Identification of Novel Regulatory Small RNAs in Acinetobacter baumannii

Small RNA (sRNA) molecules are non-coding RNAs that have been implicated in regulation of various cellular processes in living systems, allowing them to adapt to changing environmental conditions. Till date, sRNAs have not been reported in Acinetobacter baumannii (A. baumannii), which has emerged as a significant multiple drug resistant nosocomial pathogen. In the present study, a combination of bioinformatic and experimental approach was used for identification of novel sRNAs. A total of 31 putative sRNAs were predicted by a combination of two algorithms, sRNAPredict and QRNA. Initially 10 sRNAs were chosen on the basis of lower E- value and three sRNAs (designated as AbsR11, 25 and 28) showed positive signal on Northern blot. These sRNAs are novel in nature as they do not have homologous sequences in other bacterial species. Expression of the three sRNAs was examined in various phases of bacterial growth. Further, the effect of various stress conditions on sRNA gene expression was determined. A detailed investigation revealed differential expression profile of AbsR25 in presence of varying amounts of ethidium bromide (EtBr), suggesting that its expression is influenced by environmental or internal signals such as stress response. A decrease in expression of AbsR25 and concomitant increase in the expression of bioinformatically predicted targets in presence of high EtBr was reverberated by the decrease in target gene expression when AbsR25 was overexpressed. This hints at the negative regulation of target genes by AbsR25. Interestingly, the putative targets include transporter genes and the degree of variation in expression of one of them (A1S_1331) suggests that AbsR25 is involved in regulation of a transporter. This study provides a perspective for future studies of sRNAs and their possible involvement in regulation of antibiotic resistance in bacteria specifically in cryptic A. baumannii.     

Evolution,Expression Differentiation and Interaction Specificity of Heterotrimeric G-Protein Subunit Gene Family in the Mesohexaploid Brassica rapa

Heterotrimeric G-proteins, comprising of Gα, Gβ, and Gγ subunits, are important signal transducers which regulate many aspects of fundamental growth and developmental processes in all eukaryotes. Initial studies in model plants Arabidopsis and rice suggest that the repertoire of plant G-protein is much simpler than that observed in metazoans. In order to assess the consequence of whole genome triplication events within Brassicaceae family, we investigated the multiplicity of G-protein subunit genes in mesohexaploid Brassica rapa, a globally important vegetable and oilseed crop. We identified one Gα (BraA.Gα1), three Gβ (BraA.Gβ1, BraA.Gβ2, and BraA.Gβ3), and five Gγ (BraA.Gγ1, BraA.Gγ2, BraA.Gγ3, BraA.Gγ4, and BraA.Gγ5) genes from B. rapa, with a possibility of 15 Gαβγ heterotrimer combinations. Our analysis suggested that the process of genome triplication coupled with gene-loss (gene-fractionation) phenomenon have shaped the quantitative and sequence diversity of G-protein subunit genes in the extant B. rapa genome. Detailed expression analysis using qRT-PCR assays revealed that the G-protein genes have retained ubiquitous but distinct expression profiles across plant development. The expression of multiple G-protein genes was differentially regulated during seed-maturation and germination stages, and in response to various phytohormone treatments and stress conditions. Yeast-based interaction analysis showed that G-protein subunits interacted in most of the possible combinations, with some degree of subunit-specific interaction specificity, to control the functional selectivity of G-protein heterotrimer in different cell and tissue-types or in response to different environmental conditions. Taken together, this research identifies a highly diverse G-protein signaling network known to date from B. rapa, and provides a clue about the possible complexity of G-protein signaling networks present across globally important Brassica species.     

Effects of Radiofrequency Catheter Ablation of Atrial Fibrillation on Soluble P-Selectin,Von Willebrand Factor and IL-6 in the Peripheral and Cardiac Circulation

Background

Catheter ablation (CA) of atrial fibrillation (AF) is associated with inflammatory response, endothelial damage and with increased risk of thrombosis. However, whether these processes differ in peripheral and cardiac circulation is unknown.

Methods

Plasma markers (von Willebrand factor (vWf), soluble P-selectin (sPsel) and interleukin-6 (IL-6)) were measured by ELISA at three time points in 80 patients (62±10 years, 63% males, 41% paroxysmal AF) undergoing CA. These were at baseline – from femoral vein (FV) and left atrium (LA) before ablation; directly after ablation – from the pulmonary vein (PV), LA and FV; and 24 hours after procedure – from a cubital vein (CV).

Results

The levels of vWF and IL6 – but not sP-sel – increased significantly 24h after procedure (p<0.001). Baseline vWF was significantly associated with persistent AF (Beta = .303, p = 0.006 and Beta = .300, p = 0.006 for peripheral and cardiac levels, respectively), while persistent AF (Beta = .250, p = 0.031) and LAA flow pattern (Beta = .386, p<0.001) remained associated with vWF in cardiac blood after ablation. Advanced age was significantly associated with IL6 levels at baseline and after ablation in peripheral and cardiac blood. There were no clinical, procedural or anti-coagulation characteristics associated with sP-sel levels in cardiac blood, while peripheral sP-sel levels were associated with hypertension before (Beta = −.307, p = 0.007) and with persistent AF after ablation (Beta = −.262, p = 0.020).

Conclusions

vWF levels are higher in persistent AF and are associated with LAA rheological pattern after AF ablation. Increase of peripheral vWF and IL6 levels after procedure supports current AF ablation management with careful control of post-procedural anticoagulation to avoid ablation-related thromboembolism.     

