MicroRNA-21 orchestrates high glucose-induced signals to TOR complex 1, resulting in renal cell pathology in diabetes

Hyperglycemia induces a wide array of signaling pathways in the kidney that lead to hypertrophy and matrix expansion, eventually culminating in progressive kidney failure. High glucose-induced reduction of the tumor suppressor protein phosphatase and tensin homolog deleted in chromosome 10 (PTEN) contributes to renal cell hypertrophy and matrix expansion. We identified microRNA-21 (miR-21) as the molecular link between high glucose and PTEN suppression. Renal cortices from OVE26 type 1 diabetic mice showed significantly elevated levels of miR-21 associated with reduced PTEN and increased fibronectin content. In renal mesangial cells, high glucose increased the expression of miR-21, which targeted the 3'-UTR of PTEN mRNA to inhibit PTEN protein expression. Overexpression of miR-21 mimicked the action of high glucose, which included a reduction in PTEN expression and a concomitant increase in Akt phosphorylation. In contrast, expression of miR-21 Sponge, to inhibit endogenous miR-21, prevented down-regulation of PTEN and phosphorylation of Akt induced by high glucose. Interestingly, high glucose-stimulated miR-21 inactivated PRAS40, a negative regulator of TORC1. Finally, miR-21 enhanced high glucose-induced TORC1 activity, resulting in renal cell hypertrophy and fibronectin expression. Thus, our results identify a previously unrecognized function of miR-21 that is the reciprocal regulation of PTEN levels and Akt/TORC1 activity that mediate critical pathologic features of diabetic kidney disease.     

Intermittent Right Ventricular Outflow Tract Capture due to Chronic Right Atrial Lead Dislodgement

A 58 year old male, known case of type 2 diabetes and hypertension, had undergone implantation of a dual chamber pacemaker(DDDR) in 2007 for complaints of recurrent syncope and trifascicular block with a normal ejection fraction andnormal coronaries. His post implantation parameters were normal at that time.He now presented to our pacemaker clinic where his ECG done showed two types o fpaced complexes. The first few complexes were consistent with atrial sensed right ventricular apical pacing with left superior axis. Later complexes showed loss of atrial sensing with pacing from right ventricular outflow tract(inferior axis) with subtle oscillation in it''s axis. On application of magnet, two pacemaker spikes were visible withinterspike interval of 120 ms and paced complexes with inferior axis starting from the first spike suggesting that the atrial lead was responsible for RVOT depolarization. On interrogation of the pacemaker, atrial EGM showed sensed activity from atrium followed by large sensed ventricular complex. Fluoroscopy confirmed that the atrial lead was dislodged and was intermittently prolapsing into the RVOT. Since the patient was asymptomatic, he refused any intervention and subsequentlyhis atrial lead was switched off by telemetry. The above case signifies that asymptomatic lead dislodgement is no talways manifested as loss of capture and even subtle variation of the axis o fthe paced complexes can provide us with a clue that can be confirmed by telemetry of the pacemaker and fluoroscopy.     

Sialylation of Outer Membrane Porin Protein D: A Mechanistic Basis of Antibiotic Uptake in Pseudomonas aeruginosa

