditions. Patients in the Mini-lap group were less likely to have undergone previous abdominal surgery (11.5% vs. 50.6%, p < 0.001) and had more severe apical prolapse (stage 4 prolapse, 40% vs. 21.2%, p < 0.001) than their counterparts in the Lap/robot group. Regarding intraoperative parameters, length of surgery was significantly shorter in the Mini-lap group compared to the Lap/robot group (97.3 ± 35.0 min vs. 242.0 ± 52.6 min, p < 0.001). When focusing on the primary outcome, postoperative complications within the first 30 days after surgery, there were no differences noted between groups. Additionally, the number of unanticipated healthcare encounters, such as phone calls, clinic visits, emergency department visits, urgent care visits, readmissions and reoperations were similar between groups. CONCLUSIONS: Mini-laparotomy approach for SCP is safe with comparable intra- and postoperative complications, and unanticipated healthcare encounters compared to conventional minimally invasive methods.

Assessment of Urinary Dysfunction After Midurethral Sling Placement: A Comparison of Two Voiding Trial Methods.

STUDY OBJECTIVE: Temporary urinary retention after midurethral sling (MUS) surgery requiring indwelling catheter or self-catheterization usage is common. Different methods for assessment of immediate postoperative urinary retention have been described. This study aimed to compare postoperative voiding trial (VT) success after active vs passive VT in women undergoing MUS surgery. DESIGN: Comparative retrospective cohort study. SETTING: Female pelvic medicine and reconstructive surgery practice at a university-affiliated tertiary medical center. PATIENTS: Patients with stress urinary incontinence who underwent surgical treatment during the study period were eligible for inclusion. Excluded were patients younger than the age of 18 years, combined cases with other surgical services, planned laparotomy, and a history of urinary retention and patients for whom their VT was performed on postoperative day 1. The cohort was divided into 2 groups: (1) patients who underwent an active retrofill of their bladder using a Foley catheter and (2) patients who were allowed to have a spontaneous void. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: A total of 285 patients met the inclusion criteria for the study. Of these subjects, 94 underwent an active VT and 191 underwent a passive VT. There were no statistically significant differences in immediate postoperative urinary retention (30.8% vs 29.3%; p = .79) or time from surgery end to VT (233.0 ± 167.6 minutes vs 203.1 ± 147.8 minutes; p = .13) between groups. Urinary retention, as defined by a failed VT, increased from 10% to 29.3% when MUS placement was accompanied by concomitant prolapse repair procedure. Multivariate logistic regression analysis revealed that undergoing a combined anterior and posterior colporrhaphy (odds ratio [OR], 5.13; p <.001) and undergoing an apical prolapse procedure (OR, 2.75; p = .004) were independently associated with immediate postoperative urinary retention whereas increased body mass index (OR, 0.89; p <.001) lowered likelihood of retention. CONCLUSION: The method used to assess immediate postoperative urinary retention did not affect VT success. Concomitant combined anterior and posterior colporrhaphy and apical suspension were correlated with greater likelihood of VT failure whereas increased body mass index decreased odds of retention.

Impact of fed-batch process intensification on the productivity and product quality of two CHO cell lines expressing unique novel molecular format proteins.

While monospecific antibodies have long been the foundational offering of protein therapeutics, recent advancements in antibody engineering have allowed for the development of far more complex antibody structures. Novel molecular format (NMF) proteins, such as bispecific antibodies (BsAbs), are structures capable of multispecific binding, allowing for expanded therapeutic functionality. As demand for NMF proteins continues to rise, biomanufacturers face the challenge of increasing bioreactor process productivity while simultaneously maintaining consistent product quality. This challenge is exacerbated when producing structurally complex proteins with asymmetric modalities, as seen in NMFs. In this study, the impact of a high inoculation density (HID) fed-batch process on the productivity and product quality attributes of two CHO cell lines expressing unique NMFs, a monospecific antibody with an Fc-fusion protein and a bispecific antibody, compared to low inoculation density (LID) platform fed-batch processes was evaluated. It was observed that an intensified platform fed-batch process increased product concentrations by 33 and 109% for the two uniquely structured complex proteins in a shorter culture duration while maintaining similar product quality attributes to traditional fed-batch processes.

