A simple, rapid and precise method was developed for the quantitative estimation of prasugrel hydrochloride in pharmaceutical dosage form. A chromatographic separation of prasugrel and its degradants was achieved with Zorbax XDB C8, 150 × 4.6 mm, 3.5μm analytical column using aqueous solution of 0.05 M ammonium acetate pH 4.5 with acetic acid-acetonitrile (40:60 v/v). The instrumental settings include flow rate of 1.0 ml/min, column temperature at 30°C and detector wavelength of 254 nm using a photodiode array detector. Theoretical plates for prasugrel were 7023. Tailing factor for prasugrel was 1.11. Prasugrel was exposed to thermal, photolytic, hydrolytic and oxidative stress conditions, and the stressed samples were analyzed by the proposed method. Peak homogeneity data of prasugrel was obtained using photodiode array detector in the stressed sample chromatograms, which demonstrated the specificity of the method for the estimation in presence of degradants. The described method showed excellent linearity over a range of 10–300 μg/ml for prasugrel. The correlation coefficient is 0.999. The relative standard deviation of peak area for six measurements is always less than 2%. Overall, the proposed method was found to be suitable and accurate for quantitative determination and stability study of prasugrel in pharmaceutical dosage form.
Liquid chromatography; Method validation; Pharmaceutical preparation; Prasugrel hydrochloride
Tannerella forsythia is a pathogen implicated in periodontitis, an inflammatory disease of the tooth supporting tissues often leading to tooth loss. This key periodontal pathogen is decorated with a unique glycan core O-glycosidically linked to the bacterium’s proteinacious surface(S)-layer lattice and other glycoproteins. Herein we show that the terminal motif of this glycan core acts to modulate dendritic cell effector functions to suppress Th17 responses. In contrast to the wild-type bacterial strain, infection with a mutant strain lacking the complete S-layer glycan core induced robust Th17 and reduced periodontal bone loss in mice. Our findings demonstrate that surface glycosylation of this pathogen acts to ensure its persistence in the host by suppressing Th17 responses. In addition our data suggest that the bacterium then induces the TLR2-Th2 inflammatory axis that has previously shown to cause bone destruction. Our study provides a biological basis for pathogenesis and opens opportunities in exploiting bacterial glycans as therapeutic targets against periodontitis and a range of other infectious diseases.
Processing is an important and essential component to enhance the digestibility of essential nutrients of grains. Dietary fibres play an important role in bringing health advantages in chickpea and help in lowering plasma cholesterol. Changes during soaking and soaking followed by cooking on cellulose, hemicellulose, lignin and pectin contents of four genotypes of desi type (KWR 108, JG 74, DCP 92-3 and BG 256), four genotypes of kabuli types (KAK 2, JKG 1, BG 1053, and L 550) and two genotypes of green seed type (BGD 112 and Sadabahar) of chickpeas (Cicer arietinum, L.) was studied. Cellulose, lignin and pectin increased during soaking and cooking, whereas hemicellulose increased during soaking but decreased drastically during cooking. Cellulose recorded an overall increase of 40% during cooking, followed by 15.7% and 15.2% increase in pectin and lignin, respectively during cooking of chickpea grain. Hemicellulose, on the contrary showed a decrease of 26.8% during cooking.
Chickpea; Cooking; Cellulose; Hemicellulose; Lignin; Pectin
In many high-risk populations, access to tuberculosis (TB) diagnosis and treatment is limited and pockets of high prevalence persist. We estimated the cost-effectiveness of an extensive active case finding program in areas of Cambodia where TB notifications and household poverty rates are highest and access to care is restricted. Thirty operational health districts with high TB incidence and household poverty were randomized into intervention and control groups. In intervention operational health districts, all household and symptomatic neighborhood contacts of registered TB patients of the past two years were encouraged to attend screening at mobile centers. In control districts, routine passive case finding activities continued. The program screened more than 35,000 household and neighborhood contacts and identified 810 bacteriologically confirmed cases. The cost-effectiveness analysis estimated that in these cases the reduction in mortality from 14% to 2% would result in a cost per daily adjusted life year averted of $330, suggesting that active case finding was highly cost-effective.
