Abstract
We present the first high-resolution determination of transcriptome architecture in the priority pathogen Acinetobacter baumannii. Pooled RNA from 16 laboratory conditions was used for ...differential RNA-seq (dRNA-seq) to identify 3731 transcriptional start sites (TSS) and 110 small RNAs, including the first identification in A. baumannii of sRNAs encoded at the 3′ end of coding genes. Most sRNAs were conserved among sequenced A. baumannii genomes, but were only weakly conserved or absent in other Acinetobacter species. Single nucleotide mapping of TSS enabled prediction of −10 and −35 RNA polymerase binding sites and revealed an unprecedented base preference at position +2 that hints at an unrecognized transcriptional regulatory mechanism. To apply functional genomics to the problem of antimicrobial resistance, we dissected the transcriptional regulation of the drug efflux pump responsible for chloramphenicol resistance, craA. The two craA promoters were both down-regulated >1000-fold when cells were shifted to nutrient limited medium. This conditional down-regulation of craA expression renders cells sensitive to chloramphenicol, a highly effective antibiotic for the treatment of multidrug resistant infections. An online interface that facilitates open data access and visualization is provided as 'AcinetoCom' (http://bioinf.gen.tcd.ie/acinetocom/).
Aims/hypothesis
The angiotensin II receptor type 2 (AT
2
R) may be a potential therapeutic target for the treatment of hypertension and chronic kidney disease (CKD). The expression and function of AT
...2
R in the vasculature and kidney appear sexually dimorphic. We hypothesised that
Agtr2
knockout dams (AT
2
RKO) with gestational diabetes would program their offspring for subsequent hypertension and CKD in a sex-dependent manner.
Methods
Age- and sex-matched offspring of non-diabetic and diabetic dams of wild-type (WT) and AT
2
RKO mice were followed from 4 to 20 weeks of age and were monitored for development of hypertension and nephropathy; a mouse podocyte cell line (mPOD) was also studied.
Results
Body weight was progressively lower in female compared with male offspring throughout the lifespan. Female but not male offspring from diabetic AT
2
RKO dams developed insulin resistance. Compared with the offspring of non-diabetic dams, the progeny of diabetic dams had developed more hypertension and nephropathy (apparent glomerulosclerosis with podocyte loss) at 20 weeks of age; this programming was more pronounced in the offspring of AT
2
RKO diabetic dams, particularly female AT
2
RKO progeny. Female AT
2
RKO offspring had lower basal ACE2 glomerular expression, resulting in podocyte loss. The aberrant ACE2/ACE ratio was far more diminished in glomeruli of female progeny of diabetic AT
2
RKO dams than in male progeny. Knock-down of
Agtr2
in mPODs confirmed the in vivo data.
Conclusions/interpretation
AT
2
R deficiency accelerated kidney programming in female progeny of diabetic dams, possibly due to loss of protective effects of ACE2 expression in the kidney.
Graphical abstract
We investigated the relationship between Ang-(1-7) angiotensin-(1-7) action, sHTN (systolic hypertension), oxidative stress, kidney injury, ACE2 (angiotensin-converting enzyme-2) and MasR Ang-(1-7) ...receptor expression in Type 1 diabetic Akita mice. Ang-(1-7) was administered daily 500 μg/kg of BW (body weight) per day, subcutaneously to male Akita mice from 14 weeks of age with or without co-administration of an antagonist of the MasR, A779 (10 mg/kg of BW per day). The animals were killed at 20 weeks of age. Age-matched WT (wild-type) mice served as controls. Ang-(1-7) administration prevented sHTN and attenuated kidney injury (reduced urinary albumin/creatinine ratio, glomerular hyperfiltration, renal hypertrophy and fibrosis, and tubular apoptosis) without affecting blood glucose levels in Akita mice. Ang-(1-7) also attenuated renal oxidative stress and the expression of oxidative stress-inducible proteins (NADPH oxidase 4, nuclear factor erythroid 2-related factor 2, haem oxygenase 1), pro-hypertensive proteins (angiotensinogen, angiotensin-converting enzyme, sodium/hydrogen exchanger 3) and profibrotic proteins (transforming growth factor-β1 and collagen IV), and increased the expression of anti-hypertensive proteins (ACE2 and MasR) in Akita mouse kidneys. These effects were reversed by A779. Our data suggest that Ang-(1-7) plays a protective role in sHTN and RPTC (renal proximal tubular cell) injury in diabetes, at least in part, through decreasing renal oxidative stress-mediated signalling and normalizing ACE2 and MasR expression.
