Mouse all-cone retina models of Cav1.4 synaptopathy

The voltage-gated calcium channel, Cav1.4 is localized to photoreceptor ribbon synapses and functions both in molecular organization of the synapse and in regulating release of synaptic vesicles. Mutations in Cav1.4 subunits typically present as either incomplete congenital stationary night blindness or a progressive cone-rod dystrophy in humans. We developed a cone-rich mammalian model system to further study how different Cav1.4 mutations affect cones. RPE65 R91W KI; Nrl KO “Conefull” mice were crossed to Cav1.4 α1F or α2δ4 KO mice to generate the “Conefull:α1F KO” and “Conefull:α2δ4 KO” lines. Animals were assessed using a visually guided water maze, electroretinogram (ERG), optical coherence tomography (OCT), and histology. Mice of both sexes and up to six-months of age were used. Conefull: α1F KO mice could not navigate the visually guided water maze, had no b-wave in the ERG, and the developing all-cone outer nuclear layer reorganized into rosettes at the time of eye opening with degeneration progressing to 30% loss by 2-months of age. In comparison, the Conefull: α2δ4 KO mice successfully navigated the visually guided water maze, had a reduced amplitude b-wave ERG, and the development of the all-cone outer nuclear layer appeared normal although progressive degeneration with 10% loss by 2-months of age was observed. In summary, new disease models for studying congenital synaptic diseases due to loss of Cav1.4 function have been created

The outer segment serves as a default destination for the trafficking of membrane proteins in photoreceptors

Photoreceptors are compartmentalized neurons in which all proteins responsible for evoking visual signals are confined to the outer segment. Yet, the mechanisms responsible for establishing and maintaining photoreceptor compartmentalization are poorly understood. Here we investigated the targeting of two related membrane proteins, R9AP and syntaxin 3, one residing within and the other excluded from the outer segment. Surprisingly, we have found that only syntaxin 3 has targeting information encoded in its sequence and its removal redirects this protein to the outer segment. Furthermore, proteins residing in the endoplasmic reticulum and mitochondria were similarly redirected to the outer segment after removing their targeting signals. This reveals a pattern where membrane proteins lacking specific targeting information are delivered to the outer segment, which is likely to reflect the enormous appetite of this organelle for new material necessitated by its constant renewal. This also implies that every protein residing outside the outer segment must have a means to avoid this “default” trafficking flow

Expression of the Flp proteins by Haemophilus ducreyi is necessary for virulence in human volunteers

<p>Abstract</p> <p>Background</p> <p><it>Haemophilus ducreyi</it>, the causative agent of the sexually transmitted disease chancroid, contains a <it>flp </it>(fimbria like protein) operon that encodes proteins predicted to contribute to adherence and pathogenesis. <it>H. ducreyi </it>mutants that lack expression of Flp1 and Flp2 or TadA, which has homology to NTPases of type IV secretion systems, have decreased abilities to attach to and form microcolonies on human foreskin fibroblasts (HFF). A <it>tadA </it>mutant is attenuated in its ability to cause disease in human volunteers and in the temperature dependent rabbit model, but a <it>flp1flp2 </it>mutant is virulent in rabbits. Whether a <it>flp </it>deletion mutant would cause disease in humans is not clear.</p> <p>Results</p> <p>We constructed 35000HPΔ<it>flp1-3</it>, a deletion mutant that lacks expression of all three Flp proteins but has an intact <it>tad </it>secretion system. 35000HPΔ<it>flp1-3 </it>was impaired in its ability to form microcolonies and to attach to HFF in vitro when compared to its parent (35000HP). Complementation of the mutant with <it>flp1-3 </it>in trans restored the parental phenotype. To test whether expression of Flp1-3 was necessary for virulence in humans, ten healthy adult volunteers were experimentally infected with a fixed dose of 35000HP (ranging from 54 to 67 CFU) on one arm and three doses of 35000HPΔ<it>flp1-3 </it>(ranging from 63 to 961 CFU) on the other arm. The overall papule formation rate for the parent was 80% (95% confidence interval, CI, 55.2%-99.9%) and for the mutant was 70.0% (95% CI, 50.5%-89.5%) (<it>P </it>= 0.52). Mutant papules were significantly smaller (mean, 11.2 mm²) than were parent papules (21.8 mm²) 24 h after inoculation (<it>P </it>= 0.018). The overall pustule formation rates were 46.7% (95% CI 23.7-69.7%) at 30 parent sites and 6.7% (95% CI, 0.1-19.1%) at 30 mutant sites (<it>P </it>= 0.001).</p> <p>Conclusion</p> <p>These data suggest that production and secretion of the Flp proteins contributes to microcolony formation and attachment to HFF cells in vitro. Expression of <it>flp1-3 </it>is also necessary for <it>H. ducreyi </it>to initiate disease and progress to pustule formation in humans. Future studies will focus on how Flp proteins contribute to microcolony formation and attachment in vivo.</p

