Whole-Exome Sequencing Efficiently Detects Rare Mutations in Autosomal Recessive Nonsyndromic Hearing Loss

Identification of the pathogenic mutations underlying autosomal recessive nonsyndromic hearing loss (ARNSHL) is difficult, since causative mutations in 39 different genes have so far been reported. After excluding mutations in the most common ARNSHL gene, GJB2, via Sanger sequencing, we performed whole-exome sequencing (WES) in 30 individuals from 20 unrelated multiplex consanguineous families with ARNSHL. Agilent SureSelect Human All Exon 50 Mb kits and an Illumina Hiseq2000 instrument were used. An average of 93%, 84% and 73% of bases were covered to 1X, 10X and 20X within the ARNSHL-related coding RefSeq exons, respectively. Uncovered regions with WES included those that are not targeted by the exome capture kit and regions with high GC content. Twelve homozygous mutations in known deafness genes, of which eight are novel, were identified in 12 families: MYO15A-p.Q1425X, -p.S1481P, -p.A1551D; LOXHD1-p.R1494X, -p.E955X; GIPC3-p.H170N; ILDR1-p.Q274X; MYO7A-p.G2163S; TECTA-p.Y1737C; TMC1-p.S530X; TMPRSS3-p.F13Lfs*10; TRIOBP-p.R785Sfs*50. Each mutation was within a homozygous run documented via WES. Sanger sequencing confirmed co-segregation of the mutation with deafness in each family. Four rare heterozygous variants, predicted to be pathogenic, in known deafness genes were detected in 12 families where homozygous causative variants were already identified. Six heterozygous variants that had similar characteristics to those abovementioned variants were present in 15 ethnically-matched individuals with normal hearing. Our results show that rare causative mutations in known ARNSHL genes can be reliably identified via WES. The excess of heterozygous variants should be considered during search for causative mutations in ARNSHL genes, especially in small-sized families.     

A conformational intermediate between the resting and desensitized states of the nicotinic acetylcholine receptor

The structural changes induced in the nicotinic acetylcholine receptor by two noncompetitive channel blockers, proadifen and phencyclidine, have been studied by infrared difference spectroscopy and using the conformationally sensitive photoreactive noncompetitive antagonist 3-(trifluoromethyl)-3-m-([(125)I]iodophenyl)diazirine. Simultaneous binding of proadifen to both the ion channel pore and neurotransmitter sites leads to the loss of positive markers near 1663, 1655, 1547, 1430, and 1059 cm(-)(1) in carbamylcholine difference spectra, suggesting the stabilization of a desensitized conformation. In contrast, only the positive markers near 1663 and 1059 cm(-)(1) are maximally affected by the binding of either blocker to the ion channel pore suggesting that the conformationally sensitive residues vibrating at these two frequencies are stabilized in a desensitized-like conformation, whereas those vibrating near 1655 and 1430 cm(-)(1) remain in a resting-like state. The vibrations at 1547 cm(-)(1) are coupled to those at both 1663 and 1655 cm(-)(1) and thus exhibit an intermediate pattern of band intensity change. The formation of a structural intermediate between the resting and desensitized states in the presence of phencyclidine is further supported by the pattern of 3-(trifluoromethyl)-3-m-([(125)I]iodophenyl)diazirine photoincorporation. In the presence of phencyclidine, the subunit labeling pattern is distinct from that observed in either the resting or desensitized conformations; specifically, there is a concentration-dependent increase in the extent of photoincorporation into the delta-subunit. Our data show that domains of the nicotinic acetylcholine receptor interconvert between the resting and desensitized states independently of each other and suggest a revised model of channel blocker action that involves both low and high affinity agonist binding conformational intermediates.     

A computational approach to characterization of bovine sperm chromatin alterations

We describe here a computational morphology-based approach to the investigation of possible causes of chromatin alterations in sperm. A comprehensive set of state-of-the-art and geometric measures are computationally extracted from toluidine blue stained images and analyzed to infer the possible processes leading to normal and abnormal chromatin formation while seeking a possible taxonomy of chromatin alterations and their influence on sperm head morphology. Using this methodology, we have identified higher chromatin fragility at some specific points of the sperm head. Despite the lack of correlation between morphologies of sperm head and chromatin structure, four main morphological types of chromatin alterations in bull spermatozoa have been identified and their possible causes discussed.     

Characterizing the role benthos plays in large coastal seas and estuaries: A modular approach

Ecologists studying coastal and estuarine benthic communities have long taken a macroecological view, by relating benthic community patterns to environmental factors across several spatial scales. Although many general ecological patterns have been established, often a significant amount of the spatial and temporal variation in soft-sediment communities within and among systems remains unexplained. Here we propose a framework that may aid in unraveling the complex influence of environmental factors associated with the different components of coastal systems (i.e. the terrestrial and benthic landscapes, and the hydrological seascape) on benthic communities, and use this information to assess the role played by benthos in coastal ecosystems. A primary component of the approach is the recognition of system modules (e.g. marshes, dendritic systems, tidal rivers, enclosed basins, open bays, lagoons). The modules may differentially interact with key forcing functions (e.g. temperature, salinity, currents) that influence system processes and in turn benthic responses and functions. Modules may also constrain benthic characteristics and related processes within certain ecological boundaries and help explain their overall spatio-temporal variation. We present an example of how benthic community characteristics are related to the modular structure of 14 coastal seas and estuaries, and show that benthic functional group composition is significantly related to the modular structure of these systems. We also propose a framework for exploring the role of benthic communities in coastal systems using this modular approach and offer predictions of how benthic communities may vary depending on the modular composition and characteristics of a coastal system.     