A large fraction of non-small cell lung cancers (NSCLC) are dependent on defined oncogenic driver mutations. Although targeted agents exist for EGFR- and EML4-ALK-driven NSCLC, no therapies target the most frequently found driver mutation, KRAS. Furthermore, acquired resistance to the currently targetable driver mutations is nearly universally observed. Clearly a novel therapeutic approach is needed to target oncogene driven NSCLC. We recently demonstrated that the basic helix-loop-helix transcription factor Twist1 cooperates with mutant Kras to induce lung adenocarcinoma in transgenic mouse models and that inhibition of Twist1 in these models led to Kras-induced senescence. In the current study, we examine the role of TWIST1 in oncogene driven human NSCLC. Silencing of TWIST1 in KRAS mutant human NSCLC cell lines resulted in dramatic growth inhibition and either activation of a latent oncogene-induced senescence program or in some cases, apoptosis. Similar effects were observed in EGFR mutation driven and c-Met amplified NSCLC cell lines. Growth inhibition by silencing of TWIST1 was independent of p53 or p16 mutational status and did not require previously defined mediators of senescence, p21 and p27, nor could this phenotype be rescued by overexpression of SKP2. In xenograft models, silencing of TWIST1 resulted in significant growth inhibition of KRAS mutant, EGFR mutant and c-Met amplified NSCLC. Remarkably, inducible silencing of TWIST1 resulted in significant growth inhibition of established KRAS mutant tumors. Together these findings suggest that silencing of TWIST1 in oncogene driver dependent NSCLC represents a novel and promising therapeutic strategy.
TWIST1; OIS; KRAS; NSCLC; EGFR
Outcomes for poor-risk localized prostate cancers treated with radiation are still insufficient. Targeting the “non-oncogene” addiction or stress response machinery is an appealing strategy for cancer therapeutics. Heat-shock-protein-90 (Hsp90), an integral member of this machinery, is a molecular chaperone required for energy-driven stabilization and selective degradation of misfolded “client” proteins, that is commonly overexpressed in tumor cells. Hsp90 client proteins include critical components of pathways implicated in prostate cancer cell survival and radioresistance, such as androgen receptor signaling and the PI3K-Akt-mTOR pathway. We examined the effects of a novel non-geldanamycin Hsp90 inhibitor, AUY922, combined with radiation (RT) on two prostate cancer cell lines, Myc-CaP and PC3, using in vitro assays for clonogenic survival, apoptosis, cell cycle distribution, γ-H2AX foci kinetics and client protein expression in pathways important for prostate cancer survival and radioresistance. We then evaluated tumor growth delay and effects of the combined treatment (RT-AUY922) on the PI3K-Akt-mTOR and AR pathways in a hind-flank tumor graft model. We observed that AUY922 caused supra-additive radiosensitization in both cell lines at low nanomolar doses with enhancement ratios between 1.4–1.7 (p < 0.01). RT-AUY922 increased apoptotic cell death compared with either therapy alone, induced G2-M arrest and produced marked changes in client protein expression. These results were confirmed in vivo, where RT-AUY922 combination therapy produced supra-additive tumor growth delay compared with either therapy by itself in Myc-CaP and PC3 tumor grafts (both p < 0.0001). Our data suggest that combined RT-AUY922 therapy exhibits promising activity against prostate cancer cells, which should be investigated in clinical studies.
prostate cancer; Hsp90; NVP-AUY922; radiosensitizer; DNA damage response
Protein modification with complex glycans is increasingly being recognized in many pathogenic and non-pathogenic bacteria, and is now thought to be central to the successful life-style of those species in their respective hosts. This review aims to convey current knowledge on the extent of protein glycosylation in periodontal pathogenic bacteria and its role in the modulation of the host immune responses. The available data show that surface glycans of periodontal bacteria orchestrate dendritic cell cytokine responses to drive T cell immunity in ways that facilitate bacterial persistence in the host and induce periodontal inflammation. In addition, surface glycans may help certain periodontal bacteria protect against serum complement attack or help them escape immune detection through glycomimicry. In this review we will focus mainly on the generalized surface-layer protein glycosylation system of the periodontal pathogen Tannerella forsythia in shaping innate and adaptive host immunity in the context of periodontal disease. In addition, we will also review the current state of knowledge of surface protein glycosylation and its potential for immune modulation in other periodontal pathogens.
protein glycosylation; periodontal bacteria; immune response
Sortase A from S. pneumoniae has been crystallized in two crystal forms: diamond-shaped (construct ΔN59SrtA) and rod-shaped (construct ΔN81SrtA). The diamond-shaped crystals diffracted poorly to 4.0 Å resolution and belonged to a tetragonal system, whereas the rod-shaped crystals diffracted to 2.91 Å resolution and belonged to space group P21.
