DDX41 is required for cGAS-STING activation against DNA virus infection.

Upon binding double-stranded DNA (dsDNA), cyclic GMP-AMP synthase (cGAS) is activated and initiates the cGAS-stimulator of IFN genes (STING)-type I interferon pathway. DEAD-box helicase 41 (DDX41) is a DEAD-box helicase, and mutations in DDX41 cause myelodysplastic syndromes (MDSs) and acute myeloid leukemia (AML). Here, we show that DDX41-knockout (KO) cells have reduced type I interferon production after DNA virus infection. Unexpectedly, activations of cGAS and STING are affected in DDX41 KO cells, suggesting that DDX41 functions upstream of cGAS. The recombinant DDX41 protein exhibits ATP-dependent DNA-unwinding activity and ATP-independent strand-annealing activity. The MDS/AML-derived mutant R525H has reduced unwinding activity but retains normal strand-annealing activity and stimulates greater cGAS dinucleotide-synthesis activity than wild-type DDX41. Overexpression of R525H in either DDX41-deficient or -proficient cells results in higher type I interferon production. Our results have led to the hypothesis that DDX41 utilizes its unwinding and annealing activities to regulate the homeostasis of dsDNA and single-stranded DNA (ssDNA), which, in turn, regulates cGAS-STING activation.

Structure-function analysis of DEAD-box helicase DDX43.

DDX43 (DEAD-box helicase 43), also known as HAGE (helicase antigen gene), is a member of the DEAD-box protein family. It contains a K homology (KH) domain in its N terminus, a helicase core domain in its C terminus, and a flexible linker domain in between. DDX43 expression is low or undetectable in normal tissue, but is overexpressed in many tumors; therefore, it is considered a potential target molecule for cancer therapy. We, along with other groups, have shown that DDX43 is an ATP-dependent RNA and DNA helicase, and the KH domain is required for its ATPase and unwinding activity. Electrophoretic mobility shift assay (EMSA), SELEX (systematic evolution of ligands by exponential enrichment), chromatin immunoprecipitation (ChIP)-seq, crosslinking immunoprecipitation (CLIP)-seq, and nuclear magnetic resonance (NMR) showed that the KH domain prefers to bind pyrimidine-rich ssDNA and ssRNA, such as TTGT in the promoter regions of genes. Moreover, the KH domain facilitates the substrate specificity and processivity of the DDX43 helicase. No animal model has been generated for DDX43; cellular studies have revealed that DDX43 has roles in piRNA amplification, tumorigenesis, RAS signaling, and innate immunity. Structural and functional studies of DDX43 will not only advance our understanding of DEAD-box helicases and KH domains, but also shed light on the application of DDX43 as therapeutics, where its key binding sites can be targeted by small molecules and natural products as an alternative approach in treating DDX43 overexpressed cancers.

Synthetic lethal interactions of DEAD/H-box helicases as targets for cancer therapy.

DEAD/H-box helicases are implicated in virtually every aspect of RNA metabolism, including transcription, pre-mRNA splicing, ribosomes biogenesis, nuclear export, translation initiation, RNA degradation, and mRNA editing. Most of these helicases are upregulated in various cancers and mutations in some of them are associated with several malignancies. Lately, synthetic lethality (SL) and synthetic dosage lethality (SDL) approaches, where genetic interactions of cancer-related genes are exploited as therapeutic targets, are emerging as a leading area of cancer research. Several DEAD/H-box helicases, including DDX3, DDX9 (Dbp9), DDX10 (Dbp4), DDX11 (ChlR1), and DDX41 (Sacy-1), have been subjected to SL analyses in humans and different model organisms. It remains to be explored whether SDL can be utilized to identity druggable targets in DEAD/H-box helicase overexpressing cancers. In this review, we analyze gene expression data of a subset of DEAD/H-box helicases in multiple cancer types and discuss how their SL/SDL interactions can be used for therapeutic purposes. We also summarize the latest developments in clinical applications, apart from discussing some of the challenges in drug discovery in the context of targeting DEAD/H-box helicases.

A Dynamic Quantitative Systems Pharmacology Model of Inflammatory Bowel Disease: Part 2 - Application to Current Therapies in Crohn's Disease.

