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Inherited macular dystrophies (iMDs) are a group of genetic disorders, which affect the central region of the retina. To investigate the genetic basis of iMDs, we used single-molecule Molecular Inversion Probes to sequence 105 maculopathy-associated genes in 1352 patients diagnosed with iMDs. Within this cohort, 39.8% of patients were considered genetically explained by 460 different variants in 49 distinct genes of which 73 were novel variants, with some affecting splicing. The top five most frequent causative genes were ABCA4 (37.2%), PRPH2 (6.7%), CDHR1 (6.1%), PROM1 (4.3%) and RP1L1 (3.1%). Interestingly, variants with incomplete penetrance were revealed in almost one-third of patients considered solved (28.1%), and therefore, a proportion of patients may not be explained solely by the variants reported. This includes eight previously reported variants with incomplete penetrance in addition to CDHR1:c.783G>A and CNGB3:c.1208G>A. Notably, segregation analysis was not routinely performed for variant phasing-a limitation, which may also impact the overall diagnostic yield. The relatively high proportion of probands without any putative causal variant (60.2%) highlights the need to explore variants with incomplete penetrance, the potential modifiers of disease and the genetic overlap between iMDs and age-related macular degeneration. Our results provide valuable insights into the genetic landscape of iMDs and warrant future exploration to determine the involvement of other maculopathy genes.

Neural differentiation, synaptic transmission, and action potential propagation depend on membrane sphingolipids, whose metabolism is tightly regulated. Mutations in the ceramide transporter CERT (CERT1), which is involved in sphingolipid biosynthesis, are associated with intellectual disability, but the pathogenic mechanism remains obscure. Here, we characterize 31 individuals with de novo missense variants in CERT1. Several variants fall into a previously uncharacterized dimeric helical domain that enables CERT homeostatic inactivation, without which sphingolipid production goes unchecked. The clinical severity reflects the degree to which CERT autoregulation is disrupted, and inhibiting CERT pharmacologically corrects morphological and motor abnormalities in a Drosophila model of the disease, which we call ceramide transporter (CerTra) syndrome. These findings uncover a central role for CERT autoregulation in the control of sphingolipid biosynthetic flux, provide unexpected insight into the structural organization of CERT, and suggest a possible therapeutic approach for patients with CerTra syndrome.

Blood cell formation is classically thought to occur through a hierarchical differentiation process, although recent studies have shown that lineage commitment may occur earlier in hematopoietic stem and progenitor cells (HSPCs). The relevance to human blood diseases and the underlying regulation of these refined models remain poorly understood. By studying a genetic blood disorder, Diamond-Blackfan anemia (DBA), where the majority of mutations affect ribosomal proteins and the erythroid lineage is selectively perturbed, we are able to gain mechanistic insight into how lineage commitment is programmed normally and disrupted in disease. We show that in DBA, the pool of available ribosomes is limited, while ribosome composition remains constant. Surprisingly, this global reduction in ribosome levels more profoundly alters translation of a select subset of transcripts. We show how the reduced translation of select transcripts in HSPCs can impair erythroid lineage commitment, illuminating a regulatory role for ribosome levels in cellular differentiation. A global reduction in ribosome levels in Diamond-Blackfan anemia profoundly alters translation of a select subset of transcripts, thereby impeding erythroid lineage commitment. Keyword: hematopoiesis; lineage commitment; GATA1; genetics; ribosome; translation; Diamond-Blackman anemia; erythropoiesis, National Institutes of Health (U.S.) (Grants R01 DK103794), National Institutes of Health (U.S.) (Grant R33 HL120791)

