Human microvascular dysfunction and apoptotic injury induced by AL amyloidosis light chain proteins

Light chain amyloidosis (AL) involves overproduction of amyloidogenic light chain proteins (LC) leading to heart failure, yet the mechanisms underlying tissue toxicity remain unknown. We hypothesized that LC induces endothelial dysfunction in non-AL human microvasculature and apoptotic injury in human coronary artery endothelial cells (HCAECs). Adipose arterioles (n = 34, 50 ± 3 yr) and atrial coronary arterioles (n = 19, 68 ± 2 yr) from non-AL subjects were cannulated. Adipose arteriole dilator responses to acetylcholine/papaverine were measured at baseline and 1 h exposure to LC (20 μg/ml) from biopsy-proven AL subjects (57 ± 11 yr) without and with antioxidant cotreatment. Coronary arteriole dilation to bradykinin/papaverine was measured post-LC exposure. HCAECs were exposed to 1 or 24 h of LC. LC reduced dilation to acetylcholine (10(-4) M: 41.6 ± 7 vs. 85.8 ± 2.2% control, P < 0.001) and papaverine (81.4 ± 4.6 vs. 94.8 ± 1.3% control, P < 0.01) in adipose arterioles and to bradykinin (10(-6) M: 68.6 ± 6.2 vs. 90.9 ± 1.6% control, P < 0.001) but not papaverine in coronary arterioles. There was an increase in superoxide and peroxynitrite in arterioles treated with LC. Adipose arteriole dilation was restored by cotreatment with polyethylene glycol-superoxide dismutase and tetrahydrobiopterin but only partially restored by mitoquinone (mitochondria-targeted antioxidant) and gp91ds-tat (NADPH oxidase inhibitor). HCAECs exposed to LC showed reduced NO and increased superoxide, peroxynitrite, annexin-V, and propidium iodide compared with control. Brief exposure to physiological amounts of LC induced endothelial dysfunction in human adipose and coronary arterioles and increased apoptotic injury in coronary artery endothelial cells likely as a result of oxidative stress, reduced NO bioavailability, and peroxynitrite production. Microvascular dysfunction and injury is a novel mechanism underlying AL pathobiology and is a potential target for therapy.     

Native and recombinant Slc26a3 (downregulated in adenoma, Dra) do not exhibit properties of 2Cl-/1HCO3- exchange

The recent proposal that Dra/Slc26a3 mediates electrogenic 2Cl(-)/1HCO(3)(-) exchange suggests a required revision of classical concepts of electroneutral Cl(-) transport across epithelia such as the intestine. We investigated 1) the effect of endogenous Dra Cl(-)/HCO(3)(-) activity on apical membrane potential (V(a)) of the cecal surface epithelium using wild-type (WT) and knockout (KO) mice; and 2) the electrical properties of Cl(-)/(OH(-))HCO(3)(-) exchange by mouse and human orthologs of Dra expressed in Xenopus oocytes. Ex vivo (36)Cl(-) fluxes and microfluorometry revealed that cecal Cl(-)/HCO(3)(-) exchange was abolished in the Dra KO without concordant changes in short-circuit current. In microelectrode studies, baseline V(a) of Dra KO surface epithelium was slightly hyperpolarized relative to WT but depolarized to the same extent as WT during luminal Cl(-) substitution. Subsequent studies indicated that Cl(-)-dependent V(a) depolarization requires the anion channel Cftr. Oocyte studies demonstrated that Dra-mediated exchange of intracellular Cl(-) for extracellular HCO(3)(-) is accompanied by slow hyperpolarization and a modest outward current, but that the steady-state current-voltage relationship is unaffected by Cl(-) removal or pharmacological blockade. Further, Dra-dependent (36)Cl(-) efflux was voltage-insensitive in oocytes coexpressing the cation channels ENaC or ROMK. We conclude that 1) endogenous Dra and recombinant human/mouse Dra orthologs do not exhibit electrogenic 2Cl(-)/1HCO(3)(-) exchange; and 2) acute induction of Dra Cl(-)/HCO(3)(-) exchange is associated with secondary membrane potential changes representing homeostatic responses. Thus, participation of Dra in coupled NaCl absorption and in uncoupled HCO(3)(-) secretion remains compatible with electroneutrality of these processes, and with the utility of electroneutral transport models for predicting epithelial responses in health and disease.     

Synthesis and biophysical characterization of R-6'-Me-α-L-LNA modified oligonucleotides

The synthesis and biophysical properties of R-6′-Me-α-l-LNA, which has a methyl group in the (R) configuration on the 2′,4′-bridging substituent of α-l-LNA, is reported. The synthesis of the uracil nucleobase phosphoramidite was efficiently accomplished in 14 steps and 8 chromatographic purifications starting from a known sugar intermediate. Biophysical evaluation revealed that substitution along the edge of the major groove does not impair the high affinity duplex forming ability of α-l-LNA modified oligonucleotides.     