Sortases are cell-membrane-anchored cysteine transpeptidases that are essential for the assembly and anchoring of cell-surface adhesins in Gram-positive bacteria. Thus, they play critical roles in virulence, infection and colonization by pathogens. Sortases have been classified into four types based on their primary sequence and the target-protein motifs that they recognize. All Gram-positive bacteria express a class A housekeeping sortase (SrtA). Sortase A from Streptococcus pneumoniae (NP_358691) has been crystallized in two crystal forms. Diamond-shaped crystals of ΔN59SrtA diffracted to 4.0 Å resolution and belonged to a tetragonal system with unit-cell parameters a = b = 122.8, c = 86.5 Å, α = β = γ = 90°, while rod-shaped crystals of ΔN81SrtA diffracted to 2.91 Å resolution and belonged to the monoclinic space group P21 with unit-cell parameters a = 66.8, b = 103.47, c = 74.79 Å, α = γ = 90, β = 115.65°. The Matthews coefficient (V
M = 2.77 Å3 Da−1) with ∼56% solvent content suggested the presence of four molecules in the asymmetric unit for ΔN81SrtA. Also, a multi-copy search using a monomer as a probe in the molecular-replacement method resulted in the successful location of four sortase molecules in the asymmetric unit, with statistics R = 41.61, R
free = 46.44, correlation coefficient (CC) = 64.31, CCfree = 57.67.
sortases; cysteine transpeptidases; Streptococcus pneumoniae
Pathogenesis of many bacterially-induced inflammatory diseases is driven by toll- like receptor (TLR) mediated immune responses following recognition of bacterial factors by different TLRs. Periodontitis is a chronic inflammation of the tooth supporting apparatus often leading to tooth loss, and is caused by a Gram-negative bacterial consortium that includes Tannerella forsythia. This bacterium expresses a virulence factor, the BspA, which drives periodontal inflammation by activating TLR2. The N- terminal portion of the BspA protein comprises a leucine-rich repeat (LRR) domain previously shown to be involved in the binding and activation of TLR2. The objective of the current study was to identify specific epitopes in the LRR domain of BspA that interact with TLR2. Our results demonstrate that a sequence motif GC(S/T)GLXSIT is involved in mediating the interaction of BspA with TLR2. Thus, our study has identified a peptide motif that mediates the binding of a bacterial protein to TLR2 and highlights the promiscuous nature of TLR2 with respect to ligand binding. This work could provide a structural basis for designing peptidomimetics to modulate the activity of TLR2 in order to block bacterially-induced inflammation.
leucine-rich repeat protein; BspA; TLR-2; Tannerella forsythia
The recent discovery of a small-molecule benzosuberene-based phenol that demonstrates remarkable picomolar cytotoxicity against selected human cancer cell lines and strongly inhibits tubulin polymerization (1–2 µM) inspired the design and synthesis of a variety of new, structurally diverse benzosuberene derivatives. An efficient synthetic route to functionalized benzosuberenes was developed. This methodology utilized a Wittig reaction, followed by a selective alkene reduction and ring-closing cyclization to form the core benzosuberone structure. This synthetic route facilitated the preparation of a 6-nitro-1-(3′,4′,5′-trimethoxyphenyl) benzosuberene derivative and its corresponding 6-amino analogue in good yield. The 6-amino analogue was a strong inhibitor of tubulin polymerization (1.2 µM), demonstrated enhanced cytotoxicity against the human cancer cell lines examined (GI50 = 33 pM against SK-OV-3 ovarian cancer, for example), and exhibited a concentration dependent disruption of a pre-established capillary-like network of tubules formed from human umbilical vein endothelial cells.