The RESOLVE and ECO Gas in Galaxy Groups Initiative: The Group Finder and the Group HI–Halo Mass Relation

We present a four-step group-finding algorithm for the Gas in Galaxy Groups (G3) initiative, a spin-off of the z ∼ 0 REsolved Spectroscopy Of a Local VolumE (RESOLVE) and Environmental COntext (ECO) surveys. In preparation for future comparisons to intermediate redshift (e.g., the LADUMA survey), we design the group finder to adapt to incomplete, shallow, or nonuniform data. We use mock catalogs to optimize the group finder’s performance. Compared to friends-of-friends (with false-pair splitting), the G3 algorithm offers improved completeness and halo-mass recovery with minimal loss of purity. Combining it with the volume-limited H I census data for RESOLVE and ECO, we examine the H I content of galaxy groups as a function of group halo mass. Group-integrated H I mass M rises monotonically over halo masses M ∼ 10–10 M, pivoting in slope at M ∼ 10M, the gas-richness threshold scale. We present the first measurement of the scatter in this relation, which has a median of ∼0.3 dex and is asymmetric toward lower M I,grp. We discuss interesting tensions with theoretical predictions and prior measurements of the M–M relation. In an appendix, we release RESOLVE DR4 and ECO DR3, including updates to survey redshifts, photometry, and group catalogs, as well as a major expansion of the ECO H I inventory with value-added data products. © 2023. The Author(s). Published by the American Astronomical SocietyWe are grateful to the anonymous referee, whose feedback has improved the quality of this work. We also thank Adrienne Erickcek, Andrew Mann, Mugdha Polimera, Matthew Bershady, Joshua Oppor, Jeremy Darling, Hayley Roberts, and Amir Kazemi-Moridani for valuable feedback at varying stages of the project. Z.L.H., S.J.K., and E.R.C. acknowledge support for this research from National Science Foundation (NSF) grant AST-1814486. Z.L.H. and D.S.C. are also supported through a North Carolina Space Grant Graduate Research Fellowship. S.J.K. and D.S.C. acknowledge support from NSF grant AST-2007351. A.J.B. acknowledges support from NSF grant AST-1814421. K.M.H. acknowledges financial support from the State Agency for Research of the Spanish Ministry of Science, Innovation and Universities through the "Center of Excellence Severo Ocho" awarded to the Instituto de Astrofisica de Andalucia (SEV-2017-0709), via participation in SKA-SPAIN, funded by the Ministry of Science and Innovation (MCIN), and financial support from grant RTI2018-096228-B-C31 (MCIU/AEI/FEDER,UE)

