Rat primary cortical cell tri-culture to study effects of amyloid-beta on microglia function.

Introduction: The etiology and progression of sporadic Alzheimer's Disease (AD) have been studied for decades. One proposed mechanism is that amyloid-beta (Aß) proteins induce neuroinflammation, synapse loss, and neuronal cell death. Microglia play an especially important role in Aß clearance, and alterations in microglial function due to aging or disease may result in Aß accumulation and deleterious effects on neuronal function. However, studying these complex factors in vivo , where numerous confounding processes exist, is challenging, and until recently, in vitro models have not allowed sustained culture of microglia, astrocytes and neurons in the same culture. Here, we employ a tri-culture model of rat primary neurons, astrocytes, and microglia and compare it to co-culture (neurons and astrocytes) and mono-culture enriched for microglia to study microglial function (i.e., motility and Aß clearance) and proteomic response to exogenous Aß. Methods: We established cortical co-culture (neurons and astrocytes), tri-culture (neurons, astrocytes, and microglia), and mono-culture (microglia) from perinatal rat pups. On days in vitro (DIV) 7 - 14, the cultures were exposed to fluorescently-labeled Aß (FITC-Aß) particles for varying durations. Images were analyzed to determine the number of FITC-Aß particles after specific lengths of exposure. A group of cells were stained for ßIII-tubulin, GFAP, and Iba1 for morphological analysis via quantitative fluorescence microscopy. Cytokine profiles from conditioned media were obtained. Live-cell imaging with images acquired every 5 minutes for 4 hours was employed to extract microglia motility parameters (e.g., Euclidean distance, migration speed, directionality ratio). Results and discussion: FITC-Aß particles were more effectively cleared in the tri-culture compared to the co-culture. This was attributed to microglia engulfing FITC-Aß particles, as confirmed via epifluorescence and confocal microscopy. Adding FITC-Aß significantly increased the size of microglia, but had no significant effect on neuronal surface coverage or astrocyte size. Analysis of the cytokine profile upon FITC-Aß addition revealed a significant increase in proinflammatory cytokines (TNF-α, IL-1α, IL-1ß, IL-6) in tri-culture, but not co-culture. In addition, Aß addition altered microglia motility marked by swarming-like motion with decreased Euclidean distance yet unaltered speed. These results highlight the importance of cell-cell communication in microglia function (e.g., motility and Aß clearance) and the utility of the tri-culture model to further investigate microglia dysfunction in AD.

Effects of Spermine Synthase Deficiency in Mesenchymal Stromal Cells Are Rescued by Upstream Inhibition of Ornithine Decarboxylase.

Despite the well-known relevance of polyamines to many forms of life, little is known about how polyamines regulate osteogenesis and skeletal homeostasis. Here, we report a series of in vitro studies conducted with human-bone-marrow-derived pluripotent stromal cells (MSCs). First, we show that during osteogenic differentiation, mRNA levels of most polyamine-associated enzymes are relatively constant, except for the catabolic enzyme spermidine/spermine N1-acetyltransferase 1 (SAT1), which is strongly increased at both mRNA and protein levels. As a result, the intracellular spermidine to spermine ratio is significantly reduced during the early stages of osteoblastogenesis. Supplementation of cells with exogenous spermidine or spermine decreases matrix mineralization in a dose-dependent manner. Employing N-cyclohexyl-1,3-propanediamine (CDAP) to chemically inhibit spermine synthase (SMS), the enzyme catalyzing conversion of spermidine into spermine, also suppresses mineralization. Intriguingly, this reduced mineralization is rescued with DFMO, an inhibitor of the upstream polyamine enzyme ornithine decarboxylase (ODC1). Similarly, high concentrations of CDAP cause cytoplasmic vacuolization and alter mitochondrial function, which are also reversible with the addition of DFMO. Altogether, these studies suggest that excess polyamines, especially spermidine, negatively affect hydroxyapatite synthesis of primary MSCs, whereas inhibition of polyamine synthesis with DFMO rescues most, but not all of these defects. These findings are relevant for patients with Snyder-Robinson syndrome (SRS), as the presenting skeletal defects-associated with SMS deficiency-could potentially be ameliorated by treatment with DFMO.

