Sticking It to Conventional Models; Micropatterned hiPSC Culture as a Novel Approach for Studying Complex Molecular and Cellular Systems

Studying complex molecular and cellular systems in humans presents a crucial challenge that demands specialized models for visualization and experimentation. While human induced pluripotent stem cells (hiPSCs) offer a valuable tool due to their ability to maintain a pluripotent state and differentiate upon direction, they can yield irreproducible results. To overcome this limitation, better-controlled and more sophisticated hiPSC differentiation models are needed. One promising approach to the study of tissue-scale cell fate patterning involves pre-patterned geometrical confinement (Warmflash et al., 2014; Etoc et al, 2016). In this study, we aimed to test a modified methodology for producing hiPSC micropatterned adhesive islands to investigate complex molecular systems, specifically intermediate filament protein vimentin and the effects of molecular chaperones. We were able to construct two CRISPR-Cas9 plasmids that targeted the human vimentin gene and showed that upon transfection into HeLa cells, they induced a vimentin-null phenotype. We then successfully adapted the Warmflash et al. protocol to produce adhesive islands in HeLa and hiPSC which constrained the cells using pre-patterned geometric constraints. This is a crucial step in studying complex molecular systems and genetic manipulations in differentiating human cells. Our modifications also enhanced the cost-effectiveness of our model potentially making it more applicable to a teaching setting (CU MCDB Skills Center) . In the long term, this approach has the potential to shed light on the role(s) of vimentin and the effects of molecular chaperones on hiPSC differentiation and development. Keywords: human induced pluripotent stem cells; adhesive islands; micropatterning; intermediate filament proteins; vimentin; differentiation; chaperones.</p

Does place of residence affect risk of suicide? a spatial epidemiologic investigation in Kentucky from 1999 to 2008

Background Approximately 32,000 people take their own lives every year in the United States. In Kentucky, suicide mortality rates have been steadily increasing since 1999. Few studies in the United States have assessed spatial clustering of suicides. The purpose of this study was to identify high-risk clusters of suicide at the county level in Kentucky and assess the characteristics of those suicide cases within the clusters. Methods A spatial epidemiological study was undertaken using suicide data for the period January 1, 1999 to December 31, 2008, obtained from the Kentucky Office of Vital Statistics. Descriptive analyses using Pearson\u27s chi-square test and t-test were performed to determine whether differences existed in age, marital status, year, season, and suicide method between males and females, and between cases inside and outside high-risk spatial clusters. Annual age-adjusted cumulative incidence rates were also calculated. Suicide incidence rates were spatially smoothed using the Spatial Empirical Bayesian technique. Kulldorff\u27s spatial scan statistic was applied on all suicide cases at the county level to identify counties with the highest risks of suicide. Temporal cluster analysis was also performed. Results There were a total of 5,551 suicide cases in Kentucky from 1999 to 2008, of which 5,237 (94%) were included in our analyses. The majority of suicide cases were males (82%). The average age of suicide victims was 45.4 years. Two statistically significant (p \u3c 0.05) high-risk spatial clusters, involving 15 counties, were detected. The county level cumulative incidence rate in the most likely high-risk cluster ranged from 12.4 to 21.6 suicides per 100,000 persons. The counties inside both high-risk clusters had relative risks ranging from 1.24 to 1.38. Conclusions Statistically significant high-risk spatial clusters of suicide were detected at the county level. This study may be useful for guiding future research and intervention efforts. Future studies will need to focus on these high-risk clusters to investigate reasons for these occurrences