Retinitis pigmentosa (RP) is a major cause of blindness that affects 1.5 million people worldwide. Mutations in cyclic nucleotide-gated channel β 1 (CNGB1) cause approximately 4% of autosomal recessive RP. Gene augmentation therapy shows promise for treating inherited retinal degenerations; however, relevant animal models and biomarkers of progression in patients with RP are needed to assess therapeutic outcomes. Here, we evaluated RP patients with CNGB1 mutations for potential biomarkers of progression and compared human phenotypes with those of mouse and dog models of the disease. Additionally, we used gene augmentation therapy in a CNGβ1-deficient dog model to evaluate potential translation to patients. CNGB1-deficient RP patients and mouse and dog models had a similar phenotype characterized by early loss of rod function and slow rod photoreceptor loss with a secondary decline in cone function. Advanced imaging showed promise for evaluating RP progression in human patients, and gene augmentation using adeno-associated virus vectors robustly sustained the rescue of rod function and preserved retinal structure in the dog model. Together, our results reveal an early loss of rod function in CNGB1-deficient patients and a wide window for therapeutic intervention. Moreover, the identification of potential biomarkers of outcome measures, availability of relevant animal models, and robust functional rescue from gene augmentation therapy support future work to move CNGB1-RP therapies toward clinical trials.

Thesis (M.S.)--Massachusetts Institute of Technology, Program in Media Arts & Sciences, 1995., Includes bibliographical references (leaves 64-68)., by Lee Winston Campbell., M.S.

Inflammatory cytokines have been proposed to regulate epithelial homeostasis during intestinal inflammation. We report here that interferon-gamma (IFN-gamma) regulates the crucial homeostatic functions of cell proliferation and apoptosis through serine-threonine protein kinase AKT-beta-catenin and Wingless-Int (Wnt)-beta-catenin signaling pathways. Short-term exposure of intestinal epithelial cells to IFN-gamma resulted in activation of beta-catenin through AKT, followed by induction of the secreted Wnt inhibitor Dkk1. Consequently, we observed an increase in Dkk1-mediated apoptosis upon extended IFN-gamma treatment and reduced proliferation through depletion of the Wnt coreceptor LRP6. These effects were enhanced by tumor necrosis factor-alpha (TNF-alpha), suggesting synergism between the two cytokines. Consistent with these results, colitis in vivo was associated with decreased beta-catenin-T cell factor (TCF) signaling, loss of plasma membrane-associated LRP6, and reduced epithelial cell proliferation. Proliferation was partially restored in IFN-gamma-deficient mice. Thus, we propose that IFN-gamma regulates intestinal epithelial homeostasis by sequential regulation of converging beta-catenin signaling pathways., Nava, Porfirio Koch, Stefan Laukoetter, Mike G Lee, Winston Y Kolegraff, Keli Capaldo, Christopher T Beeman, Neal Addis, Caroline Gerner-Smidt, Kirsten Neumaier, Irmgard Skerra, Arne Li, Linheng Parkos, Charles A Nusrat, Asma eng R01 DK059888-09/DK/NIDDK NIH HHS/ R01 DK079392/DK/NIDDK NIH HHS/ R01 DK061379-08/DK/NIDDK NIH HHS/ DK55679/DK/NIDDK NIH HHS/ R29 DK055679/DK/NIDDK NIH HHS/ T32 GM008169/GM/NIGMS NIH HHS/ DK72564/DK/NIDDK NIH HHS/ DK64399/DK/NIDDK NIH HHS/ R01 DK072564/DK/NIDDK NIH HHS/ R01 DK055679-12/DK/NIDDK NIH HHS/ DK53202/DK/NIDDK NIH HHS/ R24 DK064399/DK/NIDDK NIH HHS/ R24 DK064399-019003/DK/NIDDK NIH HHS/ DK59888/DK/NIDDK NIH HHS/ DK79392/DK/NIDDK NIH HHS/ R01 DK055679/DK/NIDDK NIH HHS/ R01 DK079392-07/DK/NIDDK NIH HHS/ R24 DK064399-019002/DK/NIDDK NIH HHS/ R01 DK061379/DK/NIDDK NIH HHS/ R01 DK059888/DK/NIDDK NIH HHS/ DK61379/DK/NIDDK NIH HHS/ R01 DK072564-15/DK/NIDDK NIH HHS/ Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't 2010/03/23 06:00 Immunity. 2010 Mar 26;32(3):392-402. doi: 10.1016/j.immuni.2010.03.001. Epub 2010 Mar 18.