Pseudomonas aeruginosa (PA) is an environmentally ubiquitous, extracellular, opportunistic pathogen, associated with severe infections of immune-compromised host. We demonstrated earlier the presence of both α2,3- and α2,6-linked sialic acids (Sias) on PA (PA^+Sias) and normal human serum is their source of Sias. PA^+Sias showed decreased complement deposition and exhibited enhanced association with immune-cells through sialic acid binding immunoglobulin like lectins (Siglecs). Such Sias-siglec-9 interaction between PA^+Sias and neutrophils helped to subvert host immunity. Additionally, PA^+Sias showed more resistant to β-lactam antibiotics as reflected in their minimum inhibitory concentration required to inhibit the growth of 50% than PA^−Sias. Accordingly, we have affinity purified sialoglycoproteins of PA^+Sias. They were electrophoresed and identified by matrix-assisted laser desorption-ionization time-of-flight/time-of-flight mass spectrometry analysis. Sequence study indicated the presence of a few α2,6-linked, α2,3-linked, and both α2,3- and α2,6-linked sialylated proteins in PA. The outer membrane porin protein D (OprD), a specialized channel-forming protein, responsible for uptake of β-lactam antibiotics, is one such identified sialoglycoprotein. Accordingly, sialylated (OprD^+Sias) and non-sialylated (OprD^−Sias) porin proteins were separately purified by using anion exchange chromatography. Sialylation of purified OprD^+Sias was confirmed by several analytical and biochemical procedures. Profiling of glycan structures revealed three sialylated N-glycans and two sialylated O-glycans in OprD^+Sias. In contrast, OprD^−Sias exhibit only one sialylated N-glycans. OprD^−Sias interacts with β-lactam antibiotics more than OprD^+Sias as demonstrated by surface plasmon resonance study. Lyposome-swelling assay further exhibited that antibiotics have more capability to penetrate through OprD^−Sias purified from four clinical isolates of PA. Taken together, it may be envisaged that sialic acids on OprD protein play important role toward the uptake of commonly used antibiotics in PA^+Sias. This might be one of the new mechanisms of PA for β-lactam antibiotic uptake.Sialic acids (Sias)¹ are nine carbon atom containing acidic residues characteristically found in the terminal position of glycoproteins and glycolipids (1–4). Structural diversity of sialic acids is because of the modification of one or more hydroxyl groups in various positions of the core structure by different groups like acetyl-, methyl-, sulfate-, lactyl-, or phosphate (1, 5–7). More than fifty derivatives of Sias has been reported both in vertebrate and invertebrate systems. It functions as ligand for various cellular communications and also act as masking element for glycoconjugates (8–12).Sialic acid binding immunoglobulins (Ig)-like lectins (siglecs) selectively expressed on the hematopoetic cells and interact with an array of linkage-specific Sias on a glycan structure express on the same cells or other cells (13). Siglecs can also recognize terminal sialylated glycoconjugates on several pathogens (14–16). After recognizing, they carry out various functions like internalization, attenuation of inflammation, restraining cellular activation along with inhibition of natural killer cell activation (17).Pseudomonas aeruginosa (PA) is a Gram-negative, rod-shaped bacterium. This human pathogen has remarkable capacity to cause diseases in immune compromised hosts. This colonizing microbial pathogen is responsible for infection in chronic cystic fibrosis, nosocomial infections; severe burn, transplantation, cancer, and AIDS and other immuno-supressed patients (18).We have reported earlier the presence of linkage-specific Sias on PA. Normal human serum (NHS) is possibly one of the sources of these Sias (19). PA utilizes these Sias to interact through siglecs present on the surface of different immune cells. PA^+Sias showed enhanced association with neutrophils through α2,3-linked Sias-siglec-9 interaction which facilitated their survival by subverting innate immune function of host (20).The treatment of PA-infected patient depends upon the extent of the disease and the concerned organs. Conventional β-lactam, cephalosporins, and aminoglycosides group of antibiotics are most common for such treatment (21). β-lactam antibiotics inhibit cell wall synthesis by disrupting the synthesis of the peptidoglycan layer of bacterial cell walls (22). When PA showed resistant to β-lactam antibiotics, new generation of β-lactam with increased doses or other broad spectrum antibiotics like tetracyclines or fluoroquinolones are prescribed (23). PA isolates from intensive care unit (ICU) patients in general showed higher rates of β-lactam resistance among other hospitalized patients (24). The increasing frequency of resistance to ceftazidime, piperacillin, imipenem, fluoroquinolone, and aminoglycoside were 36.6%, 22.3%, 22.8%, 23.8%, and 17.8% respectively in PA (25).The outer membrane of Gram-negative bacteria is, in general, semipermeable through which hydrophilic molecules including antibiotics of below exclusion limit size (0.6 kDa) can pass through the channel-forming proteins generally called porins e.g. OprD, OprF, OprG etc. (26, 27). PA shows lower outer membrane permeability with respect to many other Gram-negative bacteria like Acinetobacter baumannii, Stenotrophomonas maltophilia, Burkholderia cepacia, hence the diffusion rate of β-lactam antibiotics is decreased (27).Additionally, PA uses MexA-MexB-OprM, MexC-MexD-OprJ, MexE-MexF-OprN, and MexX-MexY-OprM as efflux pumps along with important regulatory factors MexR/NalB, NfxB, NfxC/MexT, and MexZ respectively on their membrane to pump out undesirable chemicals, detergent and antibiotics (28–32). Other Gram-negative bacteria also uses similar types of efflux pumps for such purposes. Moreover, PA produces antibiotic-resistance genes by some mutation (33). Furthermore, β-lactamase and aminoglycoside-modifying enzymes produced by PA are capable of breaking down the antibiotics (34). Alternatively, these enzymes can directly modify the drug. Hence these antibiotics become functionally ineffective (27).The presence of lipopolysaccharides (LPS) containing O-specific polysaccharides with tri-saccharide repeats of 2-acetamido-2,6-dideoxy-d-glucose, 2-acetamido-2,6-dideoxy-d-galactose, and 5-acetamido-3,5,7,9-tetyradeoxy-7-[(R)-3-hydroxybutyramidol]-3-l-glycerol-l-manno-nonulosonic acid are known for PA serogroup O11 (35). The genes for key enzymes required for complex protein glycosylation are found in the genome of PA14 (36). Moreover, glycosylation in PA1244 has been reported in the form of an O-linked glycan in pilin (37). A cluster of seven genes known as the pel genes, encode proteins with similarity to components involved in polysaccharide biogenesis. Among these genes, PelF is a putative glycosyltransferase (GT) of the type IV glycosyltransferase (GT4) family (36). PA secreted sialidase in culture medium (38). Genome search reveals that PA14 has the sialidase gene, which may be responsible for cleaving sialic acids (39). PA1 also has sialic acid transporter gene, which possibly transport sialic acids inside the cells (Gene ID: 17688338, Source: http://www.ncbi.nlm.nih.gov/gene/17688338). Additionally, CMP-sialic acid transferase, which is responsible for converting sialic acids to CMP-sialic acid, was purified from PAO12 (40). This enzyme shows close similarity with the enzyme found in E. coli.However, PA being such a notorious organism, it might have many other different mechanisms to fight against antibiotics for their survival. Therefore, it is worthwhile to explore newer mechanism to understand how antibiotics penetrate inside this bacterium. Here we addressed the following questions. Does sialylation of glycoproteins demonstrated on PA play any role in the entry of antibiotics that might facilitate their survival within host?Accordingly, we have affinity purified a few sialoglycoproteins from PA. Sequence analysis identified twenty six α2,3- and α2,6-linked sialoglycoproteins. One such identified sialoglycoprotein is OprD porin protein. The presence of Sias on OprD was conclusively confirmed. We have demonstrated that Sias on OprD protein isolated four different clinical isolates hampered its interaction with β-lactam antibiotics. This might be one of the new mechanisms for β-lactam antibiotic resistance of PA and thereby facilitates their survival in host.     