Lack of Rbl1/p107 Effects on Cell Proliferation and Maturation in the Inner Ear

Loss of postnatal mammalian auditory hair cells (HCs) is irreversible. Earlier studies have highlighted the importance of the Retinoblastoma family of proteins (pRBs) (i.e., Rb1, Rbl1/p107, and Rbl2/p130) in the auditory cells’ proliferation and emphasized our lack of information on their specific roles in the auditory system. We have previously demonstrated that lack of Rbl2/p130 moderately affects HCs’ and supporting cells’ (SCs) proliferation. Here, we present evidence supporting multiple roles for Rbl1/p107 in the developing and mature mouse organ of Corti (OC). Like other pRBs, Rbl1/p107 is expressed in the OC, particularly in the Hensen’s and Deiters’ cells. Moreover, Rbl1/p107 impacts maturation and postmitotic quiescence of HCs and SCs, as evidenced by enhanced numbers of these cells and the presence of dividing cells in the postnatal Rbl1/p107−/− OC. These findings were further supported by microarray and bioinformatics analyses, suggesting downregulation of several bHLH molecules, as well as activation of the Notch/Hes/Hey signaling pathway in homozygous Rbl1/p107 mutant mice. Physiological assessments and detection of ectopic HC marker expression in postnatal spiral ganglion neurons (SGNs) provided evidence for incomplete cell maturation and differentiation in Rbl1/p107−/− OC. Collectively, the present study highlights an important role for Rbl1/p107 in OC cell differentiation and maturation, which is distinct from other pRBs

Morphology and Dynamics Observed with Hi-C and IRIS

The third flight of the High-resolution Coronal Imager (Hi-C 2.1) occurred on May 29, 2018, with significant co-observations with both space and ground based instruments, including the Interface Region Imaging Spectrograph (IRIS). The primary science goal of this flight was to identify the connections between the lower corona, transition region, and chromosphere at the native resolution of these connections. One way to relate the emission in these two instruments that image different temperature regimes is to compare the morphology, dynamics and time scales in each data set. In this poster, we present the initial results of this study

Sounding Rocket Instrument Development at UAHuntsville/NASA MSFC

We present an overview of solar sounding rocket instruments developed jointly by NASA Marshall Space Flight Center and the University of Alabama in Huntsville. The High Resolution Coronal Imager (Hi-C) is an EUV (19.3 nm) imaging telescope which was flown successfully in July 2012. The Chromospheric Lyman-Alpha SpectroPolarimeter (CLASP) is a Lyman Alpha (121.6 nm) spectropolarimeter developed jointly with the National Astronomical Observatory of Japan and scheduled for launch in 2015. The Marshall Grazing Incidence X-ray Spectrograph is a soft X-ray (0.5-1.2 keV) stigmatic spectrograph designed to achieve 5 arcsecond spatial resolution along the slit

Is the High-resolution Coronal Imager Resolving Coronal Strands? Results from AR 12712

Following the success of the first mission, the High-Resolution Coronal Imager (Hi-C) was launched for a third time (Hi-C 2.1) on 2018 May 29 from the White Sands Missile Range, NM, USA. On this occasion, 329 s of 17.2 nm data of target active region AR 12712 were captured with a cadence of ≈4 s, and a plate scale of 0farcs129 pixel−1. Using data captured by Hi-C 2.1 and co-aligned observations from SDO/AIA 17.1 nm, we investigate the widths of 49 coronal strands. We search for evidence of substructure within the strands that is not detected by AIA, and further consider whether these strands are fully resolved by Hi-C 2.1. With the aid of multi-scale Gaussian normalization, strands from a region of low emission that can only be visualized against the contrast of the darker, underlying moss are studied. A comparison is made between these low-emission strands and those from regions of higher emission within the target active region. It is found that Hi-C 2.1 can resolve individual strands as small as ≈202 km, though the more typical strand widths seen are ≈513 km. For coronal strands within the region of low emission, the most likely width is significantly narrower than the high-emission strands at ≈388 km. This places the low-emission coronal strands beneath the resolving capabilities of SDO/AIA, highlighting the need for a permanent solar observatory with the resolving power of Hi-C

Characterization of primary human hepatocyte spheroids as a model system for drug-induced liver injury, liver function and disease