Timing of clade divergence and discordant estimates of genetic and morphological diversity in the Slender Madtom,Noturus exilis (Ictaluridae)

Phylogeographic relationships, the timing of clade diversification, and the potential for cryptic diversity in the Slender Madtom, Noturus exilis, was investigated using mitochondrial Cyt b, nuclear RAG2, shape analysis, and meristic and pigmentation data. Three well-supported and deeply divergent clades were recovered from analyses of genetic data: Little Red River (White River drainage) clade, Arkansas + Red River (Mississippi River) clade, and a large clade of populations from the rest of the range of the species. Recovered clades showed little to no diagnostic morphological differences, supporting previous hypotheses of morphological conservatism in catfishes, and indicating morphology may commonly underestimate diversity in this group of fishes. The Little Red River clade is the most distinct lineage of N. exilis with 11 POM pores (vs. 10 in other populations) and unique Cyt b haplotypes and RAG 2 alleles. However, treating it as a species separate from N. exilis would imply that the other major clades of N. exilis are more closely related to one another than they are to the Little Red River clade, which was not supported.The UCLN age estimate for Noturus was 23.9 mya (95% HPD: 13.49, 35.43), indicating a late Oligocene to early Miocene origin. The age of N. exilis was estimated as late Miocene at 9.7 mya (95% HPD: 5.32, 14.93). Diversification within the species spanned the late Miocene to mid-Pleistocene. The largest clade of N. exilis, which dates to the late Miocene, includes populations from the unglaciated Eastern and Interior Highlands as well as the previously glaciated Central Lowlands. Diversification of this clade coincides with a drastic drop in sea-level and diversification of other groups of Central Highlands fishes (Centrarchidae and Cyprinidae). Sub-clades dating to the Pleistocene show that northern populations occurring in previously glaciated regions resulted from dispersal from populations in the Ozarks up the Mississippi River following retreat of the Pleistocene glaciers. Pre-Pleistocene vicariance, such as drainage pattern changes of the Mississippi River, also played a prominent role in the history of the species. The incorporation of a temporal estimate of clade diversification revealed that in some instances, phylogeographic breaks shared with other aquatic species were best explained by different or persistent vicariant events through time, rather than a single shared event.     

NAD(P)H oxidase/nitric oxide interactions in peroxisome proliferator activated receptor (PPAR)α-mediated cardiovascular effects

Activation of peroxisome proliferator activated receptor (PPAR)α and its protective role in cardiovascular function has been reported but the exact mechanism(s) involved is not clear. As we have shown that PPARα ligands increased nitric oxide (NO) production and cardiovascular function is controlled by a balance between NO and free radicals, we hypothesize that PPARα activation tilts the balance between NO and free radicals and that this mechanism defines the protective effects of PPARα ligands on cardiovascular system. Systolic blood pressure (SBP) was greater in PPARα knockout (KO) mice compared with its wild type (WT) litter mates (130 ± 10 mmHg versus 107 ± 4 mmHg). l-NAME (100 mg/L p.o.), the inhibitor of NO production abolished the difference between PPARα KO and WT mice. In kidney homogenates, tissue lipid hydroperoxide generation was greater in KO mice (11.8 ± 1.4 pM/mg versus 8.3 ± 0.6 pM/mg protein). This was accompanied by a higher total NOS activity (46 ± 6%, p < 0.05) and a 3 fold greater Ca²⁺-dependent NOS activity in kidney homogenates of untreated PPARα WT compared with the KO mice. Clofibrate, a PPARα ligand, increased NOS activity in WT but not KO mice. Bezafibrate (30 mg/kg) reduced SBP in conscious rats (19 ± 4%, p < 0.05), increased urinary NO excretion (4.06 ± 0.53–7.07 ± 1.59 μM/24 h; p < 0.05) and reduced plasma 8-isoprostane level (45.8 ± 15 μM versus 31.4 ± 8 μM), and NADP(H) oxidase activity (16 ± 5%). Implantation of DOCA pellet (20 mg s.c.) in uninephrectomized mice placed on 1% NaCl drinking water increased SBP by a margin that was markedly greater in KO mice (193 ± 13 mmHg versus 130 ± 12 mmHg). In the rat, DOCA increased SBP and NAD(P)H oxidase activity and both effects were diminished by clofibrate. In addition, clofibrate reduced ET-1 production in DOCA/salt hypertensive rats. Thus, apart from inhibition of ET-1 production, PPARα activation exerts protective actions in hypertension via a mechanism that involves NO production and/or inhibition of NAD(P)H oxidase activity.