Genome-wide studies of copy number variation and exome sequencing identify rare variants in BAG3 as a cause of dilated cardiomyopathy

Dilated cardiomyopathy commonly causes heart failure and is the most frequent precipitating cause of heart transplantation. Familial dilated cardiomyopathy has been shown to be caused by rare variant mutations in more than 30 genes but only ~35% of its genetic cause has been identified, principally by using linkage-based or candidate gene discovery approaches. In a multigenerational family with autosomal dominant transmission, we employed whole-exome sequencing in a proband and three of his affected family members, and genome-wide copy number variation in the proband and his affected father and unaffected mother. Exome sequencing identified 428 single point variants resulting in missense, nonsense, or splice site changes. Genome-wide copy number analysis identified 51 insertion deletions and 440 copy number variants > 1 kb. Of these, a 8733 bp deletion, encompassing exon 4 of the heat shock protein cochaperone BCL2-associated athanogene 3 (BAG3), was found in seven affected family members and was absent in 355 controls. To establish the relevance of variants in this protein class in genetic DCM, we sequenced the coding exons in BAG3 in 311 other unrelated DCM probands and identified one frameshift, two nonsense, and four missense rare variants absent in 355 control DNAs, four of which were familial and segregated with disease. Knockdown of bag3 in a zebrafish model recapitulated DCM and heart failure. We conclude that new comprehensive genomic approaches have identified rare variants in BAG3 as causative of DCM.     

Identification of a chemical that inhibits the mycobacterial UvrABC complex in nucleotide excision repair

Bacterial DNA can be damaged by reactive nitrogen and oxygen intermediates (RNI and ROI) generated by host immunity, as well as by antibiotics that trigger bacterial production of ROI. Thus a pathogen's ability to repair its DNA may be important for persistent infection. A prominent role for nucleotide excision repair (NER) in disease caused by Mycobacterium tuberculosis (Mtb) was suggested by attenuation of uvrB-deficient Mtb in mice. However, it was unknown if Mtb's Uvr proteins could execute NER. Here we report that recombinant UvrA, UvrB, and UvrC from Mtb collectively bound and cleaved plasmid DNA exposed to ultraviolet (UV) irradiation or peroxynitrite. We used the DNA incision assay to test the mechanism of action of compounds identified in a high-throughput screen for their ability to delay recovery of M. smegmatis from UV irradiation. 2-(5-Amino-1,3,4-thiadiazol-2-ylbenzo[f]chromen-3-one) (ATBC) but not several closely related compounds inhibited cleavage of damaged DNA by UvrA, UvrB, and UvrC without intercalating in DNA and impaired recovery of M. smegmatis from UV irradiation. ATBC did not affect bacterial growth in the absence of UV exposure, nor did it exacerbate the growth defect of UV-irradiated mycobacteria that lacked uvrB. Thus, ATBC appears to be a cell-penetrant, selective inhibitor of mycobacterial NER. Chemical inhibitors of NER may facilitate studies of the role of NER in prokaryotic pathobiology.     

β-arrestin-2 regulation of the cAMP response element binding protein

Previous work demonstrated that cystic fibrosis (CF) cells exhibit an increase in cAMP-mediated signaling as a characteristic response to lost CFTR function. Evidence for increased cAMP-mediated signaling in CF included increased phosphorylation of the cAMP response element binding protein (CREB) and elevated β-arrestin-2 (βarr2) expression. However, subsequent studies reveal that CREB activation in CF cells is independent of protein kinase-A (PKA). The goal of this study is to test the hypothesis that elevated βarr2 expression leads to increased CREB activation in a PKA-independent mechanism. βarr2-GFP expressing tracheal epithelial cells (βarr2-GFP) exhibit an increase of pCREB content and subsequent CRE activation compared to GFP expressing control cells. βarr2 activation of the ERK cascade represents a candidate mechanism leading to CREB activation. ERK exhibits increased activation in βarr2-GFP cells compared to cont-GFP cells, and ERK inhibition diminishes CRE activation in both GFP and βarr2-GFP cells. To test directly whether CREB regulation in CF is βarr2-dependent, nasal epithelium excised from wt mice (Cftr +/+; βarr2 +/+), CF mice (Cftr -/-; βarr2 +/+), and DKO mice (Cftr -/-; βarr2 -/-) were analyzed for pCREB protein content. Removal of βarr2 expression from CF mice reduces both pCREB and pERK content to wt levels. These data indicate that CF-related CREB regulation is mediated directly through βarr2 expression via the ERK pathway.     

Inter‐specific Variation in Foliar Nutrients and Resorption of Nine Canopy‐tree Species in a Secondary Neotropical Rain Forest

The influence of environmental gradients on the foliar nutrient economy of forests has been well documented; however, we have little understanding of what drives variability among individuals within a single forest stand, especially tropical forests. We evaluated inter‐ and intra‐specific variation in nutrient resorption, foliar nutrient concentrations and physical leaf traits of nine canopy tree species within a 1‐ha secondary tropical rain forest in northeastern Costa Rica. Both nitrogen (N) and phosphorus (P) resorption efficiency (RE) and proficiency of the nine tree species varied significantly among species, but not within. Both N and P RE were significantly negatively related to leaf specific strength. Green leaf N and P concentrations were strongly negatively related to leaf mass per area, and senesced leaf nutrient concentrations were significantly positively related to green leaf nutrient concentrations. This study reveals a strong influence of physical leaf traits on foliar nutrient and resorption traits of co‐occurring species in a secondary wet tropical forest stand.     