Calpain-5 Expression in the Retina Localizes to Photoreceptor Synapses

Purpose: We characterize calpain-5 (CAPN5) expression in retinal and neuronal subcellular compartments. Methods: CAPN5 gene variants were classified using the exome variant server, and RNA-sequencing was used to compare expression of CAPN5 mRNA in the mouse and human retina and in retinoblastoma cells. Expression of CAPN5 protein was ascertained in humans and mice in silico, in mouse retina by immunohistochemistry, and in neuronal cancer cell lines and fractionated central nervous system tissue extracts by Western analysis with eight antibodies targeting different CAPN5 regions. Results: Most CAPN5 genetic variation occurs outside its protease core; and searches of cancer and epilepsy/autism genetic databases found no variants similar to hyperactivating retinal disease alleles. The mouse retina expressed one transcript for CAPN5 plus those of nine other calpains, similar to the human retina. In Y79 retinoblastoma cells, the level of CAPN5 transcript was very low. Immunohistochemistry detected CAPN5 expression in the inner and outer nuclear layers and at synapses in the outer plexiform layer. Western analysis of fractionated retinal extracts confirmed CAPN5 synapse localization. Western blots of fractionated brain neuronal extracts revealed distinct subcellular patterns and the potential presence of autoproteolytic CAPN5 domains. Conclusions: CAPN5 is moderately expressed in the retina and, despite higher expression in other tissues, hyperactive disease mutants of CAPN5 only manifest as eye disease. At the cellular level, CAPN5 is expressed in several different functional compartments. CAPN5 localization at the photoreceptor synapse and with mitochondria explains the neural circuitry phenotype in human CAPN5 disease alleles

Synthesis and in vitro and in vivo characterization of highly β1-Selective β-Adrenoceptor partial agonists

β-Adrenoceptor antagonists boast a 50-year use for symptomatic control in numerous cardiovascular diseases. One might expect highly selective antagonists are available for the human β-adrenoceptor subtype involved in these diseases, yet few truly β1-selective molecules exist. To address this clinical need, we re-evaluated LK 204-545 (1),1 a selective β1-adrenoceptor antagonist, and discovered it possessed significant partial agonism. Removal of 1’s aromatic nitrile afforded 19, a ligand with similar β1-adrenoceptor selectivity and partial agonism (log KD of −7.75 and −5.15 as an antagonist of functional β1- and β2-mediated responses, respectively, and 34% of the maximal response of isoprenaline (β1)). In vitro β-adrenoceptor selectivity and partial agonism of 19 were mirrored in vivo. We designed analogues of 19 to improve affinity, selectivity, and partial agonism. Although partial agonism could not be fully attenuated, SAR suggests that an extended alkoxyalkoxy side chain, alongside substituents at the meta- or para-positions of the phenylurea, increases ligand affinity and β1- selectivity

Understanding the impact of complicated grief on combat related posttraumatic stress disorder, guilt, suicide, and functional impairment in a clinical trial of post‐9/11 service members and veterans

BackgroundComplicated grief (CG) is a bereavement‐specific syndrome distinct from but commonly comorbid with posttraumatic stress disorder (PTSD). While bereavement is common among military personnel (Simon et al., 2018), there is little research on the impact of CG comorbidity on PTSD treatment outcomes.MethodsTo evaluate the impact of comorbid CG on PTSD treatment outcomes we analyzed data from a randomized trial comparing prolonged exposure, sertraline, and their combination in veterans with a primary diagnosis of combat‐related PTSD (n = 194). Assessment of PTSD, trauma‐related guilt, functional impairment, and suicidal ideation and behavior occurred at baseline and weeks 6, 12, and 24 during the 24‐week trial.ResultsCG was associated with lower PTSD treatment response (odds ratio (OR) = 0.29, 95% confidence interval (CI) [0.12, 0.69], p = 0.005) and remission (OR = 0.28, 95% CI [0.11, 0.71], p = 0.007). Those with CG had greater severity of PTSD (p = 0.005) and trauma‐related guilt (<0.001) at baseline and endpoint. In addition, those with CG were more likely to experience suicidal ideation during the study (CG: 35%, 14/40 vs. no CG 15%, 20/130; OR = 3.01, 95% CI [1.29, 7.02], p = 0.011).ConclusionsComorbid CG is associated with elevated PTSD severity and independently associated with poorer endpoint treatment outcomes in veterans with combat‐related PTSD, suggesting that screening and additional intervention for CG may be needed.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/153078/1/da22911_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/153078/2/da22911.pd

Exploring hypotheses of the actions of TGF-beta 1 in epidermal wound healing using a 3D computational multiscale model of the human epidermis