Changes in white matter microstructure following serial ketamine infusions in treatment resistant depression.

Ketamine produces fast-acting antidepressant effects in treatment resistant depression (TRD). Though prior studies report ketamine-related changes in brain activity in TRD, understanding of ketamine's effect on white matter (WM) microstructure remains limited. We thus sought to examine WM neuroplasticity and associated clinical improvements following serial ketamine infusion (SKI) in TRD. TRD patients (N = 57, 49.12% female, mean age: 39.9) received four intravenous ketamine infusions (0.5 mg/kg) 2-3 days apart. Diffusion-weighted scans and clinical assessments (Hamilton Depression Rating Scale [HDRS-17]; Snaith Hamilton Pleasure Scale [SHAPS]) were collected at baseline and 24-h after SKI. WM measures including the neurite density index (NDI) and orientation dispersion index (ODI) from the neurite orientation dispersion and density imaging (NODDI) model, and fractional anisotropy (FA) from the diffusion tensor model were compared voxelwise pre- to post-SKI after using Tract-Based Spatial Statistics workflows to align WM tracts across subjects/time. Correlations between change in WM metrics and clinical measures were subsequently assessed. Following SKI, patients showed significant improvements in HDRS-17 (p-value = 1.8 E-17) and SHAPS (p-value = 1.97 E-10). NDI significantly decreased in occipitotemporal WM pathways (p < .05, FWER/TFCE corrected). ΔSHAPS significantly correlated with ΔNDI in the left internal capsule and left superior longitudinal fasciculus (r = -0.614, p-value = 6.24E-09). No significant changes in ODI or FA were observed. SKI leads to significant changes in the microstructural features of neurites within occipitotemporal tracts, and changes in neurite density within tracts connecting the basal ganglia, thalamus, and cortex relate to improvements in anhedonia. NODDI may be more sensitive for detecting ketamine-induced WM changes than DTI.

NGPINT: a next-generation protein-protein interaction software.

Mapping protein-protein interactions at a proteome scale is critical to understanding how cellular signaling networks respond to stimuli. Since eukaryotic genomes encode thousands of proteins, testing their interactions one-by-one is a challenging prospect. High-throughput yeast-two hybrid (Y2H) assays that employ next-generation sequencing to interrogate complementary DNA (cDNA) libraries represent an alternative approach that optimizes scale, cost and effort. We present NGPINT, a robust and scalable software to identify all putative interactors of a protein using Y2H in batch culture. NGPINT combines diverse tools to align sequence reads to target genomes, reconstruct prey fragments and compute gene enrichment under reporter selection. Central to this pipeline is the identification of fusion reads containing sequences derived from both the Y2H expression plasmid and the cDNA of interest. To reduce false positives, these fusion reads are evaluated as to whether the cDNA fragment forms an in-frame translational fusion with the Y2H transcription factor. NGPINT successfully recognized 95% of interactions in simulated test runs. As proof of concept, NGPINT was tested using published data sets and it recognized all validated interactions. NGPINT can process interaction data from any biosystem with an available genome or transcriptome reference, thus facilitating the discovery of protein-protein interactions in model and non-model organisms.

Light-induced displacement of PLASTID MOVEMENT IMPAIRED1 precedes light-dependent chloroplast movements.