A developmental brain-wide screen identifies retrosplenial cortex as a key player in the emergence of persistent memory.

Memories formed early in life are short-lived while those formed later persist. Recent work revealed that infant memories are stored in a latent state. But why they fail to be retrieved is poorly understood. Here we investigated brain-wide circuit mechanisms underlying infantile amnesia in mice. We performed a screen that combined activity-dependent neuronal tagging at different postnatal ages, tissue clearing and light sheet microscopy. We observed striking developmental transitions in the organization of fear memory networks and changes in the activity and functional connectivity of the retrosplenial cortex (RSP) that aligned with the emergence of persistent memory. 7 days after learning, chemogenetic reactivation of tagged RSP ensembles enhanced memory in adults but not in infants. But after 33 days, reactivating infant-tagged RSP ensembles recovered forgotten memories. These studies show that RSP ensembles store latent infant memories, reveal the time course of RSP functional maturation, and suggest that immature RSP functional networks contribute to infantile amnesia.

Reducing Sialylation Enhances Electrotaxis of Corneal Epithelial Cells.

Corneal wound healing is a complex biological process that integrates a host of different signals to coordinate cell behavior. Upon wounding, there is the generation of an endogenous wound electric field that serves as a powerful cue to guide cell migration. Concurrently, the corneal epithelium reduces sialylated glycoforms, suggesting that sialylation plays an important role during electrotaxis. Here, we show that pretreating human telomerase-immortalized corneal epithelial (hTCEpi) cells with a sialyltransferase inhibitor, P-3FAX-Neu5Ac (3F-Neu5Ac), improves electrotaxis by enhancing directionality, but not speed. This was recapitulated using Kifunensine, which inhibits cleavage of mannoses and therefore precludes sialylation on N-glycans. We also identified that 3F-Neu5Ac enhanced the responsiveness of the hTCEpi cell population to the electric field and that pretreated hTCEpi cells showed increased directionality even at low voltages. Furthermore, when we increased sialylation using N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz), hTCEpi cells showed a decrease in both speed and directionality. Importantly, pretreating enucleated eyes with 3F-Neu5Ac significantly improved re-epithelialization in an ex vivo model of a corneal injury. Finally, we show that in hTCEpi cells, sialylation is increased by growth factor deprivation and reduced by PDGF-BB. Taken together, our results suggest that during corneal wound healing, reduced sialylated glycoforms enhance electrotaxis and re-epithelialization, potentially opening new avenues to promote corneal wound healing.

The evolution and spread of sulfur cycling enzymes reflect the redox state of the early Earth.

The biogeochemical sulfur cycle plays a central role in fueling microbial metabolisms, regulating the Earth's redox state, and affecting climate. However, geochemical reconstructions of the ancient sulfur cycle are confounded by ambiguous isotopic signals. We use phylogenetic reconciliation to ascertain the timing of ancient sulfur cycling gene events across the tree of life. Our results suggest that metabolisms using sulfide oxidation emerged in the Archean, but those involving thiosulfate emerged only after the Great Oxidation Event. Our data reveal that observed geochemical signatures resulted not from the expansion of a single type of organism but were instead associated with genomic innovation across the biosphere. Moreover, our results provide the first indication of organic sulfur cycling from the Mid-Proterozoic onwards, with implications for climate regulation and atmospheric biosignatures. Overall, our results provide insights into how the biological sulfur cycle evolved in tandem with the redox state of the early Earth.

Descemet membrane endothelial keratoplasty in eyes with glaucoma.

