Synthesis and antitumor activity of α,ß-unsaturated carbonyl moiety- containing oleanolic acid derivatives targeting PI3K/AKT/mTOR signaling pathway.

Oleanolic acid (OA) and its semi-synthetic derivatives have been reported to have a wide range of biological activities. The introduction of electrophilic Michael acceptor group can increase the reactivity of OA to cellular targets and thus improve the anti-tumor activity. In this work, a series of novel α,ß-unsaturated carbonyl derivatives of OA were designed and synthesized. Their in vitro cytotoxic activity against MCF-7, HepG2 and HeLa cells were tested. Most derivatives exhibited improved cell growth inhibitory activity, especially for 3d with an IC50 of 0.77 µM in MCF-7 cells. Moreover, 3d inhibited the migration of MCF-7 and HeLa cells at the concentration of 4 µM. Flow cytometric analysis revealed that 3d induced cell apoptosis and S phase arrest in a concentration-dependent manner. Western blotting experiment demonstrated that 3d inhibited the phosphorylation of AKT and mTOR. These results suggest that this series of OA derivatives bearing exocyclic methylene ketone pharmacophore are promising anticancer agents as potential PI3K/AKT/mTOR pathway inhibitors.

Dissimilarity in the Contributions of the N-Terminal Domain Hydrophobic Core to the Structural Stability of Lens ß/γ-Crystallins.

Vertebrate lens ß/γ-crystallins share a conserved tertiary structure consisting of four Greek-key motifs divided into two globular domains. Numerous inherited mutations in ß/γ-crystallins have been linked to cataractogenesis. In this research, the folding mechanism underlying cataracts caused by the I21N mutation in ßB2 was investigated by comparing the effect of mutagenesis on the structural features and stability of four ß/γ-crystallins, ßB1, ßB2, γC, and γD. Our results showed that the four ß/γ-crystallins differ greatly in solubility and stability against various stresses. The I21N mutation greatly impaired ßB2 solubility and native structure as well as its stability against denaturation induced by guanidine hydrochloride, heat treatment, and ultraviolet irradiation. However, the deleterious effects were much weaker for mutations at the corresponding sites in ßB1, γC, and γD. Molecular dynamics simulations indicated that the introduction of a nonnative hydrogen bond contributed to twisting Greek-key motif I outward, which might direct the misfolding of the I21N mutant of ßB2. Meanwhile, partial hydration of the hydrophobic interior of the domain induced by the mutation destabilized ßB1, γC, and γD. Our findings highlight the importance of nonnative hydrogen bond formation and hydrophobic core hydration in crystallin misfolding caused by inherited mutations.

Orobanone analogues from acid-promoted aromatization rearrangement of curcumol inhibit hypoxia-inducible factor-1 (HIF-1) in cell-based reporter assays.

In this paper, the mechanism of orobanone analogues formation via aromatization rearrangement of curcumol was minutely explored. Aromatization of curcumol with acetone under acidic condition was selected as the model reaction. The formation of a stable aromatic system was the driving force for this reaction. Based on the model reaction, other four new orobanone analogues were prepared through curcumol reacting with different carbonyl compounds. The results showed that the stability of carbocation, which was generated from the carbonyl compounds, and the steric hindrance were main factors affecting the aromatization. We also synthesized the analogue of aromaticane B using compound 2. In vitro anti-proliferative activity of some derivatives were tested by MTT assay. Two derivatives showed weak anti-tumor effect on two cancer cell lines (HepG2 and MCF7) under normoxia. Four orobanone analogue 2, 5, 6 and 9 significantly inhibited hypoxia-induced HIF-1 luciferase reporter activity in HeLa cells with the IC50 values of 13.6, 6.6, 2.4 and 18.2 µM, respectively.

Introduction of an extra tryptophan fluorophore by cataract-associating mutations destabilizes ßB2-crystallin and promotes aggregation.