In vivo and in vitro studies give a paradoxical picture of the actions of the key regulatory factor TGF-beta 1 in epidermal wound healing with it stimulating migration of keratinocytes but also inhibiting their proliferation. To try to reconcile these into an easily visualized 3D model of wound healing amenable for experimentation by cell biologists, a multiscale model of the formation of a 3D skin epithelium was established with TGF-beta 1 literature-derived rule sets and equations embedded within it. At the cellular level, an agent-based bottom-up model that focuses on individual interacting units ( keratinocytes) was used. This was based on literature-derived rules governing keratinocyte behavior and keratinocyte/ECM interactions. The selection of these rule sets is described in detail in this paper. The agent-based model was then linked with a subcellular model of TGF-beta 1 production and its action on keratinocytes simulated with a complex pathway simulator. This multiscale model can be run at a cellular level only or at a combined cellular/subcellular level. It was then initially challenged ( by wounding) to investigate the behavior of keratinocytes in wound healing at the cellular level. To investigate the possible actions of TGF-beta 1, several hypotheses were then explored by deliberately manipulating some of these rule sets at subcellular levels. This exercise readily eliminated some hypotheses and identified a sequence of spatial-temporal actions of TGF-beta 1 for normal successful wound healing in an easy-to-follow 3D model. We suggest this multiscale model offers a valuable, easy-to-visualize aid to our understanding of the actions of this key regulator in wound healing, and provides a model that can now be used to explore pathologies of wound healing

Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis.

Multiple sclerosis is a common disease of the central nervous system in which the interplay between inflammatory and neurodegenerative processes typically results in intermittent neurological disturbance followed by progressive accumulation of disability. Epidemiological studies have shown that genetic factors are primarily responsible for the substantially increased frequency of the disease seen in the relatives of affected individuals, and systematic attempts to identify linkage in multiplex families have confirmed that variation within the major histocompatibility complex (MHC) exerts the greatest individual effect on risk. Modestly powered genome-wide association studies (GWAS) have enabled more than 20 additional risk loci to be identified and have shown that multiple variants exerting modest individual effects have a key role in disease susceptibility. Most of the genetic architecture underlying susceptibility to the disease remains to be defined and is anticipated to require the analysis of sample sizes that are beyond the numbers currently available to individual research groups. In a collaborative GWAS involving 9,772 cases of European descent collected by 23 research groups working in 15 different countries, we have replicated almost all of the previously suggested associations and identified at least a further 29 novel susceptibility loci. Within the MHC we have refined the identity of the HLA-DRB1 risk alleles and confirmed that variation in the HLA-A gene underlies the independent protective effect attributable to the class I region. Immunologically relevant genes are significantly overrepresented among those mapping close to the identified loci and particularly implicate T-helper-cell differentiation in the pathogenesis of multiple sclerosis

The Baryonic Collapse Efficiency of Galaxy Groups in the RESOLVE and ECO Surveys

We examine the z = 0 group-integrated stellar and cold baryonic (stars + cold atomic gas) mass functions (group SMF and CBMF) and the baryonic collapse efficiency (group cold baryonic to dark matter halo mass ratio) using the RESOLVE and ECO survey galaxy group catalogs and a galform semi-analytic model (SAM) mock catalog. The group SMF and CBMF fall off more steeply at high masses and rise with a shallower low-mass slope than the theoretical halo mass function (HMF). The transition occurs at group-integrated cold baryonic mass M_coldbary ~ 10^11 Msun. The SAM, however, has significantly fewer groups at the transition mass ~ 10^11 Msun and a steeper low-mass slope than the data, suggesting that feedback is too weak in low-mass halos and conversely too strong near the transition mass. Using literature prescriptions to include hot halo gas and potential unobservable galaxy gas produces a group BMF with slope similar to the HMF even below the transition mass. Its normalization is lower by a factor of ~2, in agreement with estimates of warm-hot gas making up the remaining difference. We compute baryonic collapse efficiency with the halo mass calculated two ways, via halo abundance matching (HAM) and via dynamics (extended all the way to three-galaxy groups using stacking). Using HAM, we find that baryonic collapse efficiencies reach a flat maximum for groups across the halo mass range of M_halo ~ 10^11.4-12 Msun, which we label "nascent groups." Using dynamics, however, we find greater scatter in baryonic collapse efficiencies, likely indicating variation in group hot-to-cold baryon ratios. Similarly, we see higher scatter in baryonic collapse efficiencies in the SAM when using its true groups and their group halo masses as opposed to friends-of-friends groups and HAM masses