Inflammatory bowel disease (IBD) is a heterogeneic disease with a variety of treatments targeting different mechanisms. A multistate, mechanistic, mathematical model of IBD was developed in part 1 of this two-part article series. In this paper, application of the model to predict response of key clinical biomarkers following different treatment options for Crohn's disease was explored. Five therapies, representing four different mechanisms of action, were simulated in the model and longitudinal profiles of key clinical markers, C-reactive protein and fecal calprotectin were compared with clinical observations. Model simulations provided an accurate match with both central tendency and variability observed in biomarker profiles. We also applied the model to predict biomarker and clinical response in an experimental, combination therapy of existing therapeutic options and provide possible mechanistic basis for the increased response. Overall, we present a validated, modular, mechanistic model construct, which can be applied to explore key biomarkers and clinical outcomes in IBD.

The KH domain facilitates the substrate specificity and unwinding processivity of DDX43 helicase.

The K-homology (KH) domain is a nucleic acid-binding domain present in many proteins. Recently, we found that the DEAD-box helicase DDX43 contains a KH domain in its N-terminus; however, its function remains unknown. Here, we purified recombinant DDX43 KH domain protein and found that it prefers binding ssDNA and ssRNA. Electrophoretic mobility shift assay and NMR revealed that the KH domain favors pyrimidines over purines. Mutational analysis showed that the GXXG loop in the KH domain is involved in pyrimidine binding. Moreover, we found that an alanine residue adjacent to the GXXG loop is critical for binding. Systematic evolution of ligands by exponential enrichment, chromatin immunoprecipitation-seq, and cross-linking immunoprecipitation-seq showed that the KH domain binds C-/T-rich DNA and U-rich RNA. Bioinformatics analysis suggested that the KH domain prefers to bind promoters. Using 15N-heteronuclear single quantum coherence NMR, the optimal binding sequence was identified as TTGT. Finally, we found that the full-length DDX43 helicase prefers DNA or RNA substrates with TTGT or UUGU single-stranded tails and that the KH domain is critically important for sequence specificity and unwinding processivity. Collectively, our results demonstrated that the KH domain facilitates the substrate specificity and processivity of the DDX43 helicase.

First Report of Agrobacterium rhizogenes-induced Hairy Root Formation in Selaginella bryopteris: a Pteridophyte Recalcitrant to Genetic Transformation

Abstract we report A. rhizogenes-induced hairy root formation in S. bryopteris, a medicinally and commercially important plant. A. rhizogenes strain LBA1334 co-cultivated with explants (root, rhizophore, stem portion near the root, and stem with intact fronds) for 24 and 48 h after transformation for induction of hairy roots. The induction of hairy root was observed after 6 days of infection in case of 48 h co-cultivation only. PCR with rolA and virC gene specific primers confirmed the induced hairy roots were due to Ri T-DNA integration and not due to contaminating A. rhizogenes. The root network as explants showed the maximum transformation efficiency. We tested different media like MS, SHFR (Stage Hog Fern Root) and KNOP's during transformation for hairy root induction. The SHFR based media showed good response in transformation as well as propagation. Further, transformation efficiency was enhanced by addition of TDZ (2 mg/L) and Bevistin (0.1%) in SHFR media. The present work would be helpful in hairy roots-based in vitro production of secondary metabolites and on aspect of functional genomics of S. bryopteris.

Efficacy and Safety of Dose Escalation to Adalimumab 80 mg Every Other Week in Japanese Patients with Crohn's Disease Who Lost Response to Maintenance Therapy.

BACKGROUND: Dose escalation is often recommended for loss of response in anti-TNFα-treated patients with Crohn's disease (CD). This 52-week phase 3, multicenter study investigated the efficacy and safety of escalation to adalimumab 80 mg every other week (EOW) in Japanese patients with CD who lost response to maintenance adalimumab 40 mg EOW. METHODS: Twenty-eight patients aged ≥15 years with moderately to severely active CD who had previously attained and subsequently lost clinical response to maintenance ada limumab received open-label adalimumab 80 mg EOW during weeks 0-50. Loss of response was defined as CD activity index (CDAI) ≥200, increases in CDAI ≥50 from minimum observed value, and C-reactive protein (CRP) ≥1 mg/dL at screening. The primary endpoint was the proportion of patients achieving a CDAI decrease ≥50 (CR-50) from baseline at week 8. RESULTS: At weeks 8 and 52, 75.0 and 57.1$ of patients achieved CR-50 and 25.0 and 35.7$ achieved clinical remission (CDAI < 150), respectively; median CRP changes from baseline were -0.39 and -0.77 mg/dL, respectively. Most treatment-emergent adverse events were mild to moderate. CONCLUSIONS: Adalimumab dose escalation to 80 mg EOW improved CD activity in patients who had lost response to maintenance adalimumab, with no new safety signals. (ClinicalTrials.gov Identifier: NCT01958827.).