ß/γ-Crystallins are predominant structural proteins in vertebrate lens with unique properties of extremely high solubility, long-term stability and resistance to UV damage. Four conserved Trp residues in ß/γ-crystallins account for UV absorbance and thereafter fluorescence quenching to avoid photodamage. Herein we found that ßB2-crystallin Trp fluorescence was greatly enhanced by the introduction of an extra unquenched Trp fluorophore by cataract-associated mutations S31W and R145W. Both mutations impaired oligomerization, decreased stability and promote thermal aggregation, while S31W was more deleterious. S31W accelerated ßB2-crystallin aggregation under UV damaging conditions, whereas R145W delayed. These observations suggested that the introduction of an extra Trp fluorophore had complicated effects on ßB2-crystallin stability and aggregation against various stresses. Our findings highlight that the number of Trp fluorophores in ß/γ-crystallin is evolutionarily optimized to exquisitely perform their structural roles in the lens.

The importance of the last strand at the C-terminus in ßB2-crystallin stability and assembly.

Congenital cataract is the leading cause of childhood blindness worldwide. Investigations of the effects of inherited mutations on protein structure and function not only help us to understand the molecular mechanisms underlying congenital hereditary cataract, but also facilitate the study of complicated cataract and non-lens abnormities caused by lens-specific genes. In this research, we studied the effects of the V187M, V187E and R188H mutations on ßB2-crystallin structure and stability using a combination of biophysical, cellular and molecular dynamic simulation analysis. Both V187 and R188 are located at the last strand of ßB2-crystallin Greek-key motif 4. All of the three mutations promoted ßB2-crystallin aggregation in vitro and at the cellular level. These three mutations affected ßB2-crystallin quite differentially: V187M influenced the hydrophobic core of the C-terminal domain, V187E was a Greek-key motif breaker with the disruption of the backbone H-bonding network, while R188H perturbed the dynamic oligomeric equilibrium by dissociating the dimer and stabilizing the tetramer. Our results highlighted the importance of the last strand in the structural integrity, folding, assembly and stability of ß-crystallins. More importantly, we proposed that the perturbation of the dynamic equilibrium between ß-crystallin oligomers was an important mechanism of congenital hereditary cataract. The selective stabilization of one specific high-order oligomer by mutations might also be deleterious to the stability and folding of the ß-crystalllin homomers and heteromers. The long-term structural stability and functional maintenance of ß-crystallins are achieved by the precisely regulated oligomeric equilibrium.

Cataract-causing mutation R233H affects the stabilities of ßB1- and ßA3/ßB1-crystallins with different pH-dependence.

Disease-causing mutations can be stabilizing or destabilizing. Missense mutations of structural residues are generally destabilizing, while stabilizing mutations are usually linked to alterations in protein functions. Stabilizing mutations are rarely identified in mutations linked to congenital cataract, a disease caused by the opacification of the lens. In this research, we found that R233H mutation had little impact on ßB1-crystallin structure, solubility and thermal stability under neutral solution pH conditions. The mutation increased ßB1 stability against guanidine hydrochloride-induced denaturation, suggesting that Arg233 might be a functional residue. Further analysis indicated that the R233H mutation did not affect the formation of ßA3/ßB1 heteromer, but significantly reduced heteromer stability against heat- and guanidine hydrochloride-induced denaturation. The R233H mutation negatively affected the thermal stabilities and aggregatory propensities of ßB1 and ßA3/ßB1 with different pH-dependence, implying that the protonation of His side chains during acidification played a regulatory role in crystallin stability and aggregation. Molecular dynamic simulations indicated that Arg233 is one of the residues forming an inter-subunit ion-pairing network with intrinsically dynamic nature. Based on these observations, we proposed that the highly dynamic ion-pairing network contributed to the tradeoff among ßB1 solubility, stability, aggregatory propensity and function of protecting ßA3.

Increasing ßB1-crystallin sensitivity to proteolysis caused by the congenital cataract-microcornea syndrome mutation S129R.