Aims/hypothesis
We previously reported that renal tubule-specific deletion of heterogeneous nuclear ribonucleoprotein F (
Hnrnpf
) results in upregulation of renal angiotensinogen (
Agt
) and ...downregulation of sodium-glucose co-transporter 2 (
Sglt2
) in
Hnrnpf
RT
knockout (KO) mice. Non-diabetic
Hnrnpf
RT
KO mice develop hypertension, renal interstitial fibrosis and glycosuria with no renoprotective effect from downregulated
Sglt2
expression. Here, we investigated the effect of renal tubular
Hnrnpf
deletion on hyperfiltration and kidney injury in Akita mice, a model of type 1 diabetes.
Methods
Akita
Hnrnpf
RT
KO mice were generated through crossbreeding tubule-specific (Pax8)-
Cre
mice with Akita floxed-
Hnrnpf
mice on a C57BL/6 background. Male non-diabetic control (Ctrl), Akita, and Akita
Hnrnpf
RT
KO mice were studied up to the age of 24 weeks (
n
= 8/group).
Results
Akita mice exhibited elevated systolic blood pressure as compared with Ctrl mice, which was significantly higher in Akita
Hnrnpf
RT
KO mice than Akita mice. Compared with Akita mice, Akita
Hnrnpf
RT
KO mice had lower blood glucose levels with increased urinary glucose excretion. Akita mice developed kidney hypertrophy, glomerular hyperfiltration (increased glomerular filtration rate), glomerulomegaly, mesangial expansion, podocyte foot process effacement, thickened glomerular basement membranes, renal interstitial fibrosis and increased albuminuria. These abnormalities were attenuated in Akita
Hnrnpf
RT
KO mice. Treatment of Akita
Hnrnpf
RT
KO mice with a selective A1 adenosine receptor inhibitor resulted in an increase in glomerular filtration rate. Renal
Agt
expression was elevated in Akita mice and further increased in Akita
Hnrnpf
RT
KO mice. In contrast,
Sglt2
expression was increased in Akita and decreased in Akita
Hnrnpf
RT
KO mice.
Conclusions/interpretation
The renoprotective effect of
Sglt2
downregulation overcomes the renal injurious effect of Agt when these opposing factors coexist under diabetic conditions, at least partly via the activation of tubuloglomerular feedback.
Graphical abstract
The primary dose-limiting toxicity of stereotactic radiosurgery (SRS) is radiation necrosis (RN), which occurs after approximately 5% to 10% of treatments. This adverse event may worsen neurologic ...deficits, increase the frequency and cost of imaging, and necessitate prolonged treatment with steroids or antiangiogenic agents. Previous investigations have primarily identified lesion size and dosimetric constraints as risk factors for RN in small populations. We hypothesized that disease histology, receptor status, and mutational status are associated with RN.
All patients presenting with brain metastasis between 1997 and 2015 who underwent SRS and subsequent radiographic follow-up at a single tertiary-care institution were eligible for inclusion. The primary outcome was the cumulative incidence of radiographic RN. Multivariate competing risks regression was used to identify biological risk factors for RN.