Sorafenib (SOR) is the only systemic agent known to improve survival for hepatocellular carcinoma (HCC). However, SOR prolongs survival by less than 3 months and does not alter symptomatic progression. To improve outcomes, several phase I-II trials are currently examining SOR with radiation (RT) for HCC utilizing heterogeneous concurrent and sequential treatment regimens. Our study provides preclinical data characterizing the effects of concurrent versus sequential RT-SOR on HCC cells both in vitro and in vivo. Concurrent and sequential RT-SOR regimens were tested for efficacy among 4 HCC cell lines in vitro by assessment of clonogenic survival, apoptosis, cell cycle distribution, and γ-H2AX foci formation. Results were confirmed in vivo by evaluating tumor growth delay and performing immunofluorescence staining in a hind-flank xenograft model. In vitro, concurrent RT-SOR produced radioprotection in 3 of 4 cell lines, whereas sequential RT-SOR produced decreased colony formation among all 4. Sequential RT-SOR increased apoptosis compared to RT alone, while concurrent RT-SOR did not. Sorafenib induced reassortment into less radiosensitive phases of the cell cycle through G1-S delay and cell cycle slowing. More double-strand breaks (DSBs) persisted 24 h post-irradiation for RT alone versus concurrent RT-SOR. In vivo, sequential RT-SOR produced the greatest tumor growth delay, while concurrent RT-SOR was similar to RT alone. More persistent DSBs were observed in xenografts treated with sequential RT-SOR or RT alone versus concurrent RT-SOR. Sequential RT-SOR additionally produced a greater reduction in xenograft tumor vascularity and mitotic index than either concurrent RT-SOR or RT alone. In conclusion, sequential RT-SOR demonstrates greater efficacy against HCC than concurrent RT-SOR both in vitro and in vivo. These results may have implications for clinical decision-making and prospective trial design.
Human mesenchymal stem cells (hMSCs) present in the bone marrow are the precursors of osteoblasts, chondrocytes and adipocytes, and hold tremendous potential for osteoregenerative therapy. However, achieving directed differentiation into osteoblasts has been a major concern. The use of lithium for enhancing osteogenic differentiation has been documented in animal models but its effect in humans is not clear. We, therefore, performed high throughput transcriptome analysis of lithium-treated hMSCs to identify altered gene expression and its relevance to osteogenic differentiation. Our results show suppression of proliferation and enhancement of alkaline phosphatase (ALP) activity upon lithium treatment of hMSCs under non-osteogenic conditions. Microarray profiling of lithium-stimulated hMSC revealed decreased expression of adipogenic genes (CEBPA, CMKLR1, HSD11B1) and genes involved in lipid biosynthesis. Interestingly, osteoclastogenic factors and immune responsive genes (IL7, IL8, CXCL1, CXCL12, CCL20) were also downregulated. Negative transcriptional regulators of the osteogenic program (TWIST1 and PBX1) were suppressed while genes involved in mineralization like CLEC3B and ATF4 were induced. Gene ontology analysis revealed enrichment of upregulated genes related to mesenchymal cell differentiation and signal transduction. Lithium priming led to enhanced collagen 1 synthesis and osteogenic induction of lithium pretreated MSCs resulted in enhanced expression of Runx2, ALP and bone sialoprotein. However, siRNA-mediated knockdown of RRAD, CLEC3B and ATF4 attenuated lithium-induced osteogenic priming, identifying a role for RRAD, a member of small GTP binding protein family, in osteoblast differentiation. In conclusion, our data highlight the transcriptome reprogramming potential of lithium resulting in higher propensity of lithium “primed” MSCs for osteoblastic differentiation.