Endothelial keratoplasty has become the standard for the treatment of endothelial dysfunction. In Descemet membrane endothelial keratoplasty (DMEK), only the endothelium and Descemet membrane are transplanted, providing superior outcomes compared to Descemet stripping endothelial keratoplasty (DSEK). A substantial subset of patients who require DMEK have comorbid glaucoma. Even in eyes with complex anterior segment such as eyes with previous trabeculectomy or tube shunts, DMEK can restore meaningful vision and outperforms DSEK in terms of visual recovery, decreased rejection rate, and the need for high dose of topical steroids. However, accelerated endothelial cell loss and secondary graft failure have been described in eyes with previous glaucoma surgery, namely trabeculectomy and drainage device. During DMEK and DSEK procedures, raised intraocular pressure is required to attach the graft, which could worsen preexisting glaucoma or cause de novo glaucoma. Mechanisms of postoperative ocular hypertension include delayed air clearance, pupillary block, steroid response, and damage to angle structures. Medically treated glaucoma has increased risk for postoperative ocular hypertension. By understanding these additional complications and making appropriate modifications in surgical techniques and postoperative management, DMEK can be performed successfully and achieve very good visual outcome in eyes with glaucoma. Such modifications include precisely controlled unfolding technique, iridectomies that can help avoid pupillary block, tube shunts that can be trimmed to facilitate graft unfolding, air fill tension that can be adjusted, and postoperative steroid regimens that can be modified to decrease the risk for steroid response. Long-term survival of the DMEK graft, however, is shorter in eyes with previous glaucoma surgery than those without, as observed after other types of keratoplasty.

Baseline tumor growth rate highlights the heterogeneity of well differentiated gastroenteropancreatic neuroendocrine tumors and predicts for increases in Ki67 index over time.

Refined risk stratification for gastroenteropancreatic neuroendocrine tumors (GEP-NETs) has the potential to improve comparisons of study populations across clinical trials and facilitate drug development. Tumor growth rate (TGR) is a radiological metric with demonstrated prognostic value in well differentiated grade 1 and 2 (G1-2) GEP-NETs, but little is known about TGR in G3 NETs. In this retrospective study of 48 patients with advanced G1-3 GEP-NET, we calculated baseline TGR (TGR0 ) from radiological images of metastases acquired prior to first-line therapy and evaluated its association with disease characteristics and outcomes. The median pretreatment Ki67 proliferation index for G1-3 tumors combined was 5% (range = 0.1%-52%) and median TGR0 was 4.8%/month (m) (range = 0%-45.9%/m). TGR0 correlated with pretreatment Ki67 across G1-3 pooled and within G3 GEP-NET. Patients with higher TGR0 (>11.7%/m) tumors, which were primarily G3 pancreatic NETs, exhibited decreased time to first therapy (median, 2.2 vs. 5.3 months; p = .03) and shorter overall survival (median, 4.1 years vs. not reached; p = .003). Independent of therapies given, higher TGR0 GEP-NETs experienced a greater incidence of Ki67 increase (100 vs. 50%; p = .02) and greater magnitude of Ki67 change (median, 14.0 vs. 0.1%; p = .04) upon serial biopsy. Importantly, TGR0 , but not grade, predicted for future Ki67 increase in this series. Given the heterogeneity of well differentiated GEP-NETs, future clinical trials may benefit from stratification for TGR0 , particularly in G1-2 tumors, in which TGR0 does not correlate with Ki67. TGR0 has the potential to noninvasively identify patients with previously undiagnosed grade progression and those in whom more or less frequent monitoring may be appropriate. Additional research is needed to determine the prognostic and predictive value of TGR0 in larger and more homogeneously treated cohorts, and to ascertain if post-treatment TGR has value in previously treated patients starting a new line of therapy.

Urinary Neuroendocrine Neoplasms Treated in the "Modern Era": A Multicenter Retrospective Review.