Unprecedented C-2 arylation of indole with diazonium salts: Syntheses of 2,3-disubstituted indoles and their antimicrobial activity

A novel reaction of indole with aryldiazonium salts leading to the formation of 2-aryl-3-(arylazo)indoles was discovered. The products were found to possess potent anti-MRSA and anti-LLVRE activities. The SAR studies indicate that the potentially metabolically labile azo functionality can be replaced with ether oxygen and thioether sulfur atoms without any loss of activity.     

Removal of enzymatic and fermentation inhibitory compounds from biomass slurries for enhanced biorefinery process efficiencies

Within the biorefinery paradigm, many non-monomeric sugar compounds have been shown to be inhibitory to enzymes and microbial organisms that are used for hydrolysis and fermentation. Here, two novel separation technologies, polyelectrolyte polymer adsorption and resin-wafer electrodeionization (RW-EDI), have been evaluated to detoxify a dilute acid pretreated biomass slurry. Results showed that detoxification of a dilute acid pretreated ponderosa pine slurry by sequential polyelectrolyte and RW-EDI treatments was very promising, with significant removal of acetic acid, 5-hydroxymethyl furfural, and furfural (up to 77%, 60%, and 74% removed, respectively) along with >97% removal of sulfuric acid. Removal of these compounds increased the cellulose conversion to 94% and elevated the hydrolysis rate to 0.69 g glucose/L/h. When using Saccharomyces cerevisiae D₅A for fermentation of detoxified slurry, the process achieved 99% of the maximum theoretical ethanol yield and an ethanol production rate nearly five-times faster than untreated slurry.     

dachsous and frizzled contribute separately to planar polarity in the Drosophila ventral epidermis

Cells that comprise tissues often need to coordinate cytoskeletal events to execute morphogenesis properly. For epithelial tissues, some of that coordination is accomplished by polarization of the cells within the plane of the epithelium. Two groups of genes--the Dachsous (Ds) and Frizzled (Fz) systems--play key roles in the establishment and maintenance of such polarity. There has been great progress in uncovering the how these genes work together to produce planar polarity, yet fundamental questions remain unanswered. Here, we study the Drosophila larval ventral epidermis to begin to address several of these questions. We show that ds and fz contribute independently to polarity and that they do so over spatially distinct domains. Furthermore, we find that the requirement for the Ds system changes as field size increases. Lastly, we find that Ds and its putative receptor Fat (Ft) are enriched in distinct patterns in the epithelium during embryonic development.     

BIN1 Is Decreased in Sporadic but Not Familial Alzheimer’s Disease or in Aging

Bridging integrator 1 (BIN1) has been implicated in sporadic Alzheimer’s disease (AD) by a number of genome wide association studies (GWAS) in a variety of populations. Here we measured BIN1 in frontal cortex samples from 24 sporadic AD and 24 age-matched non-dementia brains and correlated the expression of this protein with markers of AD. BIN1 was reduced by 87% (p=0.007) in sporadic AD compared to non-dementia controls, but BIN1 in sporadic AD did not correlate with soluble Aβ (r_s=-0.084, p=0.698), insoluble Aβ (r_s=0.237, p=0.269), Aβ plaque load (r_s=0.063, p=0.771) or phospho-tau load (r_s=-0.160, p=0.489). In contrast to our findings in sporadic AD, BIN1 was unchanged in the hippocampus from 6 cases of familial AD compared to 6 age-matched controls (p=0.488). BIN1 declined with age in a cohort of non-dementia control cases between 25 and 88 years but the correlation was not significant (r_s=-0.449, p=0.081). Although BIN1 is known to have a role in endocytosis, and the processing of the amyloid precursor protein (APP) to form amyloid-β (Aβ) peptides is dependent on endocytosis, knockdown of BIN1 by targeted siRNA or the overexpression of BIN1 in a human neuroblastoma cell line (SH-SY5Y) had no effect on APP processing. These data suggest that the alteration in BIN1 is involved in the pathogenesis of sporadic, but not familial AD and is not a consequence of AD neurodegeneration or the ageing process, a finding in keeping with the numerous GWAS that implicate BIN1 in sporadic AD. However, the mechanism of its contribution remains to be established.     