1939 patients (5747 lesions) were eligible for inclusion; 285 patients (15%) experienced radiographic RN after the treatment of 427 (7%) lesions. After SRS, the median time to RN was 7.6 months. After multivariate analysis, graded prognostic assessment, renal pathology, lesion diameter, and the heterogeneity index remained independently predictive of RN in the pooled cohort. In subset analyses of individual pathologies, HER2-amplified status (hazard ratio HR 2.05, P=.02), BRAF V600+ mutational status (HR 0.33, P=.04), lung adenocarcinoma histology (HR 1.89, P=.04), and ALK rearrangement (HR 6.36, P<.01) were also associated with RN.
In the present investigation constituting the largest series of RN, several novel risk factors were identified, including renal histology, lung adenocarcinoma histology, HER2 amplification, and ALK/BRAF mutational status. These risk factors may be used to guide clinical trial design incorporating biological risk stratification or dose escalation. Future studies determining the optimal timing of targeted therapies are warranted to further define the risk of RN.
We investigated the mechanism of heterogeneous nuclear ribonucleoprotein F (hnRNP F) renoprotective action in a type 2 diabetes (T2D) mouse model (
/
). Immortalized rat renal proximal tubular cells ...(IRPTCs) and kidneys from humans with T2D were also studied. The
/
mice developed hyperglycemia, oxidative stress, and nephropathy at age 20 weeks compared with their
/
littermates. These abnormalities, with the exception of hyperglycemia, were attenuated in
/
-transgenic (Tg) mice specifically overexpressing hnRNP F in their RPTCs. Sirtuin-1, Foxo3α, and catalase expression were significantly decreased in RPTCs from
/
mice and normalized in
/
-Tg mice. In vitro, hnRNP F overexpression stimulated Sirtuin-1 and Foxo3α with downregulation of acetylated p53 expression and prevented downregulation of Sirtuin-1 and Foxo3α expression in IRPTCs by high glucose plus palmitate. Transfection of
small interfering RNA prevented hnRNP F stimulation of Foxo3α and downregulation of acetylated p53 expression. hnRNP F stimulated
transcription via
-responsive element in the
promoter. Human T2D kidneys exhibited more RPTC apoptosis and lower expression of hnRNP F, SIRTUIN-1, and FOXO3α than nondiabetic kidneys. Our results demonstrate that hnRNP F protects kidneys against oxidative stress and nephropathy via stimulation of
expression and signaling in diabetes.
Nitric oxide (NO) is synthesized in skeletal muscle by neuronal-type NO synthase (nNOS), which is localized to sarcolemma of fast-twitch fibers. Synthesis of NO in active muscle opposes contractile ...force. We show that nNOS partitions with skeletal muscle membranes owing to association of nNOS with dystrophin, the protein mutated in Duchenne muscular dystrophy (DMD). The dystrophin complex interacts with an N-terminal domain of nNOS that contains a GLGF motif.
mdx mice and humans with DMD evince a selective loss of nNOS protein and catalytic activity from muscle membranes, demonstrating a novel role for dystrophin in localizing a signaling enzyme to the myocyte sarcolemma. Aberrant regulation of nNOS may contribute to preferential degeneration of fast-twitch muscle fibers in DMD.