BACKGROUND: Primary urinary neuroendocrine neoplasms (U-NENs) are extremely rare thus optimal treatment is unknown. Grading and treatment are typically extrapolated from other primary sites. Since 2010, the clinical landscape for NENs has changed substantially. We performed a retrospective review of U-NENs to assess treatment patterns and oncologic outcomes of patients treated in the recent era of NEN therapy. PATIENTS AND METHODS: A multicenter retrospective review of patients diagnosed after 2005 and alive after 2010. Time to treatment failure (TTF) was used to evaluate progression and toxicity for systemic therapy. Tumors were categorized as having either well-differentiated neuroendocrine tumor (WDNET) or poorly differentiated neuroendocrine carcinoma (PDNEC) histology. RESULTS: A total of 134 patients from 6 centers were included in our analysis, including 94 (70%) bladder, 32 (24%) kidney, 2 (1.5%) urethra and 4 other urinary primaries (3.0%). Poorly-differentiated neuroendocrine carcinoma was more common in bladder (92%) than non-bladder tumors (8%). Median Ki-67 available in bladder primary was 90% (n = 24), kidney 10% (n = 23), ureter 95% (n = 1), urethra 54% (n = 2), and others 90% (n = 3). Patients received a median of 2 therapies (range 0-10). Median time to death was not reached in locoregional WDNETs versus 8.2 years (95% CI, 3.5-noncalculable) in metastatic WDNETs (predominantly renal primary). Median time to death was 3.6 years (95% CI, 2.2-9.2) in locoregional PDNECs versus 1 year (95% CI, 0.8-1.3) in metastatic PDNECs (predominantly bladder primary). CONCLUSION: This is the most extensive series examining treatment patterns in patients with U-NENs in the recent era of NEN therapy. The apparent inferior survival for bladder NENs is likely due to the preponderance of PDNECs in this group. As predicted, treatments for U-NENs mirrored that of other more common NENs. In our retrospective cohort, we observed that patients with WD-UNETs treated with peptide receptor radionuclide therapy (PRRT) and everolimus suggested potential activity for disease control in WD-UNETs. Prospective studies are needed to assess the activity of new oncology drugs in UNENs.

Autologous Serum Eye Drops in the Management of Limbal Stem Cell Deficiency Associated With Glaucoma Surgery.

PURPOSE: To evaluate safety and efficacy of autologous serum eye drops (AS) in the treatment of limbal stem cell deficiency (LSCD) associated with glaucoma surgery. METHODS: Retrospective case series of eyes with glaucoma surgery-induced LSCD treated with AS. Diagnosis of LSCD was confirmed by anterior segment optical coherence tomography, in vivo confocal microscopy, and/or impression cytology. Limbal stem cell deficiency severity was staged using a clinical scoring system (2-10 points). Outcome measures were changes (≥2 points) of the LSCD score and best-corrected visual acuity (BCVA) from the baseline to the last follow-up. RESULTS: Thirteen eyes of 12 consecutive patients treated with 50% AS for at least 3 months were included. The mean age was 78.9±7.5 years and the mean duration of AS use was 20.9±16.8 months. Indications of AS included LSCD progression in eight eyes (61.5%) and visual axis threatening in five eyes (38.5%). The mean LSCD score at baseline (6.7±1.6) was similar to that at last follow-up (6.5±2.2, P =0.625). Two eyes (15.4%) showed improvement, nine eyes (69.2%) were stable, and two eyes (15.4%) worsened. The mean baseline BCVA (0.89±0.64 logMAR) was similar to the mean final BCVA (1.05±0.63 logMAR, P =0.173). There were no serious adverse complications related to AS. CONCLUSION: AS appears to be well tolerated and may stabilize the progression of LSCD with limited effects. A larger study is necessary to confirm the findings.

Rib Fractures in Professional Baseball Pitchers: Mechanics, Epidemiology, and Management.

Pitching is a complex kinetic chain activity requiring the transfer of energy from the lower body, through the core and trunk, and finally through the arm to generate explosive acceleration of the baseball. As a result, large forces are generated in the trunk musculature and rib attachments from the late cocking phase of pitching through deceleration. The repetitive cumulative load and high pitch velocities put professional pitchers at risk of rib stress fracture. Given the potential for a prolonged recovery course and high rate of recurrence, early recognition of rib bone stress injury is critical to optimize care. Identifying torso strength imbalances, suboptimal pitching biomechanics (such as late or inadequate pelvic rotation), as well as metabolic deficiencies that may adversely affect bone health are essential to expedite safe return to play and prevent future injury. In this review, we discuss risk factors, mechanism of injury, typical clinical presentation, diagnostic imaging findings, and propose treatment and prevention strategies for rib stress fractures in overhand pitchers.

Preoperative risk stratification of lymph node metastasis for non-functional pancreatic neuroendocrine neoplasm: An international dual-institutional study.