Chromatin dynamics play a central role in maintaining genome integrity, but how this is achieved remains largely unknown. Here, we report that microrchidia CW-type zinc finger 2 (MORC2), an uncharacterized protein with a derived PHD finger domain and a conserved GHKL-type ATPase module, is a physiological substrate of p21-activated kinase 1 (PAK1), an important integrator of extracellular signals and nuclear processes. Following DNA damage, MORC2 is phosphorylated on serine 739 in a PAK1 dependent manner, and phosphorylated MORC2 regulates its DNA-dependent ATPase activity to facilitate chromatin remodeling. Moreover, MORC2 associates with chromatin and promotes gamma-H2AX induction in a PAK1 phosphorylation-dependent manner. Consequently, cells expressing MORC2-S739A mutation displayed a reduction in DNA repair efficiency and were hypersensitive to DNA-damaging agent. These findings suggest that the PAK1-MORC2 axis is critical for orchestrating the interplay between chromatin dynamics and the maintenance of genomic integrity through sequentially integrating multiple essential enzymatic processes.
Chromatin remodeling; DNA damage response; Genomic stability; Modifier of radiosensitivity; MORC2
Tannerella forsythia is strongly associated with chronic periodontitis, an inflammatory disease of the tooth-supporting tissues, leading to tooth loss. Fusobacterium nucleatum, an opportunistic pathogen, is thought to promote dental plaque formation by serving as a bridge bacterium between early- and late-colonizing species of the oral cavity. Previous studies have shown that F. nucleatum species synergize with T. forsythia during biofilm formation and pathogenesis. In the present study, we showed that coinfection of F. nucleatum and T. forsythia is more potent than infection with either species alone in inducing NF-κB activity and proinflammatory cytokine secretion in monocytic cells and primary murine macrophages. Moreover, in a murine model of periodontitis, mixed infection with the two species induces synergistic alveolar bone loss, characterized by bone loss which is greater than the additive alveolar bone losses induced by each species alone. Further, in comparison to the single-species infection, mixed infection caused significantly increased inflammatory cell infiltration in the gingivae and osteoclastic activity in the jaw bones. These data show that F. nucleatum subspecies and T. forsythia synergistically stimulate the host immune response and induce alveolar bone loss in a murine experimental periodontitis model.
The natural products combretastatin A-4 (CA4) and combretastatin A-1 (CA1) are potent cancer vascular disrupting agents (VDAs) and inhibitors of tubulin assembly (IC50 = 1–2 μM). The phosphorylated prodrugs CA4P and CA1P are undergoing human clinical trials against cancer. CA1 is unique due to its incorporation of a vicinal phenol, which has afforded the opportunity to prepare both diphosphate and regioisomeric monophosphate derivatives. Here, we describe the first synthetic routes suitable for the regiospecific preparation of the CA1-monophosphates, CA1MPA (8a/b) and CA1MPB (4a/b). The essential regiochemistry necessary to distinguish between the two vicinal phenolic groups was accomplished with a tosyl protecting group strategy. Each of the four monophosphate analogues (including Z and E isomers) demonstrated in vitro cytotoxicity against selected human cancer cell lines comparable to their corresponding diphosphate congeners. Furthermore, Z-CA1MPA (8a) and Z-CA1MPB (4a) were inactive as inhibitors of tubulin assembly (IC50 > 40 μM), as anticipated in this pure protein assay.
Periodontal disease (PD) is a chronic inflammation of the tooth supporting soft tissue and alveolar bone due to infection by a select group of gram negative microbes, and leads to tooth loss if untreated. Since mice deficient in CD4+ cells are resistant to infection-induced alveolar bone loss, Th cells have been implicated in bone destructive processes during PD. However, the extent to which different Th-cell subtypes play roles in pathogenesis or host protection remains to be defined, and is likely to vary depending on the dominant microorganism involved. By far the best studied periodontal microbe in PD is Porphyromonas gingivalis. Even though the gram negative anaerobe Tannerella forsythia is also a vital contributor to periodontal bone loss, almost nothing is known about immune responses to this organism. Previous studies from our laboratory have revealed that T. forsythia induces periodontal bone loss in mice, and that this bone loss depends on the bacterially-expressed BspA protein. In this study, we show that T. forsythia activates murine APCs primarily through TLR2-dependent signaling via BspA. Furthermore, T. forsythia infection causes a pronounced Th2 bias, evidenced by T cell expression of IL-5 but not IFN-γ or IL-17 in draining LN. Consistently, deficiencies in TLR2 or STAT6 result in resistance to T. forsythia-induced alveolar bone loss. Thus, TLR2 signaling and Th2 cells play pathogenic roles in T. forsythia-induced alveolar bone destruction.
