Measurement of Giardia lamblia adhesion force using an integrated microfluidic assay.

The mechanisms how Giardias attach to the intestinal epithelium remain unclear. None of the methods currently being used to measure the attachment force could provide a continuous nutrition supply and a micro-aerobic atmosphere to the Giardia. Besides, they are all labor-intensive. In the present research, a microfluidic method based on electric circuit analogy was developed. The input fluid flowed through the inlet channel with different lengths and was distributed in four assay chambers. Shear force gradients were generated in chambers, too. This allowed an easy control of fluids and the shear forces. Most importantly, the shear stress large enough to detach Giardia could be generated in laminar flow regime. Moreover, analysis could be accomplished in one single test. By applying inlet flow rates of 30, 60, and 120 µL ml-1, shear force gradients ranging from 19.47 to 60.50 Pa were generated. The adhesion forces of trophozoites were analyzed and the EC50 of the force that caused 50% trophozoites detachment was calculated as 36.60 Pa. This paper presents a novel method for measurement of Giardia adhesion force. Graphical Abstract Measurement of Giardia adhesion force. Various of flow rates were applied to generate different shear forces and Giardia trophozoites remaining attached were counted (a-c). The percentages of attachment vs shear stress were plotted and the EC50 of adhesion force was calculated (d).

An on-chip pollutant toxicity determination based on marine microalgal swimming inhibition.

We report the use of microalgal swimming behavior as a sensor signal integrated into microfluidics for a rapid and high-throughput determination of pollutant toxicity. There are two types of chip. A poly(dimethylsiloxane) (PDMS) 12-well chip, used for optimization of experimental conditions (i.e. light level, temperature, initial cellular density and exposure time), can perform twelve parallel tests simultaneously. In a concentration gradient generator (CGG) chip, a CGG connected with diffusible chambers enables a large number of dose-response bioassays to be performed in a simple way. Microalgal swimming was set as a microfluidic bioassay signal and was evaluated as swimming manner, motile percentage (%MOT), curvilinear velocity (VCL), average path velocity (VAP) and straight line velocity (VSL). Under optimized physical conditions, the toxicities of Cu, Pb, phenol and nonylphenol (NP) towards four mobile marine microalgae, Platymonas subcordiformis, Platymonas helgolandica var. tsingtaoensis, Isochrysis galbana and Isochrysis zhanjiangensis sp. nov, were investigated. In all cases, a toxic response (i.e. a dose-related inhibition of swimming) was detected, and a time of only 2 h was needed to predict EC50 values. The 2h-EC50s showed that I. galbana was the most tolerant and that P. subcordiformis was one of the most sensitive. Based on the relative motile percentage data, the EC50 values for Cu of I. galbana and P. subcordiformis were 6.04 and 1.67 µM, respectively, while for Pb the EC50 values were 15.30 and 3.87 µM, for phenol the EC50 values were 8.69 and 6.08 mM, and for NP the EC50 values were 29.65 and 14.47 µM, respectively. Taking into account all the swimming inhibition parameters, MOT provided more sensitive EC results. The sensitivity differences between the velocity parameters (VCL, VAP and VSL) were ascribed to differences in swimming manner of the different classes of microalgae.

Giardia duodenalis GlSir2.2, homolog of SIRT1, is a nuclear-located and NAD(+)-dependent deacethylase.

Sir2 family proteins are highly conserved and catalyze Nicotinamide Adenine Dinucleotide (NAD(+))-dependent protein deacetylation reaction that regulates multiple cellular processes. Little is known about Sir2 family proteins in Giardia. In this research, Sir2 homologs of Giardia were Phylogenetically analyzed. GL50803_10707 (GlSIR2.2) showed strong homology to SIRT1 and was the only parasite SIRT1 homolog being reported to date. Recombinant GlSIR2.2 (rGlSIR2.2) was expressed and purified. The renaturied recombinant protein showed a typical NAD-dependent protein deacetylase activity that could be inhibited by nicotinamide, with IC50 of 4.47 mM rGlSIR2.2 displayed deacetylase activity under varied NAD(+), with Km, kcat and kcat/Km values of 31.71 µM, 1.4 × 10(-3) s(-1), and 4.42 × 10(-5) µM(-1) s(-1). Similarly, the steady-state kinetic parameters with varied ZMAL, yielded Km, kcat and kcat/Km values of 96.89 µM, 4.7 × 10(-3) s(-1), and 4.85 × 10(-5) µM(-1) s(-1). Anti-rGlSIR2.2 serum was used to probe subcellular localization of GlSIR2.2 and strong staining was found predominantly in the nucleus. So we demonstrated that GlSIR2.2 was a SIRT1-like, nuclear-located, NAD(+)-dependent deacetylase. This is the first report of deacetylase activity of Sir2 family protein in Giardia.