BACKGROUND: /Objectives: Although the presence of lymph node metastasis (LNM) defines malignant potential, preoperative prediction of LNM has not been established for non-functional pancreatic neuroendocrine neoplasm (NF-PNEN). We sought to develop a prediction system using only preoperatively available factors that would stratify the risk of LNM for NF-PNEN. METHODS: We retrospectively reviewed patients who underwent R0/1 resection of NF-PNEN at Kyoto University (2007-2019) and the University of California, San Francisco (2010-2019). Risk stratification of LNM was developed using preoperative factors by the logistic regression analysis. Long-term outcomes were compared across the risk groups. RESULTS: A total of 131 patients were included in this study. Lymph nodes were pathologically examined in 116 patients, 23 (20%) of whom had LNM. Radiological tumor size [1.5-3.5 cm (odds ratio: 13.5, 95% confidence interval: 1.77-398) and >3.5 cm (72.4, 9.06-2257) against ≤1.5 cm], <50% cystic component (8.46 × 10^6, 1.68 × 10^106-), and dilatation of main pancreatic duct ≥5 mm (31.2, 3.94-702) were independently associated with LNM. When patients were classified as the low-risk (43 patients), intermediate-risk (44 patients), and high-risk groups (29 patients), proportions of LNM differed significantly across the groups (0%, 14%, and 59%, respectively). Recurrence-free survival (RFS) of the low- and intermediate-risk groups were significantly better than that of the high-risk group (5-year RFS rates of 92.2%, 85.4%, and 47.1%, respectively). CONCLUSIONS: The prediction system using preoperative radiological factors stratifies the risk of LNM for NF-PNEN. This stratification helps to predict malignant potential and determine the surgical procedure and necessity of regional lymphadenectomy.

Student Outcomes From a Large-Enrollment Introductory Course-Based Undergraduate Research Experience on Soil Microbiomes.

In recent years, national reports have called for undergraduate laboratory education that engages students in authentic research experiences. As a result, a number of course-based undergraduate research experiences (CUREs) have been developed in biological sciences and some specifically in microbiology. Students benefit from CUREs much like in traditional mentored research experiences, where students carry out independent projects in faculty laboratories. These benefits include increased self-efficacy in research skills, enhanced identification as scientists, and higher graduation rates in science, technology, engineering, and mathematics majors. Because mentored research experiences are not readily available to every student, CUREs represent a potential mechanism to democratize the research experience by providing such opportunities to all students. However, many of existing CUREs described in the literature are designed for advanced undergraduates or are limited to a small number of students. Here, we report student outcomes from a large-enrollment introductory CURE on soil microbiomes that engages students in a real-world context with microbiology. In pre- and post-course surveys, students reported significant gains in self-efficacy on a number of research skills. These results are triangulated with post-course survey data on project ownership, sense of community, and CURE design elements such as collaboration, iteration, discovery, and relevance.

Mesenchymal Stromal Cells Regulate Sialylations of N-Glycans, Affecting Cell Migration and Survival.

N-Glycosylations are an important post-translational modification of proteins that can significantly impact cell function. Terminal sialic acid in hybrid or complex N-glycans has been shown to be relevant in various types of cancer, but its role in non-malignant cells remains poorly understood. We have previously shown that the motility of human bone marrow derived mesenchymal stromal cells (MSCs) can be modified by altering N-glycoforms. The goal of this study was to determine the role of sialylated N-glycans in MSCs. Here, we show that IFN-gamma or exposure to culture media low in fetal bovine serum (FBS) increases sialylated N-glycans, while PDGF-BB reduces them. These stimuli alter mRNA levels of sialyltransferases such as ST3Gal1, ST6Gal1, or ST3Gal4, suggesting that sialylation of N-glycans is regulated by transcriptional control of sialyltransferases. We next show that 2,4,7,8,9-pentaacetyl-3Fax-Neu5Ac-CO2Me (3F-Neu5Ac) effectively inhibits sialylations in MSCs. Supplementation with 3F-Neu5Ac increases adhesion and migration of MSCs, as assessed by both videomicroscopy and wound/scratch assays. Interestingly, pre-treatment with 3F-Neu5Ac also increases the survival of MSCs in an in vitro ischemia model. We also show that pre-treatment or continuous treatment with 3F-Neu5Ac inhibits both osteogenic and adipogenic differentiation of MSCs. Finally, secretion of key trophic factors by MSCs is variably affected upon exposure to 3F-Neu5Ac. Altogether, our experiments suggest that sialylation of N-glycans is tightly regulated in response to environmental cues and that glycoengineering MSCs to reduce sialylated N-glycans could be beneficial to increase both cell migration and survival, which may positively impact the therapeutic potential of the cells.

