Splicing error in E1alpha pyruvate dehydrogenase mRNA caused by novel intronic mutation responsible for lactic acidosis and mental retardation

An intronic point mutation was identified in the E1alpha PDH gene from a boy with delayed development and lactic acidosis, an X-linked disorder associated with a partial defect in pyruvate dehydrogenase (PDH) activity. Protein analysis demonstrated a corresponding decrease in immunoreactivity of the alpha and beta subunits of the PDH complex. In addition to the normal spliced mRNA product of the E1alpha PDH gene, patient samples contained significant levels of an aberrantly spliced mRNA with the first 45 nucleotides of intron 7 inserted in-frame between exons 7 and 8. The genomic DNA analysis found no mutation in the coding regions but revealed a hemizygous intronic G to A substitution 26 nucleotides downstream from the normal exon 7 5'-splice site. Splicing experiments in COS-7 cells demonstrated that this point mutation at intron 7 position 26 is responsible for the aberrant splicing phenotype, which involves a switch from the use of the normal 5'-splice site (intron 7 position 1) to the cryptic 5'-splice site downstream of the mutation (intron 7 position 45). The intronic mutation is unusual in that it generates a consensus binding motif for the splicing factor, SC35, which normally binds to exonic enhancer elements resulting in increased exon inclusion. Thus, the aberrant splicing phenotype is most likely explained by the generation of a de novo splicing enhancer motif, which activates the downstream cryptic 5'-splice site. The mutation documented here is a novel case of intron retention responsible for a human genetic disease.     

Ursodesoxycholic acid and heme-arginate are unable to improve hematopoiesis and liver injury in an erythropoietic protoporphyria mouse model

Erythropoietic protoporphyria (EPP) is an inherited disorder of heme biosynthesis caused by partial ferrochelatase deficiency, resulting in protoporphyrin overproduction which is responsible for painful skin photosensitivity. Chronic liver disease is the most severe complication of EPP, requiring liver transplantation in some patients. Data from a mouse model suggest that cytotoxic bile formation with high concentrations of bile salts and protoporphyrin may cause biliary fibrosis by damaging bile duct epithelium. In humans, cholestasis is a result of intracellular and canalicular precipitation of protoporphyrin. To limit liver damage two strategies may be considered: the first is to reduce protoporphyrin production and the second is to enhance protoporphyrin excretion. Bile salts are known to increase protoporphyrin excretion via the bile, while heme arginate is used to decrease the production of porphyrins in acute attacks of hepatic porphyrias. The Griseofulvin-induced protoporphyria mouse model has been used to study several aspects of human protoporphyria including the effects of bile salts. However, the best EPP animal model is an ethylnitrosourea-induced point mutation with fully recessive transmission, named ferrochelatase deficiency (Fech(m1Pas)). Here we investigate the effect of early ursodesoxycholic acid (UDCA) administration and heme-arginate injections on the ferrochelatase deficient EPP mouse model. In this model UDCA administration and heme-arginate injections do not improve the protoporphyric condition of Fech(m1Pas)/Fech(m1Pas) mice.     

Lineage-tracing and translatomic analysis of damage-inducible mitotic cochlear progenitors identifies candidate genes regulating regeneration

Cochlear supporting cells (SCs) are glia-like cells critical for hearing function. In the neonatal cochlea, the greater epithelial ridge (GER) is a mitotically quiescent and transient organ, which has been shown to nonmitotically regenerate SCs. Here, we ablated Lgr5⁺ SCs using Lgr5-DTR mice and found mitotic regeneration of SCs by GER cells in vivo. With lineage tracing, we show that the GER houses progenitor cells that robustly divide and migrate into the organ of Corti to replenish ablated SCs. Regenerated SCs display coordinated calcium transients, markers of the SC subtype inner phalangeal cells, and survive in the mature cochlea. Via RiboTag, RNA-sequencing, and gene clustering algorithms, we reveal 11 distinct gene clusters comprising markers of the quiescent and damaged GER, and damage-responsive genes driving cell migration and mitotic regeneration. Together, our study characterizes GER cells as mitotic progenitors with regenerative potential and unveils their quiescent and damaged translatomes.

Cochlear supporting cells are glia-like cells essential for hearing. Genetic ablation of Lgr5+ supporting cells reveals a population of damage inducible, mitotically activated progenitors in the greater epithelial ridge, which can divide and migrate into the organ of Corti to replenish ablated supporting cells. Translatomic analyses provide insights into the genes that may regulate this regenerative potential.     