Congenital hereditary cataract, which is mainly caused by the deposition of crystallins in light-scattering particles, is one of the leading causes of newborn blindness in human beings. Recently, an autosomal dominant congenital cataract-microcornea syndrome in a Chinese family has been associated with the S129R mutation in ßB1-crystallin. To investigate the underlying molecular mechanism, we examined the effect of the mutation on ßB1-crystallin structure and thermal stability. Biophysical experiments indicated that the mutation impaired the oligomerization of ßB1-crystallin and shifted the dimer-monomer equilibrium to monomer. Molecular dynamic simulations revealed that the mutation altered the hydrogen-bonding network and hydrophobic interactions in the subunit interface of the dimeric protein, which resulted in the opening of the tightly associated interacting sites to allow the infiltration of the solvent molecules into the interface. Despite the disruption of ßB1-crystallin assembly, the thermal stability of ßB1-crystallin was increased by the mutation accompanied by the reduction of thermal aggregation at high temperatures. Further analysis indicated that the mutation significantly increased the sensitivity of ßB1-crystallin to trypsin hydrolysis. The digested fragments of the mutant were prone to aggregate and unable to protect ßA3-crystallin against aggregation. These results indicated that the thermal stability-beneficial mutation S129R in ßB1-crystallin provided an excellent model for discovering molecular mechanisms apart from solubility and stability. Our results also highlighted that the increased sensitivity of mutated crystallins towards proteases might play a crucial role in the pathogenesis of congenital hereditary cataract and associated syndrome.

[Study on antithrombotic activities and structure-activity relationships of Veratrum nigrum Alkaloids].

OBJECTIVE: To explore antithrombotic effects and mechanisms of total alkaloids (VnA) and single alkaloids (Al - A6) from Veratrum nigrum. METHODS: The effects of VnA and Al - A6 on thrombosis and coagulation parameters (APTT, PT and TT) were examined in rat in vivo, while their effects on platelet aggregation induced by ADP and thrombin were determined in rabbit in vitro. RESULTS: VnA and Al ~ A5 decreased thrombus wet weight and platelet aggregation, increased TT, while they had no influence on APTT or PT. A6 had no obvious effect on thrombus wet weight, platelet aggregation or coagulation parameters. CONCLUSIONS: VnA and Al ~ A5 have antithrombotic activities on venous thrombosis. The effective component in VnA is A2 probably. The antithrombotic effects are possibly related to the inhibition of platelet aggregation and the extension of TT. The cevanine type skeleton of steroidal alkaloids is necessary for antithrombotic activities and the ester substitute at C3 can enhance antithrombotic activities.

Hyperinsulinemia impairs decidualization via AKT-NR4A1 signaling: new insight into polycystic ovary syndrome (PCOS)-related infertility.

BACKGROUND: Investigating the underlying molecular mechanisms responsible for endometrial dysfunction in women with PCOS is essential, particularly focusing on the role of hyperinsulinemia. METHODS: We explored the role of insulin in the decidualization process using a synthetic decidualization assay. To dissect the effects of PI3K/AKT-NR4A signaling, we employed small interfering RNAs (siRNAs) targeting the NR4A genes and inhibitors of the PI3K/AKT pathway. We also investigated the disruption of AKT-NR4A1 signaling in the endometrium of PCOS female rats induced with dehydroepiandrosterone (DHEA). Quantitative real-time PCR (qRT-PCR) and Western blot (WB) analyses were utilized to evaluate gene expression regulation. RESULTS: Insulin was found to suppress the expression of decidualization markers in human endometrial stromal cells (hESC) in a dose-dependent manner, concurrently triggering an inappropriate activation of the PI3K/AKT pathway. Members of the NR4A family, as downstream effectors in the PI3K/AKT pathway, were implicated in the insulin-induced disruptions during the decidualization process. Moreover, the endometrium of PCOS models showed significantly elevated levels of phosphorylated (Ser473) AKT, with a corresponding reduction in Nr4a1 protein. CONCLUSIONS: Our research demonstrates that insulin negatively regulates decidualization in hESC via the PI3K/AKT-NR4A pathway. In vivo analysis revealed a significant dysregulation of the AKT-NR4A1 pathway in the endometrium of PCOS rats. These findings offer novel insights into the pathogenesis of infertility and endometrial disorders associated with hyperinsulinemia in PCOS.

[Photochemotherapy with psoralen and ultraviolet A induced apoptosis of NB4 cells and its effects on caspase-8 and caspase-8 protein expressions].