AvrRps4 effector family processing and recognition in lettuce

During pathogenesis, effector proteins are secreted from the pathogen to the host plant to provide virulence activity for invasion of the host. However, once the host plant recognizes one of the delivered effectors, effector‐triggered immunity activates a robust immune and hypersensitive response (HR). In planta, the effector AvrRps4 is processed into the N‐terminus (AvrRps4^N) and the C‐terminus (AvrRps4^C). AvrRps4^C is sufficient to trigger HR in turnip and activate AtRRS1/AtRPS4‐mediated immunity in Arabidopsis; on the other hand, AvrRps4^N induces HR in lettuce. Furthermore, AvrRps4^N‐mediated HR requires a conserved arginine at position 112 (R112), which is also important for full‐length AvrRps4 (AvrRps4^F) processing. Here, we show that effector processing and effector recognition in lettuce are uncoupled for the AvrRps4 family. In addition, we compared effector recognition by lettuce of AvrRps4 and its homologues, HopK1 and XopO. Interestingly, unlike for AvrRps4 and HopK1, mutation of the conserved R111 in XopO by itself was insufficient to abolish recognition. The combination of amino acid substitutions arginine 111 to leucine with glutamate 114 to lysine abolished the XopO‐mediated HR, suggesting that AvrRps4 family members have distinct structural requirements for perception by lettuce. Together, our results provide an insight into the processing and recognition of AvrRps4 and its homologues.     

Psychedelic-inspired drug discovery using an engineered biosensor

1. Download : Download high-res image (206KB)
2. Download : Download full-size image

     

Polymorphism of the 3'' open reading frame of the virus associated with the acquired immune deficiency syndrome, human T-lymphotropic virus type III.

The genome of the virus associated with the acquired immune deficiency syndrome (AIDS), human T-lymphotropic virus type III (HTLV-III), includes two open reading frames, not found in other retroviruses. One of these, designated 3' open reading frame (3'orf) is 648 base pairs (bp) in length, and overlaps with the 3' long terminal repeat (LTR) sequences. Sequences of additional HTLV-III clones were determined in order to estimate the level and location of variation within 3'orf, to gain some insight into the function of its protein product. Newly determined sequences are reported for 3'orf of two unintegrated clones of HTLV-III and three cDNA clones made from virion RNA derived from the same cell line infected with pooled blood samples of different patients with AIDS or AIDS-related complex symptoms (ARC). In addition, sequences for 3'orf were derived from an unintegrated viral clone derived from a different cell line infected with a distinct isolate from a single patient. These sequences are compared to those previously reported for six other viral clones. Sequences of 3'orf differ among clones by 1.1-10.4% bp and 2.4-17.0% of predicted amino acids. This represents significantly greater sequence variation than is found in the entire genome on average. Moreover, a functional proviral clone has a termination codon at amino acid residue 124 of this open reading frame. This raises questions concerning the structure, and regulation of expression of the protein encoded by 3'orf.     

A Study of Differential Expression of Testicular Genes in Various Reproductive Phases of Hemidactylus flaviviridis (Wall Lizard) to Derive Their Association with Onset of Spermatogenesis and Its Relevance to Mammals

Testis of Hemidactylus flaviviridis, commonly known as Indian wall lizard, displays a lack of cellular and metabolic activity in regressed phase of testis during non-breeding season of the year. Retracted Sertoli cells (Sc), fibroid myoid cells and pre-meiotic resting spermatogonia are observed in such testis. This situation is akin to certain forms of infertility in men where hormone supplementation fails to generate sperm despite the presence of Sc and germ cells (Gc) in testis. In testis of lizard, spermatogenesis is reinitiated upon increased level of hormones during appropriate season (phase of recrudescence). Study of genes associated with generation of sperm, from regressed adult testis in lizard, may provide valuable information for understanding certain forms of male idiopathic infertility. Subtractive hybridization using testicular RNA obtained from the regressed and active phases of lizard reproductive cycle led to identify eight partial mRNA sequences that showed sequence homology with mice genes. We further evaluated the gene expression prolife by real-time PCR in three different reproductive phases of H. flaviviridis: regressed (pre-meiotic), recrudescent (meiotic) and active (post meiotic), for comparison with the corresponding testicular phases found in testis of 5 days (pre-meiotic), 20 days (meiotic) and 60 days (post-meiotic) old mouse. This is the first report where genes associated with progression of spermatogenesis during active phase, which follows a regressed state of adult testis, were identified in lizard and found to be conserved in mouse. Six important genes, Hk1, Nme5, Akap4, Arih1, Rassf7 and Tubb4b were found to be strictly associated with active spermatogenesis in both mouse and lizard. Factors interfering with the expression of any of these genes may potentially abrogate the process of spermatogenesis leading to infertility. Such information may shed light on unknown causes of idiopathic male infertility.     

Reversible changes in the testes and epididymides of dog treated with alpha-chlorohydrin.

1. Chronic administration of alpha-chlorohydrin (8 mg/kg body wt for 30 days) caused lesions in the testis of dog. The changes in the germ cells were degenerative. The seminiferous tubule and Leydig cell nuclear diameter were reduced. 2. Epididymal cell height was greatly reduced and the stereocilia had disappeared completely. The lumen was devoid of spermatozoa. 3. Alpha-chlorohydrin administration inhibited the RNA and sialic acid contents in the testes and epididymides of dog. Total cholesterol and lipids/g of testes were increased significantly after alpha-chlorohydrin administration. 4. These effects were reversible. Repopulation of testis tubules occurred following a period of 100 days recovery in dog. Numerous spermatogonia and sperm develop and traverse the epididymides. The RNA, sialic acid, cholesterol and total lipids of testes and epididymides returned to subnormal levels. 5. The possibility of using alpha-chlorohydrin as male contraception is indicated.