PRKX critically regulates endothelial cell proliferation, migration, and vascular-like structure formation

Angiogenesis is a fundamental step in several important physiological events and pathological conditions including embryonic development, wound repair, tumor growth and metastasis. PRKX was identified as a novel type-I cAMP-dependent protein kinase gene expressed in multiple developing tissues. PRKX has also been shown to be phylogenetically and functionally distinct from PKA. This study presents the first evidence that PRKX stimulates endothelial cell proliferation, migration, and vascular-like structure formation, which are the three essential processes for angiogenesis. In contrast, classic PKA demonstrated an inhibitory effect on endothelia vascular-like structure formation. Our findings suggest that PRKX is an important protein kinase engaged in the regulation of angiogenesis and could play critical roles in various physiological and pathological conditions involving angiogenesis. PRKX binds to Pin-1, Magi-1 and Bag-3, which regulate cell proliferation, apoptosis, differentiation and tumorigenesis. The interaction of PRKX with Pin-1, Magi-1 and Bag-3 could contribute to the stimulating role of PRKX in angiogenesis.     

High resolution scanning electron microscopy of plasmodesmata

Symplastic transport occurs between neighbouring plant cells through functionally and structurally dynamic channels called plasmodesmata (PD). Relatively little is known about the composition of PD or the mechanisms that facilitate molecular transport into neighbouring cells. While transmission electron microscopy (TEM) provides 2-dimensional information about the structural components of PD, 3-dimensional information is difficult to extract from ultrathin sections. This study has exploited high-resolution scanning electron microscopy (HRSEM) to reveal the 3-dimensional morphology of PD in the cell walls of algae, ferns and higher plants. Varied patterns of PD were observed in the walls, ranging from uniformly distributed individual PD to discrete clusters. Occasionally the thick walls of the giant alga Chara were fractured, revealing the surface morphology of PD within. External structures such as spokes, spirals and mesh were observed surrounding the PD. Enzymatic digestions of cell wall components indicate that cellulose or pectin either compose or stabilise the extracellular spokes. Occasionally, the PD were fractured open and desmotubule-like structures and other particles were observed in their central regions. Our observations add weight to the argument that Chara PD contain desmotubules and are morphologically similar to higher plant PD.     

Antimicrobial susceptibility of bacterial isolates from sea otters (Enhydra lutris)

Bacterial infections are an important cause of sea otter (Enhydra lutris) mortality, and some of these infections may originate from terrestrial and anthropogenic sources. Antimicrobials are an important therapeutic tool for management of bacterial infections in stranded sea otters and for prevention of infection following invasive procedures in free-ranging otters. In this study, susceptibility to commonly used antimicrobials was determined for 126 isolates of 15 bacterial species or groups from necropsied, live-stranded injured or sick, and apparently healthy wild sea otters examined between 1998 and 2005. These isolates included both gram-positive and gram-negative strains of primary pathogens, opportunistic pathogens, and environmental flora, including bacterial species with proven zoonotic potential. Minimal evidence of antimicrobial resistance and no strains with unusual or clinically significant multiple-drug resistance patterns were identified. Collectively, these findings will help optimize selection of appropriate antimicrobials for treatment of bacterial diseases in sea otters and other marine species.     

A homozygous <Emphasis Type="Italic">RAB3GAP2</Emphasis> mutation causes Warburg Micro syndrome

Warburg Micro syndrome and Martsolf syndrome are clinically overlapping autosomal recessive conditions characterized by congenital cataracts, microphthalmia, postnatal microcephaly, and developmental delay. The neurodevelopmental and ophthalmological phenotype is more severe in Warburg Micro syndrome in which cerebral malformations and severe motor and mental retardation are common. While biallelic loss-of-function mutations in RAB3GAP1 are present in the majority of patients with Warburg Micro syndrome; a hypomorphic homozygous splicing mutation of RAB3GAP2 has been reported in a single family with Martsolf syndrome. Here, we report a novel homozygous RAB3GAP2 small in-frame deletion, c.499_507delTTCTACACT (p.Phe167_Thr169del) that causes Warburg Micro syndrome in a girl from a consanguineous Turkish family presenting with congenital cataracts, microphthalmia, absent visually evoked potentials, microcephaly, polymicrogyria, hypoplasia of the corpus callosum, and severe developmental delay. No RAB3GAP2 mutations were detected in ten additional unrelated patients with RAB3GAP1-negative Warburg Micro syndrome, consistent with further genetic heterogeneity. In conclusion, we provide evidence that RAB3GAP2 mutations are not specific to Martsolf syndrome. Rather, our findings suggest that loss-of-function mutations of RAB3GAP1 as well as functionally severe RAB3GAP2 mutations cause Warburg Micro syndrome while hypomorphic RAB3GAP2 mutations can result in the milder Martsolf phenotype. Thus, a phenotypic severity gradient may exist in the RAB3GAP-associated disease continuum (the “Warburg–Martsolf syndrome”) which is presumably determined by the mutant gene and the nature of the mutation.