Expression and clinical significance of nestin in astrocytic tumors.

PURPOSE: This study aimed to investigate the expression and clinical significance of nestin in human astrocytic tumors. METHODS: Indirect immunofluorescent staining and flow cytometry were used to quantitatively detect the nestin content in 35 specimens, including 3 normal brain tissues, 29 astrocytic tumor (AT) tissues, and 3 peritumoral tissues. RESULTS: In normal brain tissues, nestin expression was extremely low. Nestin expression was significantly positively correlated with the histological grade of astrocytic tumors (p<0.05, rs=0.83). Nestin content in the peritumoral tissues was between the levels of nestin in tumor tissue and in normal brain tissue (p<0.01). Nestin expression was unrelated to the patient's gender, age, tumor location, size, etc. (p>0.05). CONCLUSION: The application of flow cytometry in the determination of nestin content could improve the accuracy of early cancer diagnosis. This method would be helpful for developing a reference range that is closely related to the pathological grading of ATs through routine assessments of nestin in many patients. Additionally, through examining nestin levels in peritumoral tissues, the invasiveness of ATs can be clarified.

Paper-based three-dimensional microfluidic device for monitoring of heavy metals with a camera cell phone.

A 3D paper-based microfluidic device has been developed for colorimetric determination of selected heavy metals in water samples by stacking layers of wax patterned paper and double-sided adhesive tape. It has the capability of wicking fluids and distributing microliter volumes of samples from single inlet into affrays of detection zones without external pumps, thus a range of metal assays can be simply and inexpensively performed. We demonstrate a prototype of four sample inlets for up to four heavy metal assays each, with detection limits as follows: Cu (II) = 0.29 ppm, Ni(II) = 0.33 ppm, Cd (II) = 0.19 ppm, and Cr (VI) = 0.35 ppm, which provided quantitative data that were in agreement with values gained from atomic absorption. It has the ability to identify these four metals in mixtures and is immune to interferences from either nontoxic metal ions such as Na(I) and K(I) or components found in reservoir or beach water. With the incorporation of a portable detector, a camera mobile phone, this 3D paper-based microfluidic device should be useful as a simple, rapid, and on-site screening approach of heavy metals in aquatic environments.

An integrated microfludic device for culturing and screening of Giardia lamblia.

In vitro culturing of trophozoites was important for research of Giardia lamblia (G. lamblia), especially in discovery of anti-Giardia agents. The current culture methods mainly suffer from lab-intension or the obstacle in standardizing the gas condition. Thus, it could benefit from a more streamlined and integrated approach. Microfluidics offers a way to accomplish this goal. Here we presented an integrated microfluidic device for culturing and screening of G. lamblia. The device consisted of a polydimethylsiloxane (PDMS) microchip with an aerobic culture system. In the microchip, the functionality of integrated concentration gradient generator (CGG) with micro-scale cell culture enables dose-response experiment to be performed in a simple and reagent-saving way. The diffusion-based culture chambers allowed growing G. lamblia at the in vivo like environment. It notable that the highly air permeable material of parallel chambers maintain uniform anaerobic environment in different chambers easily. Using this device, G. lamblia were successfully cultured and stressed on-chip. In all cases, a dose-related inhibitory response was detected. The application of this device for these purposes represents the first step in developing a completely integrated microfluidic platform for high-throughput screening and might be expanded to other assays based on in vitro culture of G. lamblia with further tests.

Association of ERCC1 rs3212986 & ERCC2 rs13181 polymorphisms with the risk of glioma.