Two-Photon Microperimetry: A Media Opacity-Independent Retinal Function Assay.

Purpose: Compare results obtained using infrared two-photon microperimetry (2PM-IR) with conventional visual function tests in healthy subjects of varying ages with and without simulated media opacities. Methods: Subjects from two separate cohort studies completed cone contrast threshold (CCT) testing, conventional microperimetry, visible light microperimetry from a novel device (2PM-Vis), and infrared two-photon microperimetry. The first cohort study, which consisted of six healthy volunteers (23 to 29 years of age), evaluated the effects of simulated media opacities on visual performance testing. Subjects underwent testing on four visual function devices nine separate times under the following conditions: no filter, red filter, green filter, blue filter, light brown filter, dark brown filter, polarized black filter (0° rotation), and polarized black filter (90° rotation). Subjects subsequently performed 2PM-IR and 2PM-Vis testing without a filter in the mydriatic state. The second cohort study evaluated the effect of age on visual test performance in 42 healthy subjects split between two groups (ages 20-40 years and 60-80 years). Results: Retinal sensitivity measured by 2PM-IR demonstrated lower variability than all other devices relying on visible spectrum stimuli. Retinal sensitivity decreased proportionally with the transmittance of light through each filter. CCT scores and retinal sensitivity decreased with age in all testing modalities. Visible spectrum testing modalities demonstrated larger test result differences between young and old patient cohorts; this difference was inversely proportional to the wavelength of the visual function test. Conclusions: 2PM-IR mitigates media opacities that may mask small differences in retinal sensitivity when tested with conventional visual function testing devices. Translational Relevance: Conventional visual function tests that emit visible light may not detect differences in retinal function during the early stages of age-related diseases due to the confounding effects of cataracts. Infrared light, which has greater transmittance through ocular tissue, may reliably quantify retinal sensitivity and thereby detect degenerative changes early on.

Prenylated xanthones from mangosteen (Garcinia mangostana) activate the AhR and Nrf2 pathways and protect intestinal barrier integrity in HT-29 cells.

Xanthones from the tropical fruit mangosteen (Garcinia mangostana) display anti-inflammatory and anti-oxidative activities. Here, we isolate and identify potential inducers of the aryl hydrocarbon receptor (AhR) and nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathways from mangosteen using a bioassay-guided strategy. Mangosteen fruit pericarp extracts were subjected to sequential solvent extractions, followed by chromatography coupled with NMR spectroscopy and mass spectrometric analyses for identification and isolation of pure compounds. Isolation of active fractions led to seven prenylated xanthones that were identified and subsequently evaluated for bioactivity. In vitro luciferase reporter cellular assays using H1L6.1c3 (AhR induction) and HepG2-ARE (Nrf2 induction) were used to identify AhR and Nrf2 activators. All seven prenylated xanthones displayed AhR inducing activity, whereas only five xanthones activated Nrf2. Garcinone D (GarD) significantly upregulated AhR/Cyp1a1 and Nrf2/HO-1 protein expression and enhanced zonula occludens-1 and occludin protein levels in HT-29 cells. In addition, GarD inhibited oxidative stress-induced intestinal epithelial barrier dysfunction by enhancing tight junction (TJ) proteins and inhibition of reactive oxygen species production. Inhibition of AhR by pretreating cells with an AhR antagonist revealed that the AhR pathway is required for the improved epithelial barrier functions of GarD. These results highlight a dual mechanism by GarD that confers protection against intestinal epithelial barrier dysfunction.

Quantifying Color Vision Changes Associated With Cataracts Using Cone Contrast Thresholds.