OBJECTIVE: To study the regulatory effects of psoralen (PSO) plus ultraviolet A (UVA), which is PUVA, on cell apoptosis of human leukemia cell line NB4 and signal pathway of cell apoptosis. METHODS: Human leukemia cell line NB4 was cultured in vitro. The NB4 cells were treated with PSO extracted from Chinese medicine psoralea fruits at different concentrations (0, 5, 10, 20 and 40 microL) plus UVA of wave length 360 nm at different irradiation time points (0 and 5 min). The apoptosis ratio was detected by flow cytometry (FCM). The ultrastructure changes were observed using transmission electron microscope (TEM). The expressions of Caspase-8 and Caspase-8 protein were detected by immunocytochemical method (ICC). RESULTS: After treatment of PSO at different concentrations with a 0 and 5-min exposure of UVA, the apoptosis rate of NB4 cells increased dose-and time-dependently, and was up to peak after treatment of PSO at 40 microg/mL with 5-min exposure of UVA. An interaction was shown between the two factors (P <0. 01). There were obvious morphological apoptosis of NB4 cells under TEM after treated with PUVA. The expressions of Caspase-3 and Caspase-8 protein were up-regulated by PSO, UVA, and PUVA, but the effects of PUVA on Caspase-3 protein were stronger than PSO and UVA at 12 h time-dependently (P <0.01).An interaction was shown between the concentration of PSO and time of UVA (P <0.01). CONCLUSIONS: The optimal combination of PUVA was PSO in 40 microg/mL and 5-min exposure of UVA. PUVA could induce the apoptosis of NB4 cells and in vitro activate Caspase-3 and Caspase-8 genes.

[Quantitative models for Baicalin content using NIR technology for the study of Shang Jie plaster].

A dynamic prediction model for the content of Baicalin in Shang Jie plasters extract solutions was developed using near-infrared spectroscopy in transmission mode. Sixty five spectra were obtained through near-infrared transmission mode during extracting process. Refering to the content of Baicalin performed by reversed-phase high performance liquid chromatography (HPLC), the calibration model was developed with the application of partial least squares regression algorithm (PLSR). The constructed model was validated by 30 samples; some parameters of the calibration model were optimized by cross-validation. The root mean square error (RMSECV) of Baicalin was 0.006 8 mg x g(-1), the correlation coefficient (R) was 0.9991, and the optimal dimension factor was 8; After predicted by test set, the root mean square error (RMSEP) and correlation coefficient (R) of prediction obtained were 0.009 2 mg x g(-1) and 0.998 7 respectively. This work demonstrated that NIR spectroscopy combined with PLS could be used for the determination of Baicalin in Shang Jie plasters extract.

In Situ Forming of Nitric Oxide and Electric Stimulus for Nerve Therapy by Wireless Chargeable Gold Yarn-Dynamos.

Endogenous signals, namely nitric oxide (NO) and electrons, play a crucial role in regulating cell fate as well as the vascular and neuronal systems. Unfortunately, utilizing NO and electrical stimulation in clinical settings can be challenging due to NO's short half-life and the invasive electrodes required for electrical stimulation. Additionally, there is a lack of tools to spatiotemporally control gas release and electrical stimulation. To address these issues, an "electromagnetic messenger" approach that employs on-demand high-frequency magnetic field (HFMF) to trigger NO release and electrical stimulation for restoring brain function in cases of traumatic brain injury is introduced. The system comprises a NO donor (poly(S-nitrosoglutathione), pGSNO)-conjugated on a gold yarn-dynamos (GY) and embedded in an implantable silk in a microneedle. When subjected to HFMF, conductive GY induces eddy currents that stimulate the release of NO from pGSNO. This process significantly enhances neural stem cell (NSC) synapses' differentiation and growth. The combined strategy of using NO and electrical stimulation to inhibit inflammation, angiogenesis, and neuronal interrogation in traumatic brain injury is demonstrated in vivo.

A novel mutation impairing the tertiary structure and stability of γC-crystallin (CRYGC) leads to cataract formation in humans and zebrafish lens.

Congenital cataract is one of the leading causes of human blindness. In this study, we identified a novel, heterozygous c.385G

[A preliminary study on immune adjunctive therapy for multidrug-resistant Mycobacterium tuberculosis infection in animal models].