Light-dependent chloroplast movements are an actin-dependent cellular response to changes in the light environment that help plants maximize photosynthetic potential and reduce photodamage. Over a dozen proteins are known to be required for normal chloroplast movements, but the molecular mechanisms regulating the transformation of light perception into chloroplast motility are not fully understood. Here, we show that in Arabidopsis (Arabidopsis thaliana) the actin-bundling plasma membrane-associated proteins THRUMIN1, PLASTID MOVEMENT IMPAIRED1 (PMI1), and KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT1 (KAC1) interact through the 14-3-3 proteins KAPPA and OMEGA. We also show that the interaction of PMI1 with 14-3-3 KAPPA and OMEGA is regulated by blue light activation of the Phototropin2 photoreceptor. Live-cell confocal microscopy revealed light-induced dynamic changes in the cellular localizations of PMI1 and KAC1. In particular, PMI1 was relocated away from irradiated areas of the plasma membrane in less than a minute after blue light exposure, consistent with PMI1 playing a critical role in initiating light-dependent chloroplast movements. We present a modified conceptual model for high light-dependent chloroplast movements in which PMI1 acts as the mobile signal that initiates a coordinated sequence of changes in protein-protein and protein-plasma membrane interactions that initiate the chloroplast movement response and determine where in the cell chloroplasts are able to anchor to the plasma membrane.

Long-term outcomes after gene therapy for adenosine deaminase severe combined immune deficiency.

Patients lacking functional adenosine deaminase activity have severe combined immunodeficiency (ADA SCID), which can be treated with ADA enzyme replacement therapy (ERT), allogeneic hematopoietic stem cell transplantation (HSCT), or autologous HSCT with gene-corrected cells (gene therapy [GT]). A cohort of 10 ADA SCID patients, aged 3 months to 15 years, underwent GT in a phase 2 clinical trial between 2009 and 2012. Autologous bone marrow CD34+ cells were transduced ex vivo with the MND (myeloproliferative sarcoma virus, negative control region deleted, dl587rev primer binding site)-ADA gammaretroviral vector (gRV) and infused following busulfan reduced-intensity conditioning. These patients were monitored in a long-term follow-up protocol over 8 to 11 years. Nine of 10 patients have sufficient immune reconstitution to protect against serious infections and have not needed to resume ERT or proceed to secondary allogeneic HSCT. ERT was restarted 6 months after GT in the oldest patient who had no evidence of benefit from GT. Four of 9 evaluable patients with the highest gene marking and B-cell numbers remain off immunoglobulin replacement therapy and responded to vaccines. There were broad ranges of responses in normalization of ADA enzyme activity and adenine metabolites in blood cells and levels of cellular and humoral immune reconstitution. Outcomes were generally better in younger patients and those receiving higher doses of gene-marked CD34+ cells. No patient experienced a leukoproliferative event after GT, despite persisting prominent clones with vector integrations adjacent to proto-oncogenes. These long-term findings demonstrate enduring efficacy of GT for ADA SCID but also highlight risks of genotoxicity with gRVs. This trial was registered at www.clinicaltrials.gov as #NCT00794508.

Soluble components from mesenchymal stromal cell processing exert anti-inflammatory effects and facilitate ischemic muscle regeneration.

BACKGROUND AIMS: Skeletal muscle regeneration after severe damage is reliant on local stem cell proliferation and differentiation, processes that are tightly regulated by macrophages. Peripheral artery disease is a globally prevalent cardiovascular disease affecting millions of people. Progression of the disease leads to intermittent claudication, subsequent critical limb ischemia and muscle injury. Tissue-derived and ex vivo-expanded mesenchymal stromal cells (MSCs) for skeletal muscle regeneration have been studied, but pre-clinical and clinical results have not been consistent. As a result, the potential therapeutic efficacy and associated repair mechanisms of MSCs remain unclear. Numerous studies have demonstrated the vulnerability of delivered MSCs, with a precipitous drop in cell viability upon transplantation. This has prompted investigation into the therapeutic benefit of apoptotic cells, microvesicles, exosomes and soluble signals that are released upon cell death. METHODS: In this study, we characterized various components produced by MSCs after cell death induction under different conditions. We discovered anti-inflammatory and pro-regenerative effects produced by cell components following a freeze and thaw (F&T) process on macrophage polarization in vitro. We further investigated the underlying mechanisms of macrophage polarization by those components resulting from severe cell death induction. RESULTS: We found potent therapeutic effects from F&T-induced cell debris are dependent on the externalization of phosphatidylserine on the plasma membrane. In contrast, effects from the supernatant of F&T-induced cell death primarily depends on the released protein content. We then applied the F&T-induced cell supernatant to an animal model of peripheral artery disease to treat muscle injury caused by severe ischemia. Treatment with the F&T supernatant but not the vulnerable MSCs resulted in significantly improved recovery of muscle function, blood flow and morphology and inflammation resolution in the affected muscles 2 weeks after injury. CONCLUSIONS: This study validates the therapeutic potential of F&T-induced supernatant obviating the need for a viable population from vulnerable MSCs to treat injury, thus providing a roadmap for cell-free therapeutic approaches for tissue regeneration.