Genome-Wide Analysis of Copy Number Variation Identifies Candidate Gene Loci Associated with the Progression of Non-Alcoholic Fatty Liver Disease

Between 10 and 25% of individuals with non-alcoholic fatty liver disease (NAFLD) develop hepatic fibrosis leading to cirrhosis and hepatocellular carcinoma (HCC). To investigate the molecular basis of disease progression, we performed a genome-wide analysis of copy number variation (CNV) in a total of 49 patients with NAFLD [10 simple steatosis and 39 non-alcoholic steatohepatitis (NASH)] and 49 matched controls using high-density comparative genomic hybridization (CGH) microarrays. A total of 11 CNVs were found to be unique to individuals with simple steatosis, whilst 22 were common between simple steatosis and NASH, and 224 were unique to NASH. We postulated that these CNVs could be involved in the pathogenesis of NAFLD progression. After stringent filtering, we identified four rare and/or novel CNVs that may influence the pathogenesis of NASH. Two of these CNVs, located at 13q12.11 and 12q13.2 respectively, harbour the exportin 4 (XPO4) and phosphodiesterase 1B (PDE1B) genes which are already known to be involved in the etiology of liver cirrhosis and HCC. Cross-comparison of the genes located at these four CNV loci with genes already known to be associated with NAFLD yielded a set of genes associated with shared biological processes including cell death, the key process involved in ‘second hit’ hepatic injury. To our knowledge, this pilot study is the first to provide CNV information of potential relevance to the NAFLD spectrum. These data could prove invaluable in predicting patients at risk of developing NAFLD and more importantly, those who will subsequently progress to NASH.     

