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.     

Spectrofluorimetric analysis of the interaction of amyloid peptides with neuronal nitric oxide synthase: Implications in Alzheimer's disease

Background

The deposition of aggregated β-amyloid peptide senile plaques and the accumulation of arginine within the astrocytes in the brain of an Alzheimer's patient are classic observations in the neuropathology of the disease. It would be logical, in the aetiology and pathogenesis, to investigate arginine-metabolising enzymes and their intimate association with amyloid peptides.

Methods

Neuronal nitric oxide synthase (nNOS) was isolated, purified and shown, through fluorescence quenching spectroscopy and fluorescence resonance energy transfer (FRET), to interact with structural fragments of Aβ_1–40 and be catalytic towards amyloid fibril formation.

Results

Only one binding site on the enzyme was available for binding. Two amyloid peptide fragments of Aβ_1–40 (Aβ_17–28 and Aβ_25–35) had Stern–Volmer values (K_SV) of 0.111 μM⁻¹ and 0.135 μM⁻¹ indicating tight binding affinity to nNOS and easier accessibility to fluor molecules during binding. The polarity of this active site precludes binding of the predominantly hydrophobic amyloid peptide fragments contained within Aβ_17–28 and within two glycine zipper motifs [G-X-X-X-G-X-X-X-G] [Aβ_29–37] and bind to the enzyme at a site remote to the active region.

Conclusions

The interaction and binding of Aβ_17–28 and Aβ_25–35 to nNOS causes the movement of two critical tryptophan residues of 0.77 nm and 0.57 nm respectively towards the surface of the enzyme.

General significance

The binding of Aβ-peptide fragments with nNOS has been studied by spectrofluorimetry. The information and data presented should contribute towards understanding the mechanism for deposition of aggregated Aβ-peptides and fibrillogenesis in senile plaques in an AD brain.     

Synergism between DNA methylation and macroH2A1 occupancy in epigenetic silencing of the tumor suppressor gene p16(CDKN2A)

Promoter hypermethylation and heterochromatinization is a frequent event leading to gene inactivation and tumorigenesis. At the molecular level, inactivation of tumor suppressor genes in cancer has many similarities to the inactive X chromosome in female cells and is defined and maintained by DNA methylation and characteristic histone modifications. In addition, the inactive-X is marked by the histone macroH2A, a variant of H2A with a large non-histone region of unknown function. Studying tumor suppressor genes (TSGs) silenced in cancer cell lines, we find that when active, these promoters are associated with H2A.Z but become enriched for macroH2A1 once silenced. Knockdown of macroH2A1 was not sufficient for reactivation of silenced genes. However, when combined with DNA demethylation, macroH2A1 deficiency significantly enhanced reactivation of the tumor suppressor genes p16, MLH1 and Timp3 and inhibited cell proliferation. Our findings link macroH2A1 to heterochromatin of epigenetically silenced cancer genes and indicate synergism between macroH2A1 and DNA methylation in maintenance of the silenced state.     

Detection of naturally aerosolized <Emphasis Type="Italic">Mycoplasma hyopneumoniae</Emphasis> from the air of selected swine farms

We aimed to detect Mycoplasma hyopneumoniae from the air of selected farms through air filtration-based air sampling using polymerase chain reaction (PCR). Air samples were collected at different locations inside and outside of pig farm rooms on polyethersulfone membrane (0.22 μm), and the presence of M. hyopneumoniae DNA was analyzed by PCR. Furthermore, nasal swab and blood samples were collected from 336 pigs in the same air sampled rooms and were analyzed by PCR and IDEXX ELISA, respectively. The suitability of the air sampling method was validated with analysis of an artificially induced aerosolized avirulent M. hyopneumoniae in an enclosed box showing PCR-positive results. M. hyopneumoniae was detected from air sample of pig farm rooms using PCR. Although the probability of an airborne M. hyopneumoniae causing an infection is not yet confirmed, air sampling PCR results could serve as a tool to assess the spread of M. hyopneumoniae by bioaerosols and the infection dynamics in a herd and between herds.     