PA-824 is one of two nitroimidazoles in phase II clinical trials to treat tuberculosis. In mice, it has dose-dependent early bactericidal and sterilizing activity. In humans with tuberculosis, PA-824 demonstrated early bactericidal activity (EBA) at doses ranging from 200 to 1,200 mg per day, but no dose-response effect was observed. To better understand the relationship between drug exposure and effect, we performed a dose fractionation study in mice. Dose-ranging pharmacokinetic data were used to simulate drug exposure profiles. Beginning 2 weeks after aerosol infection with Mycobacterium tuberculosis, total PA-824 doses from 144 to 4,608 mg/kg were administered as 3, 4, 8, 12, 24, or 48 divided doses over 24 days. Lung CFU counts after treatment were strongly correlated with the free drug T>MIC (R2 = 0.87) and correlated with the free drug AUC/MIC (R2 = 0.60), but not with the free drug Cmax/MIC (R2 = 0.17), where T>MIC is the cumulative percentage of the dosing interval that the drug concentration exceeds the MIC under steady-state pharmacokinetic conditions and AUC is the area under the concentration-time curve. When the data set was limited to regimens with dosing intervals of ≤72 h, both the T>MIC and the AUC/MIC values fit the data well. Free drug T>MIC of 22, 48, and 77% were associated with bacteriostasis, a 1-log kill, and a 1.59-log kill (or 80% of the maximum observed effect), respectively. Human pharmacodynamic simulations based on phase I data predict 200 mg/day produces free drug T>MIC values near the target for maximal observed bactericidal effect. The results support the recently demonstrated an EBA of 200 mg/day and the lack of a dose-response between 200 and 1,200 mg/day. T>MIC, in conjunction with AUC/MIC, is the parameter on which dose optimization of PA-824 should be based.
A group A Streptococcus(GAS) isolate,serotypeM12,recovered from a patient with streptococcal toxic shock syndrome was analyzed for superantigen-carrying prophages, revealing 149, which encodes superantigen SSA. Sequence analysis of the att-L proximal region of 149 showed that the phage had a mosaic nature. Remarkably, we successfully obtained lysogenic conversion of GAS clinical isolates of various M serotypes (M1, M3, M5, M12, M19, M28, and M94), as well as of group C Streptococcus equisimilis (GCSE) clinical isolates, via transfer of a recombinant phage 149::Kmr. Phage149::Kmr from selected lysogenized GAS and GCSE strains could be transferred back to M12 GAS strains. Our data indicate that horizontal transfer of lysogenic phages among GAS can occur across the M-type barrier; these data also provide further support for the hypothesis that toxigenic conversion can occur via lysogeny between species. Streptococci might employ this mechanism specifically to allow more efficient adaptation to changing host challenges, potentially leading to fitter and more virulent clones.
Structural redesign of selected non-steroidal estrogen receptor binding compounds has previously been successful in the discovery of new inhibitors of tubulin assembly. Accordingly, tetra-substituted alkene analogues (21-30) were designed based in part on combinations of the structural and electronic components of tamoxifen and combretastatin A-4 (CA4). The McMurry coupling reaction was used as the key synthetic step in the preparation of these tri- and tetra-arylethylene analogues. The structural assignment of E, Z isomers was determined on the basis of 2D-NOESY experiments. The ability of these compounds to inhibit tubulin polymerization and cell growth in selected human cancer cell lines was evaluated. Although the compounds were found to be less potent than CA4, these analogues significantly advance the known structure activity relationship associated with the colchicine binding site on β-tubulin.
In the present work, orodispersible tablets of pheniramine maleate were designed with a view to enhance patient compliance by effervescent method. In the effervescent method, mixture of sodium bicarbonate and tartaric acid (each of 12% w/w concentration) were used along with super disintegrants, i.e., pregelatinized starch, sodium starch glycolate, croscarmellose sodium and crospovidone. The prepared batches of tablets were evaluated for hardness, friability, drug content uniformity and in vitro dispersion time. Based on in vitro dispersion time (approximately 60 s), three formulations were tested for in vitro drug release pattern (in pH 6.8 phosphate buffer), short-term stability (at 40±2°/75±5% RH for 3 mo) and drug-excipient interaction (IR spectroscopy). Among three promising formulations, formulation ECP4 containing 4% w/w crospovidone and mixture of sodium bicarbonate and tartaric acid (each of 12% w/w) emerged as the overall best formulation (t70% = 1.65 min) based on the in vitro drug release characteristics compared to commercial conventional tablet formulation. Short-term stability studies on the formulations indicated no significant changes in the drug content and in vitro dispersion time (P < 0.05).