OBJECTIVE: To investigate the effect of adjunctive therapy by immune agents in mice infected with multidrug-resistant tuberculosis (MDR-TB). METHODS: Sixty-eight adult male BALB/c mice were infected with multidrug-resistant Mycobacterium tuberculosis (MTB) by aerosol route. The mice were randomly divided into a control group, an immuno-treatment group, a drug treatment group and a combination treatment group (drug plus immuno-treatment). In each treatment group, 16 mice were treated at day 21 after infection, and another 4 mice were sacrificed at day 21 after infection (treatment for 0 week) as the blank control group. In the treatment group, 4 mice were sacrificed in turn at the day after treatment for 4, 8, 16 and 20 weeks. Lung and spleen mass index at the day after treatment for 8, 16 and 20 weeks, lung and spleen live bacterial count in each period, serum IFN-γ and IL-10 levels at the day after treatment for 8 weeks were measured. Comparisons of analyzed parameters among groups were performed with the one way ANOVA test, and comparisons of parameters between 2 groups were performed with SNK and Games-Howell test. RESULTS: The lung mass index in the immuno-treatment group (0.66 ± 0.09)%, drug group (0.60 ± 0.07)% and combination therapy group (0.57 ± 0.05)% at the day after treatment for 8 weeks were significantly lower than that of the control group (0.81 ± 0.09)%, (F = 7.364, P < 0.01). Spleen CFU of immuno-treatment group at 16 and 20 weeks [(3.11 ± 0.14) lg CFU and (3.15 ± 0.18) lg CFU] were significantly lower than those of the control group [(3.77 ± 0.35) lg CFU and (4.31 ± 0.06)] (F values were 446.424 and 2107.689, P < 0.01). Spleen tissues of the drug group and the combination therapy group were sterile from 4 weeks. The serum IFN-γ levels of immuno-treatment group, drug group and combination therapy were (5.3 ± 1.9) ng/L, (1.3 ± 0.5) ng/L and (0.9 ± 1.3) ng/L, respectively, being significantly lower than that of the control group (10.3 ± 2.1) ng/L (F = 32.128, P < 0.01). The lung and spleen mass index, lung and spleen CFU, serum IFN-γ and IL-10 between medication group and combination therapy showed no significant differences. CONCLUSIONS: Immuno-treatment could mitigate lung tissue inflammation, reduce the number of MTB in mouse spleen tissues and decrease serum IFN-γ levels in the MDR-TB mouse model. However immuno-treatment failed to reduce the number of MTB in lung tissues. There was no adjunctive effect of immuno treatment for MDR-TB mice in reducing the number of MTB and mitigating inflammation.

RhoB Promotes Endometrial Stromal Cells Decidualization Via Semaphorin3A/PlexinA4 Signaling in Early Pregnancy.

Endometrial decidualization refers to a series of morphological changes and functional remodeling of the uterine endometrium to accept the embryo under the effect of estrogen and progesterone secreted by ovaries after ovulation. During decidualization, endometrial stromal cells (ESCs) proliferate and differentiate into decidual stromal cells, undergoing cytoskeletal rearrangement-mediated morphological changes and expressing decidualization markers, such as insulin-like growth factor-binding protein-1 and prolactin. Ras homology (Rho) proteins, a family of small G proteins, are well known as regulators of cellular morphology and involved in multiple other cellular processes. In this study, we found ras homolog family member B (RHOB) was the most significantly upregulated gene in the Rho protein family after the in vitro decidualization of human primary ESCs. RhoB expression was induced mainly by 3',5'-cyclic adenosine 5'-monophosphate (cAMP) / protein kinase A (PKA) / cyclic adenosine monophosphate-response element binding protein signaling and partly by progesterone signaling. Knockdown of RhoB in ESCs greatly inhibited actin cytoskeletal rearrangement, cell morphological transformation, and upregulation of insulin-like growth factor-binding protein-1, suggesting an indispensable role of RhoB in decidualization. Mechanistically, the downstream target of RhoB was semaphorin3A (Sema3A), which mediated its signaling via interacting with the receptor, plexinA4. More importantly, decreased expression of RhoB, Sema3A, and plexinA4 were detected in deciduas from patients with unexplained spontaneous miscarriage. Collectively, our results indicate that RhoB/Sema3A/plexinA4 signaling plays a positive role in endometrial decidualization and relates to unexplained spontaneous miscarriage, which is worthy of further exploration so as to provide new insights into therapeutic strategies for pregnancy diseases associated with poor decidualization.