Purpose: To evaluate effects of age and simulated and real cataractous changes on color vision as measured by the high-definition cone contrast test (CCT). Methods: Twenty-four healthy volunteers from two cohort studies performed CCT using best-corrected visual acuity, filters, mydriasis, and pinhole correction. Retrospective cross-sectional study of patients seen in eye clinics evaluated the relationship between age and color vision, and age and lens status in 355 eyes. Last, 25 subjects underwent CCT before and after cataract surgery. Results: CCT scores were most reliable in the nonmydriatic condition without pinhole correction. Progressively dense brown filters produced small decreases in S-cone sensitivity. Linear regression analysis of phakic subjects showed a decline for all cone classes with age. Rate of decline was greater for S-cones (slope = -1.09; 95% confidence interval [CI], -1.30 to 0.86) than M-cones (slope = -0.80; 95% CI, -1.03 to -0.58) and L-cones (slope = -0.66; 95% CI, -0.88 to -0.44). CCT scores increased for S-cones but reduced for L- and M-cones in pseudophakic subjects compared with phakic patients. CCT scores after cataract surgery increased for S-cones, M-cones, and L-cones by 33.0 (95% CI, 8.6 to 57.4), 24.9 (95% CI, 3.8 to 46.0), and 22.0 (95% CI, -3.2 to 47.3), respectively. Conclusions: CCT assessment allows for clinically practical quantitation of color and contrast vision improvement after cataract surgery and aging patients who note poor vision despite good visual acuity. Translational Relevance: CCT testing, which quantifies hereditary and acquired color deficiency, can also quantify the degree of cataract severity and, combined with other parameters, can provide more precise guidance for cataract extraction to optimize patient care.

High Mannose N-Glycans Promote Migration of Bone-Marrow-Derived Mesenchymal Stromal Cells.

For hundreds of indications, mesenchymal stromal cells (MSCs) have not achieved the expected therapeutic efficacy due to an inability of the cells to reach target tissues. We show that inducing high mannose N-glycans either chemically, using the mannosidase I inhibitor Kifunensine, or genetically, using an shRNA to silence the expression of mannosidase I A1 (MAN1A1), strongly increases the motility of MSCs. We show that treatment of MSCs with Kifunensine increases cell migration toward bone fracture sites after percutaneous injection, and toward lungs after intravenous injection. Mechanistically, high mannose N-glycans reduce the contact area of cells with its substrate. Silencing MAN1A1 also makes cells softer, suggesting that an increase of high mannose N-glycoforms may change the physical properties of the cell membrane. To determine if treatment with Kifunensine is feasible for future clinical studies, we used mass spectrometry to analyze the N-glycan profile of MSCs over time and demonstrate that the effect of Kifunensine is both transitory and at the expense of specific N-glycoforms, including fucosylations. Finally, we also investigated the effect of Kifunensine on cell proliferation, differentiation, and the secretion profile of MSCs. Our results support the notion of inducing high mannose N-glycans in MSCs in order to enhance their migration potential.

Author Correction: Modeling Snyder-Robinson Syndrome in multipotent stromal cells reveals impaired mitochondrial function as a potential cause for deficient osteogenesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Modeling Snyder-Robinson Syndrome in multipotent stromal cells reveals impaired mitochondrial function as a potential cause for deficient osteogenesis.

Patients with Snyder-Robinson Syndrome (SRS) exhibit deficient Spermidine Synthase (SMS) gene expression, which causes neurodevelopmental defects and osteoporosis, often leading to extremely fragile bones. To determine the underlying mechanism for impaired bone formation, we modelled the disease by silencing SMS in human bone marrow - derived multipotent stromal cells (MSCs) derived from healthy donors. We found that silencing SMS in MSCs led to reduced cell proliferation and deficient bone formation in vitro, as evidenced by reduced mineralization and decreased bone sialoprotein expression. Furthermore, transplantation of MSCs in osteoconductive scaffolds into immune deficient mice shows that silencing SMS also reduces ectopic bone formation in vivo. Tag-Seq Gene Expression Profiling shows that deficient SMS expression causes strong transcriptome changes, especially in genes related to cell proliferation and metabolic functions. Similarly, metabolome analysis by mass spectrometry, shows that silencing SMS strongly impacts glucose metabolism. This was consistent with observations using electron microscopy, where SMS deficient MSCs show high levels of mitochondrial fusion. In line with these findings, SMS deficiency causes a reduction in glucose consumption and increase in lactate secretion. Our data also suggests that SMS deficiency affects iron metabolism in the cells, which we hypothesize is linked to deficient mitochondrial function. Altogether, our studies suggest that SMS deficiency causes strong transcriptomic and metabolic changes in MSCs, which are likely associated with the observed impaired osteogenesis both in vitro and in vivo.