Space Use Variation in Co-Occurring Sister Species: Response to Environmental Variation or Competition?

Coexistence often involves niche differentiation either as the result of environmental divergence, or in response to competition. Disentangling the causes of such divergence requires that environmental variation across space is taken into account, which is rarely done in empirical studies. We address the role of environmental variation versus competition in coexistence between two rodent species: Rhabdomys bechuanae (bechuanae) and Rhabdomys dilectus dilectus (dilectus) comparing their habitat preference and home range (HR) size in areas with similar climates, where their distributions abut (allopatry) or overlap (sympatry). Using Outlying Mean Index analyses, we test whether habitat characteristics of the species deviate significantly from a random sample of available habitats. In allopatry, results suggest habitat selection: dilectus preferring grasslands with little bare soil while bechuanae occurring in open shrublands. In sympatry, shrubland type habitats dominate and differences are less marked, yet dilectus selects habitats with more cover than bechuanae. Interestingly, bechuanae shows larger HRs than dilectus, and both species display larger HRs in sympatry. Further, HR overlaps between species are lower than expected. We discuss our results in light of data on the phylogeography of the genus and propose that evolution in allopatry resulted in adaptation leading to different habitat preferences, even at their distribution margins, a divergence expected to facilitate coexistence. However, since sympatry occurs in sites where environmental characteristics do not allow complete species separation, competition may explain reduced inter-species overlap and character displacement in HR size. This study reveals that both environmental variation and competition may shape species coexistence.     

Evolution of the sacrum in hominoids

In order to study the formation of the sacrum during the primate evolution, a new way of numbering mammalian vertebrae is presented; this demonstrates that the thoracolumbosacral complex is fixed at 22 vertebrae in 80% and at 22 +/- 1 in 100% of the cases. The shift of a vertebra from one type to another occurs either at the thoracolumbar or at the lumbosacral junction and not at the cervicothoracic junction. Rarely does the shift take place at the sacrococcygeal junction. Data from 318 primates reveal that the seven original lumbar vertebrae of the Old World monkeys are reduced in the great apes by a caudad "thoracization" of one to two lumbar vertebrae and a cephalad sacralization of one to four lumbar vertebrae. In the apes, sacralization is not total and different stages that are intermediate between lumbar and sacral are described. In Homo sapiens there is a total sacralization of the last two original lumbar vertebrae. In addition, development of the sacral wings (alae) is minimal in apes and reaches its maximum in hominids. The tendency of the hominoid sacrum to incorporate the last lumbar vertebrae and to widen markedly provides for an enhanced articulation of the sacrum with the ilium and offers a firm base of support for the trunk during erect posture. This is necessary for the support of the weight of the trunk above the sacrum and for the stabilization of the body during bipedal posture and locomotion. Encephalization did not play any major role in the widening of the sacrum since the former by far preceded the latter.     

Control of P-glycoprotein activity by membrane cholesterol amounts and their relation to multidrug resistance in human CEM leukemia cells

P-glycoprotein (P-gp) is the most well-known ATP-binding cassette (ABC) transporter involved in unidirectional substrate translocation across the membrane lipid bilayer, thereby causing the typical multidrug resistance (MDR) phenotype expressed in many cancers. We observed that in human CEM acute lymphoblastic leukemia cells expressing various degrees of chemoresistance and where P-gp was the sole MDR-related ABC transporter detected, the amount of esterified cholesterol increased linearly with the level of resistance to vinblastine while the amounts of total and free cholesterol increased in a nonlinear way. Membrane cholesterol controlled the ATPase activity of P-gp in a linear manner, whereas the P-gp-induced daunomycin efflux decreased nonlinearly with the depletion of membrane cholesterol. All these elements suggest that cholesterol controls both the ATPase and the drug efflux activities of P-gp. In addition, in CEM cell lines that expressed increasing levels of elevated chemoresistance, the amount of P-gp increases to a plateau value of 40% of the total membrane proteins and remained unvaried while the amount of membrane cholesterol increased with the elevation of the MDR level, strongly suggesting that cholesterol may be directly involved in the typical MDR phenotype. Finally, we showed that the decreased daunomycin efflux by P-gp due to the partial depletion of membrane cholesterol was responsible for the efficient chemosensitization of resistant CEM cells, which could be totally reversed after cholesterol repletion.