One-Step Exfoliation Method for Plasmonic Activation of Large-Area 2D Crystals.

Advanced exfoliation techniques are crucial for exploring the intrinsic properties and applications of 2D materials. Though the recently discovered Au-enhanced exfoliation technique provides an effective strategy for the preparation of large-scale 2D crystals, the high cost of gold hinders this method from being widely adopted in industrial applications. In addition, direct Au contact could significantly quench photoluminescence (PL) emission in 2D semiconductors. It is therefore crucial to find alternative metals that can replace gold to achieve efficient exfoliation of 2D materials. Here, the authors present a one-step Ag-assisted method that can efficiently exfoliate many large-area 2D monolayers, where the yield ratio is comparable to Au-enhanced exfoliation method. Differing from Au film, however, the surface roughness of as-prepared Ag films on SiO2 /Si substrate is much higher, which facilitates the generation of surface plasmons resulting from the nanostructures formed on the rough Ag surface. More interestingly, the strong coupling between 2D semiconductor crystals (e.g., MoS2 , MoSe2 ) and Ag film leads to a unique PL enhancement that has not been observed in other mechanical exfoliation techniques, which can be mainly attributed to enhanced light-matter interaction as a result of extended propagation of surface plasmonic polariton (SPP). This work provides a lower-cost and universal Ag-assisted exfoliation method, while at the same time offering enhanced SPP-matter interactions.

[A study on the activity of clofazimine with antituberculous drugs against Mycobacterium tuberculosis].

OBJECTIVE: To explore the role of clofazimine in combination with other antituberculous drugs against Mycobacterium tuberculosis. METHODS: The minimal inhibitory concentration (MIC) of each drug and the drugs in combination against M. tuberculosis H37Rv were determined, and the synergetic activities according to the fractional inhibitory concentration (FIC) formula was calculated. Female BALB/C mice were infected intravenously with 0.2 mg/L portions containing 5 × 105 CFU of the MDR clinical isolate 2931. One day after the infection, 6 mice were sacrificed and the numbers of CFU in the lungs and spleens were determined. The remaining mice were allocated either to untreated group or to drug-treated groups (6 mice per group). Thirty days post-infection, both untreated and treated mice were sacrificed and the CFU counted. RESULTS: Clofazimine in combination with the 10 antituberculosis drugs resulted in two- to eight fold reduction in MIC. The FIC of clofazimine (Cfz) in combination with p-aminosalicylic acid (PAS), protionamide (Pto), clarithromycin (Clr), and capreomycin were less than 0.5, suggesting a synergic interaction against M. tuberculosis H37Rv. All the drug treatments reduced the numbers of CFU in the lungs and spleens compared with the numbers in the untreated controls. Of the individual drugs, Cfz was the most effective on day 30, with a mean CFU count decrease of 1.82 and 2.32 lg10, in comparison with that of the untreated control. The Cfz-Clr (clarithromycin) combination showed significantly greater activity than the control or Clr alone, with a mean CFU count decrease of 1.30 and 1.91 lg, in comparison with that of the Clr treatment. Cfz-Clr was slightly more active than Cfz alone, but not at a statistically significant level. CONCLUSIONS: The activity of clofazimine was enhanced by the addition of clarithromycin. These observations are important for the therapy of multidrug-resistant tuberculosis.

[Tissue distribution and deposition of clofazimine in mice following oral administration of isoniazid].