The DEAD-box protein DDX43 (HAGE) is a dual RNA-DNA helicase and has a K-homology domain required for full nucleic acid unwinding activity.

The K-homology (KH) domain is a nucleic acid-binding domain present in many proteins but has not been reported in helicases. DDX43, also known as HAGE (helicase antigen gene), is a member of the DEAD-box protein family. It contains a helicase core domain in its C terminus and a potential KH domain in its N terminus. DDX43 is highly expressed in many tumors and is, therefore, considered a potential target for immunotherapy. Despite its potential as a therapeutic target, little is known about its activities. Here, we purified recombinant DDX43 protein to near homogeneity and found that it exists as a monomer in solution. Biochemical assays demonstrated that it is an ATP-dependent RNA and DNA helicase. Although DDX43 was active on duplex RNA regardless of the orientation of the single-stranded RNA tail, it preferred a 5' to 3' polarity on RNA and a 3' to 5' direction on DNA. Truncation mutations and site-directed mutagenesis confirmed that the KH domain in DDX43 is responsible for nucleic acid binding. Compared with the activity of the full-length protein, the C-terminal helicase domain had no unwinding activity on RNA substrates and had significantly reduced unwinding activity on DNA. Moreover, the full-length DDX43 protein, with single amino acid change in the KH domain, had reduced unwinding and binding activates on RNA and DNA substrates. Our results demonstrate that DDX43 is a dual helicase and the KH domain is required for its full unwinding activity.

Cavitary penetration of levofloxacin among patients with multidrug-resistant tuberculosis.

A better understanding of second-line drug (SLD) pharmacokinetics, including cavitary penetration, may help optimize SLD dosing. Patients with pulmonary multidrug-resistant tuberculosis (MDR-TB) undergoing adjunctive surgery were enrolled in Tbilisi, Georgia. Serum was obtained at 0, 1, 4, and 8 h and at the time of cavitary removal to measure levofloxacin concentrations. After surgery, microdialysis was performed using the ex vivo cavity, and levofloxacin concentrations in the collected dialysate fluid were measured. Noncompartmental analysis was performed, and a cavitary-to-serum levofloxacin concentration ratio was calculated. Twelve patients received levofloxacin for a median of 373 days before surgery (median dose, 11.8 mg/kg). The median levofloxacin concentration in serum (Cmax) was 6.5 µg/ml, and it was <2 µg/ml in 3 (25%) patients. Among 11 patients with complete data, the median cavitary concentration of levofloxacin was 4.36 µg/ml (range, 0.46 to 8.82). The median cavitary/serum levofloxacin ratio was 1.33 (range, 0.63 to 2.36), and 7 patients (64%) had a ratio of >1. There was a significant correlation between serum and cavitary concentrations (r = 0.71; P = 0.01). Levofloxacin had excellent penetration into chronic cavitary TB lesions, and there was a good correlation between serum and cavitary concentrations. Optimizing serum concentrations will help ensure optimal cavitary concentrations of levofloxacin, which may enhance treatment outcomes.

Comparative pharmacokinetics of purified flaxseed and associated mammalian lignans in male Wistar rats.

Consumption of flaxseed lignans is associated with various health benefits; however, little is known about the bioavailability of purified lignans in flaxseed. Data on their bioavailability and hence pharmacokinetics (PK) are necessary to better understand their role in putative health benefits. In the present study, we conducted a comparative PK analysis of the principal lignan of flaxseed, secoisolariciresinol diglucoside (SDG), and its primary metabolites, secoisolariciresinol (SECO), enterodiol (ED) and enterolactone (EL) in rats. Purified lignans were intravenously or orally administered to each male Wistar rat. SDG and its primary metabolites SECO, ED and EL were administered orally at doses of 40, 40, 10 and 10 mg/kg, respectively, and intravenously at doses of 20, 20, 5 and 1 mg/kg, respectively. Blood samples were collected at 0 (pre-dose), 5, 10, 15, 20, 30 and 45 min, and at 1, 2, 4, 6, 8, 12 and 24 h post-dosing, and serum samples were analysed. PK parameters and oral bioavailability of purified lignans were determined by non-compartmental methods. In general, administration of the flaxseed lignans SDG, SECO and ED demonstrated a high systemic clearance, a large volume of distribution and short half-lives, whereas administration of EL at the doses of 1 mg/kg (intravenously) and 10 mg/kg (orally administered) killed the rats within a few hours of dosing, precluding a PK analysis of this lignan. PK parameters of flaxseed lignans exhibited the following order: systemic clearance, SDG < SECO < ED; volume of distribution, SDG < SECO < ED; half-life, SDG < ED < SECO. The percentage of oral bioavailability was 0, 25 and < 1 % for SDG, SECO and ED, respectively.