During mucosal inflammation, a complex array of proinflammatory and protective mechanisms regulates inflammation and severity of injury. Secretion of anti-inflammatory mediators is a mechanism that is critical in controlling inflammatory responses and promoting epithelial restitution and barrier recovery. AnxA1 is a potent anti-inflammatory protein that has been implicated to play a critical immune regulatory role in models of inflammation. Although AnxA1 has been shown to be secreted in intestinal mucosal tissues during inflammation, its potential role in modulating the injury/inflammatory response is not understood. In this study, we demonstrate that AnxA1-deficient animals exhibit increased susceptibility to dextran sulfate sodium (DSS)-induced colitis with greater clinical morbidity and histopathologic mucosal injury. Furthermore, impaired recovery following withdrawal of DSS administration was observed in AnxA1 (-/-) animals compared with wild-type (WT) control mice that was independent of inflammatory cell infiltration. Since AnxA1 exerts its anti-inflammatory properties through stimulation of ALX/FPRL-1, we explored the role of this receptor-ligand interaction in regulating DSS-induced colitis. Interestingly, treatment with an ALX/FPRL-1 agonist, 15-epi-lipoxin A4 reversed the enhanced sensitivity of AnxA1 (-/-) mice to DSS colitis. In contrast, 15-epi-lipoxin A4 did not significantly improve the severity of disease in WT animals. Additionally, differential expression of ALX/FPLR-1 in control and DSS-treated WT and AnxA1-deficient animals suggested a potential role for AnxA1 in regulating ALX/FPRL-1 expression under pathophysiological conditions. Together, these results support a role of endogenous AnxA1 in the protective and reparative properties of the intestinal mucosal epithelium., Babbin, Brian A Laukoetter, Mike G Nava, Porfirio Koch, Stefan Lee, Winston Y Capaldo, Christopher T Peatman, Eric Severson, Eric A Flower, Roderick J Perretti, Mauro Parkos, Charles A Nusrat, Asma eng K08 DK074706/DK/NIDDK NIH HHS/ R01 DK059888-09/DK/NIDDK NIH HHS/ R01 DK061379-08/DK/NIDDK NIH HHS/ R29 DK055679/DK/NIDDK NIH HHS/ T32 GM008169/GM/NIGMS NIH HHS/ K08 DK074706-02/DK/NIDDK NIH HHS/ R01-DK61379/DK/NIDDK NIH HHS/ DK 55679/DK/NIDDK NIH HHS/ DK64399/DK/NIDDK NIH HHS/ R01 DK072564/DK/NIDDK NIH HHS/ T32 DK007771-09/DK/NIDDK NIH HHS/ R24 DK064399/DK/NIDDK NIH HHS/ R01 DK055679-11A1/DK/NIDDK NIH HHS/ R24 DK064399-019003/DK/NIDDK NIH HHS/ R01 DK055679/DK/NIDDK NIH HHS/ DK 59888/DK/NIDDK NIH HHS/ R01 DK061379/DK/NIDDK NIH HHS/ R01-DK72564/DK/NIDDK NIH HHS/ T32 DK007771/DK/NIDDK NIH HHS/ R01 DK059888/DK/NIDDK NIH HHS/ K08 DK074706-01/DK/NIDDK NIH HHS/ R01 DK072564-15/DK/NIDDK NIH HHS/ Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't 2008/09/20 09:00 J Immunol. 2008 Oct 1;181(7):5035-44.