OBJECTIVE: Tissue distribution and deposition of clofazimine in mice were investigated following administration of clofazimine with or without isoniazid. METHODS: Kunming mice were given clofazimine suspension orally at a daily dose of 13 mg/kg body weight either alone or with isoniazid (25 mg/kg body weight) for a single dose or for 1 or 2 months. Tissues (liver, lung, spleen, small intestine, kidneys, fat) and pooled plasma were analyzed for clofazimine in all the treated groups by high-performance liquid chromatography. RESULTS: The levels of clofazimine in fat tissues, kidneys, spleens, livers, lungs and small intestine were the highest in mice receiving the drug continuously for 2 months, and were also higher in mice receiving the drug for 1 month as compared to mice receiving a single dose administration. After continuous administration for 1 or 2 months, the clofazimine level was the highest in fat tissues as compared to other tissue samples. The clofazimine level in the lungs was higher in mice receiving concomitant administration of isoniazid [1 month (57 +/- 11) microg/g, 2 months (73 +/- 49) microg/g]than in those receiving clofazimine alone [1 month (32 +/- 8) microg/g, 2 months (47 +/- 12) microg/g], but the clofazimine level in the fat tissue was significantly lower in mice receiving concomitant isoniazid [1 month (289 +/- 30) microg/g, 2 months (275 +/- 119) microg/g], than in those receiving clofazimine alone [1 month (433 +/- 53) microg/g, 2 months (527 +/- 158) microg/g]. CONCLUSION: Concomitant administration of isoniazid reduced clofazimine levels significantly in fat tissues while resulted in an increase of its level in lung tissues.

Activities of Biapenem against Mycobacterium tuberculosis in Macrophages and Mice.

OBJECTIVE: To assess the activities of biapenem against multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis. METHODS: Biapenem/clavulanate (BP/CL) was evaluated for in vitro activity against Mycobacterium tuberculosis (Mtb) multidrug-resistant (MDR) isolates, extensively drug-resistant (XDR) isolates, and the H37RV strain. BP/CL activity against the H37Rv strain was assessed in liquid cultures, in macrophages, and in mice.. RESULTS: BP/CL exhibited activity against MDR and XDR Mtb isolates in liquid cultures. BP/CL treatment significantly reduced the number of colony forming units (CFU) of Mtb within macrophages compared with control untreated infected macrophages. Notably, BP/CL synergized in pairwise combinations with protionamide, aminosalicylate, and capreomycin to achieve a fractional inhibitory concentration for each pairing of 0.375 in vitro. In a mouse tuberculosis infection model, the efficacy of a cocktail of levofloxacin + pyrazinamide + protionamide + aminosalicylate against Mtb increased when the cocktail was combined with BP/CL, achieving efficacy similar to that of the positive control treatment (isoniazid + rifampin + pyrazinamide) after 2 months of treatment. CONCLUSION: BP/CL may provide a new option to clinically treat MDR tuberculosis.

Steroidal dimer by001 inhibits proliferation and migration of esophageal cancer cells via multiple mechanisms.

OBJECTIVE: To investigate the potential inhibitory effects of structurally novel steroidal dimer by001 in esophageal cancer in vitro. METHODS: The cytotoxicity of by001 on esophageal, gastric, neuroblastoma and prostate cancer cells was examined MTT assay and colony formation assay. By001 induced apoptosis and production of intracellular reactive oxygen species on esophageal cancer cells Ec109, TE-1 and human normal gastric epithelial cells GES-1 was detected by flow cytometry. The effect of by001 on mitochondrial membrane potential was detected by fluorescence microscope through JC-1 staining. The level of intracellular reactive oxygen species was measured by fluorescence microscope and flow cytometry via DCFH-DA staining. The effect of by001 on members of Bcl-2 family, Fas, LC3, PARP and caspases was determined by Western blot. The effect of by001 on migration was measured by transwell assay. RESULTS: By001 effectively inhibited proliferation of esophageal, gastric, neuroblastoma and prostate cancer cells in a time- and concentration-dependent manner in vitro. By001 reduced the number and the size of colonies at low micromolar concentrations, elevated cellular ROS levels and caused mitochondrial dysfunction in esophageal cancer cells. Molecular mechanistic studies showed that by001 triggered apoptosis through regulating members of Bcl-2 family and Fas. CONCLUSIONS: These findings suggested that by001 may inhibited proliferation of esophageal cancer cells through mitochondria and death receptor-mediated apoptotic pathways, autophagy induction, as well as suppressed migration of esophageal cancer cells.