Abstract
Oxidative stress induces endogenous antioxidants via nuclear factor erythroid 2–related factor 2 (Nrf2), potentially preventing tissue injury. We investigated whether insulin affects renal ...Nrf2 expression in type 1 diabetes (T1D) and studied its underlying mechanism. Insulin normalized hyperglycemia, hypertension, oxidative stress, and renal injury; inhibited renal Nrf2 and angiotensinogen (Agt) gene expression; and upregulated heterogeneous nuclear ribonucleoprotein F and K (hnRNP F and hnRNP K) expression in Akita mice with T1D. In immortalized rat renal proximal tubular cells, insulin suppressed Nrf2 and Agt but stimulated hnRNP F and hnRNP K gene transcription in high glucose via p44/42 mitogen-activated protein kinase signaling. Transfection with small interfering RNAs of p44/42 MAPK, hnRNP F, or hnRNP K blocked insulin inhibition of Nrf2 gene transcription. Insulin curbed Nrf2 promoter activity via a specific DNA-responsive element that binds hnRNP F/K, and hnRNP F/K overexpression curtailed Nrf2 promoter activity. In hyperinsulinemic-euglycemic mice, renal Nrf2 and Agt expression was downregulated, whereas hnRNP F/K expression was upregulated. Thus, the beneficial actions of insulin in diabetic nephropathy appear to be mediated, in part, by suppressing renal Nrf2 and Agt gene transcription and preventing Nrf2 stimulation of Agt expression via hnRNP F/K. These findings identify hnRNP F/K and Nrf2 as potential therapeutic targets in diabetes.
Our present study identifies an inhibitory action of insulin on renal Nrf2 gene transcription via a putative IRE in the Nrf2 gene promoter that binds hnRNP F/K.
Abstract
H-NS is a nucleoid structuring protein and global repressor of virulence and horizontally-acquired genes in bacteria. H-NS can interact with itself or with homologous proteins, but protein ...family diversity and regulatory network overlap remain poorly defined. Here, we present a comprehensive phylogenetic analysis that revealed deep-branching clades, dispelling the presumption that H-NS is the progenitor of varied molecular backups. Each clade is composed exclusively of either chromosome-encoded or plasmid-encoded proteins. On chromosomes, stpA and newly discovered hlpP are core genes in specific genera, whereas hfp and newly discovered hlpC are sporadically distributed. Six clades of H-NS plasmid proteins (Hpp) exhibit ancient and dedicated associations with plasmids, including three clades with fidelity for plasmid incompatibility groups H, F or X. A proliferation of H-NS homologs in Erwiniaceae includes the first observation of potentially co-dependent H-NS forms. Conversely, the observed diversification of oligomerization domains may facilitate stable co-existence of divergent homologs in a genome. Transcriptomic and proteomic analysis in Salmonella revealed regulatory crosstalk and hierarchical control of H-NS homologs. We also discovered that H-NS is both a repressor and activator of Salmonella Pathogenicity Island 1 gene expression, and both regulatory modes are restored by Sfh (HppH) in the absence of H-NS.
We investigated the molecular mechanism(s) by which insulin prevents Bcl2-modifying factor (Bmf)-induced renal proximal tubular cell (RPTC) apoptosis and loss in diabetic mice. Transgenic mice (Tg) ...mice specifically overexpressing human BMF in RPTCs and non-Tg littermates were studied at 10 to 20 weeks of age. Non-diabetic littermates, diabetic Akita mice +/- insulin implant, Akita Tg mice specifically overexpressing heterogeneous nuclear ribonucleoprotein F (hnRNP F) in their RPTCs and immortalized rat renal proximal tubular cells (IRPTCs) were also studied. BMF-Tg mice exhibited higher systolic blood pressure, urinary albumin/creatinine ratio, RPTC apoptosis and urinary RPTCs than non-Tg mice. Insulin treatment in Akita mice and Akita mice overexpressing hnRNP F suppressed Bmf expression and RPTC apoptosis. In hyperinsulinemic-euglycemic wild type mice, renal Bmf expression was down-regulated with up-regulation of hnRNP F. In vitro, insulin inhibited high glucose-stimulation of Bmf expression, predominantly via p44/42 mitogen-activated protein kinase (MAPK) signaling. Transfection of p44/42 MAPK or hnRNP F small interfering RNA (siRNA) prevented insulin inhibition of Bmf expression. HnRNP F inhibited Bmf transcription via hnRNP F-responsive element in the Bmf promoter. Our results demonstrate that hnRNP F suppression of Bmf transcription is an important mechanism by which insulin protects RPTCs from apoptosis in diabetes.
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