Thesis (Ph. D.)--Massachusetts Institute of Technology, School of Architecture and Planning, Program in Media Arts and Sciences, 2001., Includes bibliographical references (leaves 86-92)., A person's communicative intent can be better understood by either a human or a machine if the person's gestures are understood. This thesis project demonstrates an expansion of both the range of co-verbal gestures a machine can identify, and the range of communicative intents the machine can infer. We develop an automatic system that uses realtime video as sensory input and then segments, classifies, and responds to co-verbal gestures made by users in realtime as they converse with a synthetic character known as REA, which is being developed in parallel by Justine Cassell and her students at the MIT Media Lab. A set of 670 natural gestures, videotaped and visually tracked in the course of conversational interviews and then hand segmented and annotated according to a widely used gesture classification scheme, is used in an offline training process that trains Hidden Markov Model classifiers. A number of feature sets are extracted and tested in the offline training process, and the best performer is employed in an online HMM segmenter and classifier that requires no encumbering attachments to the user. Modifications made to the REA system enable REA to respond to the user's beat and deictic gestures as well as turntaking requests the user may convey in gesture., (cont.) The recognition results obtained are far above chance, but too low for use in a production recognition system. The results provide a measure of validity for the gesture categories chosen, and they provide positive evidence for an appealing but difficult to prove proposition: to the extent that a machine can recognize and use these categories of gestures to infer information not present in the words spoken, there is exploitable complementary information in the gesture stream., by Lee Winston Campbell., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, School of Architecture and Planning, Program in Media Arts and Sciences, 2001., Includes bibliographical references (leaves 86-92)., A person's communicative intent can be better understood by either a human or a machine if the person's gestures are understood. This thesis project demonstrates an expansion of both the range of co-verbal gestures a machine can identify, and the range of communicative intents the machine can infer. We develop an automatic system that uses realtime video as sensory input and then segments, classifies, and responds to co-verbal gestures made by users in realtime as they converse with a synthetic character known as REA, which is being developed in parallel by Justine Cassell and her students at the MIT Media Lab. A set of 670 natural gestures, videotaped and visually tracked in the course of conversational interviews and then hand segmented and annotated according to a widely used gesture classification scheme, is used in an offline training process that trains Hidden Markov Model classifiers. A number of feature sets are extracted and tested in the offline training process, and the best performer is employed in an online HMM segmenter and classifier that requires no encumbering attachments to the user. Modifications made to the REA system enable REA to respond to the user's beat and deictic gestures as well as turntaking requests the user may convey in gesture., (cont.) The recognition results obtained are far above chance, but too low for use in a production recognition system. The results provide a measure of validity for the gesture categories chosen, and they provide positive evidence for an appealing but difficult to prove proposition: to the extent that a machine can recognize and use these categories of gestures to infer information not present in the words spoken, there is exploitable complementary information in the gesture stream., by Lee Winston Campbell., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, School of Architecture and Planning, Program in Media Arts and Sciences, 2001., Includes bibliographical references (leaves 86-92)., A person's communicative intent can be better understood by either a human or a machine if the person's gestures are understood. This thesis project demonstrates an expansion of both the range of co-verbal gestures a machine can identify, and the range of communicative intents the machine can infer. We develop an automatic system that uses realtime video as sensory input and then segments, classifies, and responds to co-verbal gestures made by users in realtime as they converse with a synthetic character known as REA, which is being developed in parallel by Justine Cassell and her students at the MIT Media Lab. A set of 670 natural gestures, videotaped and visually tracked in the course of conversational interviews and then hand segmented and annotated according to a widely used gesture classification scheme, is used in an offline training process that trains Hidden Markov Model classifiers. A number of feature sets are extracted and tested in the offline training process, and the best performer is employed in an online HMM segmenter and classifier that requires no encumbering attachments to the user. Modifications made to the REA system enable REA to respond to the user's beat and deictic gestures as well as turntaking requests the user may convey in gesture., (cont.) The recognition results obtained are far above chance, but too low for use in a production recognition system. The results provide a measure of validity for the gesture categories chosen, and they provide positive evidence for an appealing but difficult to prove proposition: to the extent that a machine can recognize and use these categories of gestures to infer information not present in the words spoken, there is exploitable complementary information in the gesture stream., by Lee Winston Campbell., Ph.D.