Redox control of renal function and hypertension

Loss of redox homeostasis and formation of excessive free radicals play an important role in the pathogenesis of kidney disease and hypertension. Free radicals such as reactive oxygen species (ROS) are necessary in physiologic processes. However, loss of redox homeostasis contributes to proinflammatory and profibrotic pathways in the kidney, which in turn lead to reduced vascular compliance and proteinuria. The kidney is susceptible to the influence of various extracellular and intracellular cues, including the renin-angiotensin-aldosterone system (RAAS), hyperglycemia, lipid peroxidation, inflammatory cytokines, and growth factors. Redox control of kidney function is a dynamic process with reversible pro- and anti-free radical processes. The imbalance of redox homeostasis within the kidney is integral in hypertension and the progression of kidney disease. An emerging paradigm exists for renal redox contribution to hypertension.     

The utility of IS6110 sequence based polymerase chain reaction in comparison to conventional methods in the diagnosis of extra-pulmonary tuberculosis

IS6110 sequence based polymerase chain reaction (PCR) was compared with conventional bacteriological techniques in the laboratory diagnosis of extra-pulmonary tuberculosis (EPTB). One hundred and ninety one non-repeated clinical samples of EPTB and 17 samples from non-tuberculous cases as controls were included. All the samples were processed for Ziehl-Neelsen staining for acid fast bacilli (AFB) and 143 samples were processed by culture for M. tuberculosis . All the samples were processed for PCR amplification with primers targeting 123 bp fragment of insertion element IS6110 of M. tuberculosis complex. Of the total 191 samples processed, 34 (18%) were positive by smear for AFB. Culture for AFB was positive in 31(22%) samples among the 143 samples processed. Either smear or culture for AFB was found positive in 51(27%) samples. Of the total 191 samples processed 120 (63%) were positive by PCR. In 140 samples, wherein both the conventional techniques were found negative, 74 (53%) samples were positive by PCR alone. Among 51 samples positive by conventional techniques, 46 (90%) were found positive by PCR. PCR assay targeting IS6110 is useful in establishing the diagnosis of EPTB, where there is strong clinical suspicion, especially when the conventional techniques are negative.     

Functional characterization of tyrosine transport in fibroblast cells from healthy controls

Ependymal cilia line the ventricular system moving cerebral spinal fluid close to the brain surface. They may be exposed to fluid of increasing viscosity in certain pathological conditions such as bacterial meningitis. Our aim was to determine the effect of increasing viscosity on ciliary function. Ciliated ependyma was exposed to solutions of different viscosities (1-60cP) and ciliary function assessed by high-speed digital imaging. The mean (S.D.) ciliary beat frequency (CBF), measured after 30min incubation in Medium 199 at 37 degrees C, was 34.9 (2.9)Hz. Increased viscous loading was followed by a rapid decrease in CBF compared to baseline readings (p<0.001). After 15min of exposure to the increased viscous load, CBF reached a new stable level while the viscous load was maintained. Compared to baseline measurements of CBF, viscous loading of 3.7cP caused a 16%, 10.4cP at 34% and 24cP a 70% decrease in beat frequency. Further viscous loading at levels up to 60cP resulted in no further reduction of ependymal CBF. Solutions of 24 and 40cP had no effect on ciliary amplitude. An increase in viscosity to 60cP caused a significant (30%: p=0.001) decrease in the ciliary beat amplitude.     

Human angiogenin is a neuroprotective factor and amyotrophic lateral sclerosis associated angiogenin variants affect neurite extension/pathfinding and survival of motor neurons

Amyotrophic lateral sclerosis (ALS) is a late onset neurodegenerative disorder affecting upper and lower motor neurons (MNs). The molecular mechanisms underlying ALS are poorly understood. Mutations in SOD1 is one of the known causes of ALS but occur only in a very small number of cases of ALS. Interestingly, mutations in human angiogenin (hANG), a member of the ribonuclease A (RNase A) superfamily known to be involved in neovascularization, have been recently reported in patients with ALS, but the effects of these mutations on MN differentiation and survival has not been investigated. We have used the well-characterized pluripotent P19 embryonal carcinoma (EC) cell culture model of neuro-ectodermal differentiation to study the effects of hANG-ALS variants on MN differentiation and survival. Here we report that P19 EC cells induced to differentiate in the presence of hANG and hANG-ALS-associated variants internalize the wild-type and variant proteins. The P19 EC cells differentiate to form neurons but the ability of the neurites to extend and make contacts with neighbouring neurites is compromised when treated with the hANG-ALS variants. In addition, hANG-ALS variants also have a cytotoxic effect on MNs leading to their degeneration. hANG was able to protect neurons from hypoxia-induced cell death, but the variants of hANG implicated in ALS lacked the neuroprotective activity. Our findings show that ANG plays an important role in neurite extension/pathfinding and survival providing a causal link between mutations in hANG and ALS.     