Analysis of Genetic Diversity and Population Structure of Rice Germplasm from North-Eastern Region of India and Development of a Core Germplasm Set

The North-Eastern region (NER) of India, comprising of Arunachal Pradesh, Assam, Manipur, Meghalaya, Mizoram, Nagaland and Tripura, is a hot spot for genetic diversity and the most probable origin of rice. North-east rice collections are known to possess various agronomically important traits like biotic and abiotic stress tolerance, unique grain and cooking quality. The genetic diversity and associated population structure of 6,984 rice accessions, originating from NER, were assessed using 36 genome wide unlinked single nucleotide polymorphism (SNP) markers distributed across the 12 rice chromosomes. All of the 36 SNP loci were polymorphic and bi-allelic, contained five types of base substitutions and together produced nine types of alleles. The polymorphic information content (PIC) ranged from 0.004 for Tripura to 0.375 for Manipur and major allele frequency ranged from 0.50 for Assam to 0.99 for Tripura. Heterozygosity ranged from 0.002 in Nagaland to 0.42 in Mizoram and gene diversity ranged from 0.006 in Arunachal Pradesh to 0.50 in Manipur. The genetic relatedness among the rice accessions was evaluated using an unrooted phylogenetic tree analysis, which grouped all accessions into three major clusters. For determining population structure, populations K = 1 to K = 20 were tested and population K = 3 was present in all the states, with the exception of Meghalaya and Manipur where, K = 5 and K = 4 populations were present, respectively. Principal Coordinate Analysis (PCoA) showed that accessions were distributed according to their population structure. AMOVA analysis showed that, maximum diversity was partitioned at the individual accession level (73% for Nagaland, 58% for Arunachal Pradesh and 57% for Tripura). Using POWERCORE software, a core set of 701 accessions was obtained, which accounted for approximately 10% of the total NE India collections, representing 99.9% of the allelic diversity. The rice core set developed will be a valuable resource for future genomic studies and crop improvement strategies.     

Mutual Exclusivity of Hyaluronan and Hyaluronidase in Invasive Group A Streptococcus

A recent analysis of group A Streptococcus (GAS) invasive infections in Australia has shown a predominance of M4 GAS, a serotype recently reported to lack the antiphagocytic hyaluronic acid (HA) capsule. Here, we use molecular genetics and bioinformatics techniques to characterize 17 clinical M4 isolates associated with invasive disease in children during this recent epidemiology. All M4 isolates lacked HA capsule, and whole genome sequence analysis of two isolates revealed the complete absence of the hasABC capsule biosynthesis operon. Conversely, M4 isolates possess a functional HA-degrading hyaluronate lyase (HylA) enzyme that is rendered nonfunctional in other GAS through a point mutation. Transformation with a plasmid expressing hasABC restored partial encapsulation in wild-type (WT) M4 GAS, and full encapsulation in an isogenic M4 mutant lacking HylA. However, partial encapsulation reduced binding to human complement regulatory protein C4BP, did not enhance survival in whole human blood, and did not increase virulence of WT M4 GAS in a mouse model of systemic infection. Bioinformatics analysis found no hasABC homologs in closely related species, suggesting that this operon was a recent acquisition. These data showcase a mutually exclusive interaction of HA capsule and active HylA among strains of this leading human pathogen.     