Orodispersible tablets; pheniramine maleate; pregelatinized starch; sodium starch glycolate; croscarmellose sodium; crospovidone
Nitrofuranyl isoxazolines with increased proteolytic stability over nitrofuranyl amides were designed and synthesized leading to discovery of several compounds with potent in vitro anti-tuberculosis activity. However, their in vivo activity was limited by high protein binding and poor distribution. Consequently, a series of non-nitrofuran containing isoxazolines was prepared to determine if the core had residual anti-tuberculosis activity. This led to the discovery of novel isoxazoline 12 as anti-tuberculosis agent with a MIC90 value of 1.56 μg/mL.
Isoxazolines; Nitrofurans; Anti-tuberculosis agents
The luxS/AI-2 signaling pathway has been reported to interfere with important physiological and pathogenic functions in a variety of bacteria. In the present study, we investigated the functional role of the streptococcal luxS/AI-2 system in metabolism and diverse aspects of pathogenicity including the adaptation of the organism to stress conditions using two serotypes of Streptococcus pyogenes, M1 and M19.
Exposing wild-type and isogenic luxS-deficient strains to sulfur-limited media suggested a limited role for luxS in streptococcal activated methyl cycle metabolism. Interestingly, loss of luxS led to an increased acid tolerance in both serotypes. Accordingly, luxS expression and AI-2 production were reduced at lower pH, thus linking the luxS/AI-2 system to stress adaptation in S. pyogenes. luxS expression and AI-2 production also decreased when cells were grown in RPMI medium supplemented with 10% serum, considered to be a host environment-mimicking medium. Furthermore, interaction analysis with epithelial cells and macrophages showed a clear advantage of the luxS-deficient mutants to be internalized and survive intracellularly in the host cells compared to the wild-type parents. In addition, our data revealed that luxS influences the expression of two virulence-associated factors, the fasX regulatory RNA and the virulence gene sibA (psp).
Here, we suggest that the group A streptococcal luxS/AI-2 system is not only involved in the regulation of virulence factor expression but in addition low level of luxS expression seems to provide an advantage for bacterial survival in conditions that can be encountered during infections.
The time course of changes in apparent diffusion coefficient (ADC) and signal intensity on diffusion-weighted magnetic resonance imaging (DW MR) imaging in acute ischemic stroke is a very dynamic event. There is an initial reduction in ADCs with no change on T2-W imaging but signal intensity increase on T2-weighted takes place about 6–12 hours after onset of stroke. As necrosis begins to set in, there is a gradual reversal of ADC change, and around 3–10 days post-onset, ADC pseudonormalizes. Twenty-four patients of acute stroke underwent diffusion MR imaging in addition to conventional T1W, T2W, and Fluid Attenuated Inversion Recovery (FLAIR) sequence performed within 12 hours, at 30 days, and at 90 days. The mean signal intensity at b = 0 s/mm2 and at b = 1000 s/mm2 were significantly higher than control values for all time periods. The ratio of signal intensity at b = 0 (rSI b=0) significantly increased from 1.63 ± 0.20 in the acute stage to 2.19 ± 0.24 in the chronic stage (P < 0.001). The ratio of signal intensity on DWI (r SIDWI) decreased from 2.54 ± 0.46 to 1.54 ± 0.22. The mean ADC in the lesion was found to be 41% lower than the mean ADC in the contralateral hemisphere .Linear regression analysis between rADC and log hours showed that pseudonormalization occurred at 6.61 days (P < 0.001). We conclude that the above information could be useful in the management of very early stroke.
Apparent diffusion coefficient; B-value; DWI; FLAIR; ischemic stroke; signal intensity