Micro-architectural changes in cancellous bone differ in female and male C57BL/6 mice with high-fat diet-induced low bone mineral density.

The relationship between fat and bone mass at distinct trabecular and cortical skeletal compartments in a high-fat diet (HFD) model was studied. For this, C57BL/6 mice were assigned to four groups of eight animals each. Two groups, each of males and females, received a standard chow diet while the remaining other two groups received the HFD for a period of 10 weeks. Male mice on the HFD were heavier and gained more weight (15·8 %; P<  0·05) v. those on the control diet or when compared with the female rats fed the HFD. We observed an increased lipid profile in both males and females, with significantly higher lipid levels (about 20-25 %; P< 0·01) in males. However, glucose intolerance was more pronounced in females than males on the HFD (about 30 %; P< 0·05). The micro-architectural assessment of bones showed that compared with female mice on the HFD, male mice on the HFD showed more deterioration at the trabecular region. This was corroborated by plasma osteocalcin and carboxy-terminal collagen crosslinks (CTx) levels confirming greater loss in males (about 20 %; P< 0·01). In both sexes cortical bone parameters and strength remained unchanged after 10 weeks of HFD treatment. The direct effect of the HFD on bone at the messenger RNA level in progenitor cells isolated from femoral bone marrow was a significantly increased expression of adipogenic marker genes v. osteogenic genes. Overall, the present data indicate that obesity induced by a HFD aggravates bone loss in the cancellous bone compartment, with a greater loss in males than females, although 10 weeks of HFD treatment did not alter cortical bone mass and strength in both males and females.

A general HPLC-UV method for the quantitative determination of curcumin analogues containing the 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore in rat biomatrices.

Curcumin and its derivatives generally display favorable cytotoxic activities against a number of cancer cell types. We focus our rational antineoplastic drug design program on curcumin analogues containing the 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore. Favorable outcomes from pharmacological screens of this series demanded further pharmacokinetic evaluations to determine their suitability as effective compounds in vivo. To allow such evaluations and to provide a general, sensitive, rapid and simple method for the analysis of compounds containing the 1,5-diaryl-3-oxo-pentadienyl scaffold, we developed an HPLC method with ultraviolet detection for their detection in various biological matrices of a relevant preclinical species, i.e. the rat. Our HPLC method is specific for the analysis of many members in this series in rat blood, plasma, serum and hepatic microsomes following liquid-liquid extraction with TBME (1:30, v/v). The assay procedure involves chromatographic separation on a Zorbax-Eclipse C-18 column under isocratic conditions with the mobile phase consisting of acetonitrile and ammonium acetate buffer (pH 5.0, 10mM) in different ratios depending upon the compound. The method was validated for NC 2083 in rat serum and rat liver microsomes, a potential lead compound, to demonstrate its applicability. The standard curve was linear (r(2)≥0.997) from 50 to 5000ng/mL. Intra- and inter-day precision and accuracy of the method were within USFDA specified limits. The stability of NC 2083 was established in an auto-injector, on bench-top, during freeze-thaw cycles and long-term stability at -80°C for 40 days. The method is suitable for a number of compounds containing the 1,5-diaryl-3-oxo-pentadienyl scaffold with divergent logP values with only minor adjustments in the buffer to acetonitrile ratio of the mobile phase.

An evaluation of efficacy of balloon inflation on venous cannulation pain in children: a prospective, randomized, controlled study.

Venipuncture is the most common painful event for a hospitalized child. We evaluated the efficacy of balloon inflation for attenuating venipuncture pain in children. Seventy-five pediatric patients aged 6-12 yr, ASA physical status I-II, of either sex, undergoing elective surgery were included in this prospective and randomized study. Patients were randomly divided into 3 equal groups of 25 each; Group I (control), Group II (distraction) pressed a rubber ball, and Group III (balloon) inflated a balloon. A manual venous occlusion was applied on the forearm and venipuncture was performed with a 22-gauge venous cannula. Pain was self-reported by a pain face scale with a 10-cm visual analog scale (VAS) placed at its back, where 0 = "no pain" and 10 = "worst imaginable pain." VAS scores of 1-3 were rated as mild, 4-6 as moderate, and >6 as severe. Median (interquartile range) VAS score in the balloon group was 1 (3), which was reduced as compared with 2 (2) and 4 (2) observed in the distraction and control groups, respectively (P < 0.000). Significant reduction in the incidence and severity of venipuncture pain was also observed in the balloon group compared with the other 2 groups (P < 0.05).