Abnormal Calcium Handling and Exaggerated Cardiac Dysfunction in Mice with Defective Vitamin D Signaling

Aim

Altered vitamin D signaling is associated with cardiac dysfunction, but the pathogenic mechanism is not clearly understood. We examine the mechanism and the role of vitamin D signaling in the development of cardiac dysfunction.

Methods and Results

We analyzed 1α-hydroxylase (1α-OHase) knockout (1α-OHase^−/−) mice, which lack 1α-OH enzymes that convert the inactive form to hormonally active form of vitamin D. 1α-OHase^−/− mice showed modest cardiac hypertrophy at baseline. Induction of pressure overload by transverse aortic constriction (TAC) demonstrated exaggerated cardiac dysfunction in 1α-OHase^−/− mice compared to their WT littermates with a significant increase in fibrosis and expression of inflammatory cytokines. Analysis of calcium (Ca²⁺) transient demonstrated profound Ca²⁺ handling abnormalities in 1α-OHase^−/− mouse cardiomyocytes (CMs), and treatment with paricalcitol (PC), an activated vitamin D₃ analog, significantly attenuated defective Ca²⁺ handling in 1α-OHase^−/− CMs. We further delineated the effect of vitamin D deficiency condition to TAC by first correcting the vitamin D deficiency in 1α-OHase^−/− mice, followed then by either a daily maintenance dose of vitamin D or vehicle (to achieve vitamin D deficiency) at the time of sham or TAC. In mice treated with vitamin D, there was a significant attenuation of TAC-induced cardiac hypertrophy, interstitial fibrosis, inflammatory markers, Ca²⁺ handling abnormalities and cardiac function compared to the vehicle treated animals.

Conclusions

Our results provide insight into the mechanism of cardiac dysfunction, which is associated with severely defective Ca²⁺ handling and defective vitamin D signaling in 1α-OHase^−/− mice.     

Preferential binding of adriamycin and nogalamycin to DNase-I hypersensitive sites of Sarcoma-180 chromatin

Binding of nogalamycin and adriamycin with Sarcoma-180 ascites tumor cell chromatin was studied by a spectrofluorometric method. There was significant reduction in the number of available drug binding sites per nucleotide when the chromatin was digested with DNase I for a period which releases only 7% of the chromosomal DNA. Results indicate preferential binding of these drugs with DNase I hypersensitive sites of chromatin. The DNase-I hypersensitive sites of chromatin were shown to correlate to the sequences required for gene expression. Further digestion with DNase I increases availability of drug binding sites, probably due to relaxation of the compact chromatin.     

Changes in the Chlorophyll Content and Cytokinin Levels in the Top Three Leaves of New Plant Type Rice During Grain Filling

This paper reports the ways that the differences in leaf senescence are related to grain filling, grain yield, and the dynamics of cytokinins (CKs) in the top three leaves of four field-grown new plant type (NPT) rice, a tropical japonica developed at the International Rice Research Institute, Philippines, to increase the yield potential of rice. The chlorophyll content in leaves decreased from flowering to maturity in all the NPT lines, whereas the grain filling percentage was higher in the fast-senescing NPT line than in slow-senescing NPT line. Grain yield was positively correlated with senescence in the flag leaf. Rapid changes in the CK levels were recorded in the leaves of the fast-senescing line, whereas the CK levels were relatively stable in leaves of the slow-senescing line, suggesting that the dynamics of CKs in the fast-senescing line are vital for fast senescence. There were no significant changes in bioactive CKs, CK O-glucosides (storage CKs), and cis-zeatin derivatives in different leaves of the slow-senescing NPT line between 0 and 3 weeks after flowering, suggesting that the content of these CKs is relatively stable during grain filling. A progressive increase in levels of bioactive CKs was positively correlated with gradual accumulation of CK N-glucosides (inactive CKs) in the top three leaves of the slow-senescing NPT line, whereas the decrease of bioactive CKs in the flag leaf of the fast-senescing line was accompanied by accumulation of CK O-glucosides. These results suggest that there is a higher rate of biosynthesis and/or import of bioactive CKs as well as their turnover which may favor delay of leaf senescence in the slow-senescing NPT line.