Changes in the diet and body size of a small herbivorous mammal (hispid cotton rat,Sigmodon hispidus) following the late Pleistocene megafauna extinction

The catastrophic loss of large-bodied mammals during the terminal Pleistocene likely led to cascading effects within communities. While the extinction of the top consumers probably expanded the resources available to survivors of all body sizes, little work has focused on the responses of the smallest mammals. Here, we use a detailed fossil record from the southwestern United States to examine the response of the hispid cotton rat Sigmodon hispidus to biodiversity loss and climatic change over the late Quaternary. In particular, we focus on changes in diet and body size. We characterize diet through carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope analysis of bone collagen in fossil jaws and body size through measurement of fossil teeth; the abundance of material allows us to examine population level responses at millennial scale for the past 16 ka. Sigmodon was not present at the cave during the full glacial, first appearing at ~16 ka after ice sheets were in retreat. It remained relatively rare until ~12 ka when warming temperatures allowed it to expand its species range northward. We find variation in both diet and body size of Sigmodon hispidus over time: the average body size of the population varied by ~20% (90–110 g) and mean δ¹³C and δ¹⁵N values ranged between −13.5 to −16.5‰ and 5.5 to 7.4‰ respectively. A state–space model suggested changes in mass were influenced by diet, maximum temperature and community structure, while the modest changes in diet were most influenced by community structure. Sigmodon maintained a fairly similar dietary niche over time despite contemporaneous changes in climate and herbivore community composition that followed the megafauna extinction. Broadly, our results suggest that small mammals may be as sensitive to shifts in local biotic interactions within their ecosystem as they are to changes in climate and large-scale biodiversity loss.     

Pretreatment CT and PET radiomics predicting rectal cancer patients in response to neoadjuvant chemoradiotherapy

BackgroundThe purpose of this study was to characterize pre-treatment non-contrast computed tomography (CT) and ¹⁸F-fluorodeoxyglucose positron emission tomography (PET) based radiomics signatures predictive of pathological response and clinical outcomes in rectal cancer patients treated with neoadjuvant chemoradiotherapy (NACR T).Materials and methodsAn exploratory analysis was performed using pre-treatment non-contrast CT and PET imaging dataset. The association of tumor regression grade (TRG) and neoadjuvant rectal (NAR) score with pre-treatment CT and PET features was assessed using machine learning algorithms. Three separate predictive models were built for composite features from CT + PET.ResultsThe patterns of pathological response were TRG 0 (n = 13; 19.7%), 1 (n = 34; 51.5%), 2 (n = 16; 24.2%), and 3 (n = 3; 4.5%). There were 20 (30.3%) patients with low, 22 (33.3%) with intermediate and 24 (36.4%) with high NAR scores. Three separate predictive models were built for composite features from CT + PET and analyzed separately for clinical endpoints. Composite features with α = 0.2 resulted in the best predictive power using logistic regression. For pathological response prediction, the signature resulted in 88.1% accuracy in predicting TRG 0 vs. TRG 1–3; 91% accuracy in predicting TRG 0–1 vs. TRG 2–3. For the surrogate of DFS and OS, it resulted in 67.7% accuracy in predicting low vs. intermediate vs. high NAR scores.ConclusionThe pre-treatment composite radiomics signatures were highly predictive of pathological response in rectal cancer treated with NACR T. A larger cohort is warranted for further validation.     

Mitochondrial regulation of insect cell apoptosis: Evidence for permeability transition pore-independent cytochrome-c release in the Lepidopteran Sf9 cells

Role of cytochrome-c in insect cell apoptosis is highly controversial, with many earlier reports suggesting lack of involvement of mitochondrial factors in Drosophila while more recent studies have indicated otherwise, thus warranting more in-depth studies of insect cell apoptosis. In the present study, we investigated mitochondrial involvement during actinomycin-D induced apoptosis in Sf9 Lepidopteran cells. Cytochrome-c was released from mitochondria very early during apoptosis, and was preceded quickly by ROS generation and cardiolipin peroxidation. Albeit cytochrome-c release and apoptosis induction were inhibited by bongkrkicacid (BKA) it appears that the release is independent of permeability transition pore (PTP) as it preceded mitochondrial Ca²⁺ buildup and mitochondrial membrane potential (MMP) loss. Further, the release was found to be unaffected by PTP inhibitor cyclosporin-A. Bax inhibitory peptide BiP-P5 could effectively block both cytochrome-c release and apoptosis induction indicating dependence on Bax-channel formation. Inhibition of apoptosis by FSBA, a nucleotide analog that inhibits apoptosome formation through Apaf1 binding, suggested activity of apoptosome similar to mammalian cells. Mitochondria isolated from treated cells activated caspases in the cytosolic fraction of untreated cells while mitochondrial lysates of treated or untreated cells had similar effect. Sequestering cytochrome-c in mitochondrial lysates inhibited DEVDase activity, and addition of purified cytochrome-c and dATP to Sf9 cytosolic fraction induced DEVDase activity, suggesting that cytochrome-c may be exclusively required for Lepidopteran apoptosis. This is the first detailed study demonstrating mitochondrial regulation of Lepidopteran insect cell apoptosis, and reiterates its homology with mammalian cell apoptosis while showing distinctive differences from earlier reports in Drosophila.