Human land-uses homogenize stream assemblages and reduce animal biomass production.

Human land-use change is a major threat to natural ecosystems worldwide. Nonetheless, the effects of human land-uses on the structure of plant and animal assemblages and their functional characteristics need to be better understood. Furthermore, the pathways by which human land uses affect ecosystem functions, such as biomass production, still need to be clarified. We compiled a unique dataset of fish, arthropod and macrophyte assemblages from 61 stream ecosystems in two Neotropical biomes: Amazonian rainforest and Uruguayan grasslands. We then tested how the cover of agriculture, pasture, urbanization and afforestation affected the taxonomic richness and functional diversity of those three species assemblages, and the consequences of these effects for animal biomass production. Single trait categories and functional diversity were evaluated, combining recruitment and life-history, resource and habitat-use, and body size. The effects of intensive human land-uses on taxonomic and functional diversities were as strong as other drivers known to affect biodiversity, such as local climate and environmental factors. In both biomes, the taxonomic richness and functional diversity of animal and macrophyte assemblages decreased with increasing cover of agriculture, pasture, and urbanization. Human land-uses were associated with functional homogenization of both animal and macrophyte assemblages. Human land-uses reduced animal biomass through direct and indirect pathways mediated by declines in taxonomic and functional diversities. Our findings indicate that converting natural ecosystems to supply human demands results in species loss and trait homogenization across multiple biotic assemblages, ultimately reducing animal biomass production in streams.

A prospective, randomized trial comparing intravesical dimethyl sulfoxide (DMSO) to bupivacaine, triamcinolone, and heparin (BTH), for newly diagnosed interstitial cystitis/painful bladder syndrome (IC/PBS).

INTRODUCTION AND HYPOTHESIS: The primary aim of this study was to compare the effect of bladder instillations using dimethyl sulfoxide (DMSO) with triamcinolone versus bupivacaine, triamcinolone, and heparin (BTH) in women with newly diagnosed interstitial cystitis/painful bladder syndrome. The primary outcome was improvement in symptoms measured using the O'Leary-Sant Interstitial Cystitis Symptoms Index (ICSI) score. Secondary comparisons included changes in urinary frequency, nocturia, and bladder capacity. MATERIALS AND METHODS: This was a prospective, randomized study. Patients with a recent diagnosis of interstitial cystitis/painful bladder syndrome (IC/PBS) were randomized 1:1 to treatment with either 6 weekly bladder instillations of DMSO with triamcinolone or BTH. During follow-up visits, patients completed the ICSI questionnaire, and bladder capacity was determined through the retrograde filling of the bladder. The χ2 test or Student's t test were used for data analysis. RESULTS: A total of 83 patients were randomized, and final analysis included 70 participants who completed the 6 weekly instillations (42 DMSO, 28 BTH). The groups were similar in baseline demographics and clinical characteristics, except for cystometric maximum capacity (DMSO 338.62± 139.44 mL, BTH 447.43 ± 180.38 mL, p = 0.01). In the DMSO group, 63% of patients had a greater than 29.5% reduction in total ICSI score versus 43% in the BTH group (p = 0.15). Nocturia and pain were significantly reduced in the DMSO group. There was a significant increase from baseline in bladder capacity for both groups. CONCLUSION: In women with newly diagnosed IC/PBS, bladder instillations with DMSO and triamcinolone provide greater improvement in pain and nocturia compared to BTH.