Patterns of Brain Activation when Mothers View Their Own Child and Dog: An fMRI Study

Neural substrates underlying the human-pet relationship are largely unknown. We examined fMRI brain activation patterns as mothers viewed images of their own child and dog and an unfamiliar child and dog. There was a common network of brain regions involved in emotion, reward, affiliation, visual processing and social cognition when mothers viewed images of both their child and dog. Viewing images of their child resulted in brain activity in the midbrain (ventral tegmental area/substantia nigra involved in reward/affiliation), while a more posterior cortical brain activation pattern involving fusiform gyrus (visual processing of faces and social cognition) characterized a mother''s response to her dog. Mothers also rated images of their child and dog as eliciting similar levels of excitement (arousal) and pleasantness (valence), although the difference in the own vs. unfamiliar child comparison was larger than the own vs. unfamiliar dog comparison for arousal. Valence ratings of their dog were also positively correlated with ratings of the attachment to their dog. Although there are similarities in the perceived emotional experience and brain function associated with the mother-child and mother-dog bond, there are also key differences that may reflect variance in the evolutionary course and function of these relationships.     

Laboratory Test Surveillance following Acute Kidney Injury

Background

Patients with hospitalized acute kidney injury (AKI) are at increased risk for accelerated loss of kidney function, morbidity, and mortality. We sought to inform efforts at improving post-AKI outcomes by describing the receipt of renal-specific laboratory test surveillance among a large high-risk cohort.

Methods

We acquired clinical data from the Electronic health record (EHR) of 5 Veterans Affairs (VA) hospitals to identify patients hospitalized with AKI from January 1^st, 2002 to December 31^st, 2009, and followed these patients for 1 year or until death, enrollment in palliative care, or improvement in renal function to estimated GFR (eGFR) ≥60 L/min/1.73 m². Using demographic data, administrative codes, and laboratory test data, we evaluated the receipt and timing of outpatient testing for serum concentrations of creatinine and any as well as quantitative proteinuria recommended for CKD risk stratification. Additionally, we reported the rate of phosphorus and parathyroid hormone (PTH) monitoring recommended for chronic kidney disease (CKD) patients.

Results

A total of 10,955 patients admitted with AKI were discharged with an eGFR<60 mL/min/1.73 m². During outpatient follow-up at 90 and 365 days, respectively, creatinine was measured on 69% and 85% of patients, quantitative proteinuria was measured on 6% and 12% of patients, PTH or phosphorus was measured on 10% and 15% of patients.

Conclusions

Measurement of creatinine was common among all patients following AKI. However, patients with AKI were infrequently monitored with assessments of quantitative proteinuria or mineral metabolism disorder, even for patients with baseline kidney disease.     

Evodiamine inhibits in vitro angiogenesis: Implication for antitumorgenicity

Evodiamine, the major bioactive compound isolated from Chinese herbal drug named Wu-Chu-Yu, has been reported to exhibit anti-tumor growth and metastasis. However, the effect of evodiamine on angiogenesis remains to be investigated. We used the fresh medium containing evodiamine or human lung adenocarcinoma cell (CL1 cells) derived conditioned media free of evodiamine to test their capability to induce in vitro angiogenesis, i.e., human umbilical vein endothelial cells (HUVECs) tube formation and invasion. We demonstrated that evodiamine could directly inhibit in vitro HUVECs tube formation and invasion. Locally administered evodiamine also inhibited the in vivo angiogenesis in the chick embryo chorioallantoic membrane (CAM) assay. The gene expression of vascular endothelial growth factor (VEGF) and the p44/p42 mitogen-activated protein kinase (MAPK, ERK) that correlated with endothelial cells angiogenesis were inhibited by evodiamine. We found that the evodiamine-treated CL1 cells derived conditioned medium showed decreased VEGF release and reduced ability of inducing in vitro tube formation. After the collection of conditioned media, the VEGF expression of remaining CL1 cells were determined by Western analyses and revealed that evodiamine decreased VEGF expression. Moreover, administration of recombinant human VEGF(165) (rhVEGF(165)) induced tube formation and ERK phosphorylation by HUVECs, and partially attenuated inhibitory effect of evodiamine. From these results, we suggested that evodiamine is a potent inhibitor of angiogenesis. The mechanism might involve at least the inhibition of VEGF expression, probably through repression of ERK phosphorylation.