Multiple myeloma-associated chromosomal translocation activates orphan snoRNA ACA11 to suppress oxidative stress

The histone methyltransferase WHSC1 (also known as MMSET) is overexpressed in multiple myeloma (MM) as a result of the t(4;14) chromosomal translocation and in a broad variety of other cancers by unclear mechanisms. Overexpression of WHSC1 did not transform wild-type or tumor-prone primary hematopoietic cells. We found that ACA11, an orphan box H/ACA class small nucleolar RNA (snoRNA) encoded within an intron of WHSC1, was highly expressed in t(4;14)-positive MM and other cancers. ACA11 localized to nucleoli and bound what we believe to be a novel small nuclear ribonucleoprotein (snRNP) complex composed of several proteins involved in postsplicing intron complexes. RNA targets of this uncharacterized snRNP included snoRNA intermediates hosted within ribosomal protein (RP) genes, and an RP gene signature was strongly associated with t(4;14) in patients with MM. Expression of ACA11 was sufficient to downregulate RP genes and other snoRNAs implicated in the control of oxidative stress. ACA11 suppressed oxidative stress, afforded resistance to chemotherapy, and increased the proliferation of MM cells, demonstrating that ACA11 is a critical target of the t(4;14) translocation in MM and suggesting an oncogenic role in other cancers as well.     

Differentiation of benign periablational enhancement from residual tumor following radio-frequency ablation using contrast-enhanced ultrasonography in a rat subcutaneous colon cancer model

Benign periablational enhancement (BPE) response to thermal injury is a barrier to early detection of residual tumor in contrast enhanced imaging after radio-frequency (RF) ablation. The objective of this study was to evaluate the role of quantitative of contrast-enhanced ultrasound (CEUS) in early differentiation of BPE from residual tumor in a BD-IX rat subcutaneous colon cancer model. A phantom study was first performed to test the validity of the perfusion parameters in predicting blood flow of two US contrast imaging modes-contrast harmonic imaging (CHI) and microflow imaging (MFI). To create a simple model of BPE, a peripheral portion of the tumor was ablated along with surrounding normal tissue, leaving part of the tumor untreated. First-pass dynamic enhancement (FPDE) and MFI scans of CEUS were performed before ablation and immediately, 1, 4 and 7 days after ablation. Time-intensity-curves in regions of BPE and residual tumor were fitted to the function y = A(1-exp[-β{t-t0}])+C, in which A, β, t0 and C represent blood volume, flow speed, time to start and baseline intensity, respectively. In the phantom study, positive linear correlations were noted between A, β, Aβ and contrast concentration, speed and flow rate, respectively, in both CHI and MFI. On CEUS images of the in vivo study, the unenhanced ablated zone was surrounded by BPE and irregular peripheral enhancement consistent with residual tumor. On days 0, 4 and 7, blood volume (A) in BPE was significantly higher than that in residual tumor in both FPDE imaging and MFI. Significantly greater blood flow (Aβ) was seen in BPE compared with residual tumor tissue in FPDE on day 7 and in MFI on day 4. The results of this study demonstrate that qualitative CEUS can be potentially used for early detection of viable tumor in post-ablation assessment.     

Osteochondral interface generation by rabbit bone marrow stromal cells and osteoblasts coculture

Physiological osteochondral interface regeneration is a significant challenge. This study aims to investigate the effect of the coculture of chondrogenic rabbit bone marrow stromal cells (rBMSCs) with rabbit osteoblasts in a specially designed two-dimensional (2D)-three-dimensional (3D) co-interface culture to develop the intermediate osteochondral region in vitro. The 2D-3D coculture system was set up by first independently culturing chondrogenic rBMSCs on a scaffold and osteoblasts in cell culture plates, and subsequently placed in contact and cocultured. As control, samples not cocultured with osteoblasts were used. The regulatory effects exerted by osteoblasts on chondrogenic rBMSCs were quantified by real-time polymerase chain reaction. To study the effect of coculture on cells located in different parts of the scaffold, samples were separated into two parts and significantly different gene expression patterns were found between them. In comparison with the control group, a significant moderate downregulation of chondrogenic marker genes, such as Collagen II and Aggrecan was observed. However, the Sox-9 and Collagen I expression increased. More importantly, chondrogenic rBMSCs in the coculture system were shown to form the osteochondral interface layer by expressing calcified cartilage zone specific extracellular matrix marker Collagen X and the hypertrophic chondrocyte marker MMP-13, which were not observed in the control group. Specifically, only the chondrogenic rBMSC layer in contact with the osteoblasts expressed Collagen X and MMP-13, indicating the positive influence of the coculture upon interface formation. Biochemical analyses, histology results, and immunohistochemical staining further supported this observation. In conclusion, this study revealed that specific regulatory stimulations from osteoblasts in the 2D-3D interface coculture system could induce the formation of ostochondral interface for the purpose of osteochondral tissue engineering.