Liver biology and function, drug-induced liver injury (DILI) and liver diseases are difficult to study using current in vitro models such as primary human hepatocyte (PHH) monolayer cultures, as their rapid de-differentiation restricts their usefulness substantially. Thus, we have developed and extensively characterized an easily scalable 3D PHH spheroid system in chemically-defined, serum-free conditions. Using whole proteome analyses, we found that PHH spheroids cultured this way were similar to the liver in vivo and even retained their inter-individual variability. Furthermore, PHH spheroids remained phenotypically stable and retained morphology, viability, and hepatocyte-specific functions for culture periods of at least 5 weeks. We show that under chronic exposure, the sensitivity of the hepatocytes drastically increased and toxicity of a set of hepatotoxins was detected at clinically relevant concentrations. An interesting example was the chronic toxicity of fialuridine for which hepatotoxicity was mimicked after repeated-dosing in the PHH spheroid model, not possible to detect using previous in vitro systems. Additionally, we provide proof-of-principle that PHH spheroids can reflect liver pathologies such as cholestasis, steatosis and viral hepatitis. Combined, our results demonstrate that the PHH spheroid system presented here constitutes a versatile and promising in vitro system to study liver function, liver diseases, drug targets and long-term DILI

Statewide Systematic Evaluation of Sudden, Unexpected Infant Death Classification: Results from a National Pilot Project

The Centers for Disease Control and Prevention funded seven states, including Kentucky, to clarify statewide death certification practices in sudden, unexpected infant death and compare state performances with national expectations. Accurate assignment of the cause and manner of death in cases of sudden, unexpected infant death is critical for accurate vital statistics data to direct limited resources to appropriate targets, and to implement optimal and safe risk reduction strategies. The primary objectives are to (1) Compare SUID death certifications recommended by the KY medical examiners with the stated cause of death text field on the hard copy death electronic death certificates and (2) Compare KY and national SUID rates. Causes of death for SUID cases recommended by the medical examiners and those appearing on the hard copy and electronic death certificates in KY were collected retrospectively for 2004 and 2005. Medical examiner recommendations were based upon a classification scheme devised by them in 2003. Coroners hard copy death certificates and the cause of death rates in KY were compared to those occurring nationally. Eleven percent of infants dying suddenly and unexpectedly did not undergo autopsy during the study interval. The KY 2003 classification scheme for SIDS is at variance with the NICHD and San Diego SIDS definitions. Significant differences in causes of death recommended by medical examiners and those appearing on the hard copy and electronic death certificates were identified. SIDS rates increased in KY in contrast to decreasing rates nationally. Nationwide adoption of a widely used SIDS definition, such as that proposed in San Diego in 2004 as well as legislation by states to ensure autopsy in all cases of sudden unexpected infant death are recommended. Medical examiners’ recommendations for cause of death should appear on death certificates. Multidisciplinary pediatric death review teams prospectively evaluating cases before death certification is recommended. Research into other jurisdictions death certification process is encouraged

The Arecibo Legacy Fast ALFA Survey: III. HI Source Catalog of the Northern Virgo Cluster Region

We present the first installment of HI sources extracted from the Arecibo Legacy Fast ALFA (ALFALFA) extragalactic survey, initiated in 2005. Sources have been extracted from 3-D spectral data cubes and then examined interactively to yield global HI parameters. A total of 730 HI detections are catalogued within the solid angle 11h44m < R.A.(J2000) < 14h00m and +12deg < Dec.(J2000) < +16deg, and redshift range -1600 \kms < cz < 18000 \kms. In comparison, the HI Parkes All-Sky Survey (HIPASS) detected 40 HI signals in the same region. Optical counterparts are assigned via examination of digital optical imaging databases. ALFALFA HI detections are reported for three distinct classes of signals: (a) detections, typically with S/N > 6.5; (b) high velocity clouds in the Milky Way or its periphery; and (c) signals of lower S/N (to ~ 4.5) which coincide spatially with an optical object of known similar redshift. Although this region of the sky has been heavily surveyed by previous targeted observations based on optical flux-- or size-- limited samples, 69% of the extracted sources are newly reported HI detections. The resultant positional accuracy of HI sources is 20" (median). The median redshift of the sample is ~7000 \kms and its distribution reflects the known local large scale structure including the Virgo cluster. Several extended HI features are found in the vicinity of the Virgo cluster. A small percentage (6%) of HI detections have no identifiable optical counterpart, more than half of which are high velocity clouds in the Milky Way vicinity; the remaining 17 objects do not appear connected to or associated with any known galaxy.Comment: Astronomical Journal, in pres