OBJECTIVE: : Several previous studies have reported the role variant of ERCC1 rs3212986 and ERCC2 rs13181 polymorphisms in the risk of glioma, but the results of these studies are inconsistent. Therefore, we aimed to conduct a meta-analysis to investigate the role of ERCC1 rs3212986 and ERCC2 rs13181 on the risk of glioma. METHODS: A comprehensive research was conducted through the databases of Pubmed, EMBASE and the China National Knowledge Infrastructure (CNKI) platforms until June 1, 2014, including 14 eligible case-control studies. RESULTS: Our meta-analysis found that ERCC1 rs3212986 AA genotype was significantly associated with increased risk of glioma compared with CC genotype, and the pooled OR (95%CI) was 1.29(1.07-1.55). By subgroup analysis, ERCC1 rs3212986 AA genotype was found to be significantly correlated with increased glioma risk in Chinese population (OR=1.37, 95%CI=1.07, 1.55), Similarly, we found that ERCC2 rs13181 GT and TT genotypes were significantly associated with increased risk of glioma in Chinese population, with ORs(95%CI) of 1.47(1.17-1.85) and 1.50(1.02-2.22). But ERCC1 rs3212986 and ERCC2 rs13181 polymorphisms had no significant association with glioma risk in Caucasian populations. By begg's funnel plot, we found that no publication bias was existed in this meta-analysis. CONCLUSION: Our meta-analysis suggested that ERCC1 rs3212986 and ERCC2 rs13181 polymorphism play an important risk factor for brain tumor development in Chinese population, but no association in Caucasian populations.

[Construction of rapid tagging vector of genes in Giardia lamblia].

OBJECTIVE: To construct a recombinant vector for rapid gene tagging in Giardia lamblia. METHODS: To obtain the recombinant vector pGL gdh-Neo with the Neo selection marker, the Neo gene was put under the control of gdh promoter by overlap PCR and inserted into pGEM-5zf. A DNA fragment containing multiple cloning sites (MCS) followed by triple hemagglutinin(3HA) coding sequences was synthesized and cloned into the pGL gdh-Neo to construct a recombinant vector pGL MCS-3HA-gdh-Neo. Giardia H2A gene was selected as a tagging gene to validate the effectivity of the recombinant vector pGL MCS-3HA-gdh-Neo. The histone H2A coding sequence was amplified by PCR, digested with EcoR I and Spe I, and inserted into MCS of pGL MCS-3HA-gdh-Neo. The resulting plasmid was then linearized and transfected into Giardia trophozoites. The H2A recombinant strain selected by G418 was analyzed by PCR,Western blotting and immunofluorescence. RESULTS: A rapid tagging recombinant vector with multiple cloning sites and triple hemagglutinin (3HA) was constructed with a length of 4 260 bp. The H2A recombinant vector was transfected into Giardia trophozoites and integrate into the Giardia genome at the correct locus. The HA-tagged H2A protein was expressed with a molecular weight (Mr) of 16 900. CONCLUSION: A rapid tagging recombinant vector of genes in Giardia lamblia, pGL MCS-3HA-gdh-Neo, has been constructed.

Painful Diabetic Neuropathy Is Associated with Compromised Microglial IGF-1 Signaling Which Can Be Rescued by Green Tea Polyphenol EGCG in Mice.

Background: Painful diabetic neuropathy (PDN) is a frequent and troublesome complication of diabetes, with little effective treatment. PDN is characterized by specific spinal microglia-mediated neuroinflammation. Insulin-like growth factor 1 (IGF-1) primarily derives from microglia in the brain and serves a vital role in averting the microglial transition into the proinflammatory M1 phenotype. Given that epigallocatechin-3-gallate (EGCG) is a potent anti-inflammatory agent that can regulate IGF-1 signaling, we speculated that EGCG administration might reduce spinal microglia-related neuroinflammation and combat the development of PDN through IGF-1/IGF1R signaling. Methods: Type 1 diabetes mellitus (T1DM) was established by a single intraperitoneal (i.p.) injection of streptozotocin (STZ) in mice. The protein expression level of IGF-1, its receptor IGF1R, interleukin 1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and inducible nitric oxide synthase (iNOS) was determined by Western blot or immunofluorescence. Results: The spinal IGF-1 expression markedly decreased along with the presence of pain-like behaviors, the spinal genesis of neuroinflammation (increased IL-1ß, TNF-α, and Iba-1+ microglia), and the intensified M1 microglia polarization (increased iNOS+Iba-1+ microglia) in diabetic mice. IGF-1 could colocalize with neurons, astrocytes, and microglia, but only microglial IGF-1 was repressed in T1DM mice. Furthermore, we found that i.t. administration of mouse recombinant IGF-1 (rIGF-1) as well as i.t. or i.p. treatment with EGCG alleviated the diabetes-induced pain-like behaviors, reduced neuroinflammation (suppressed IL-1ß, TNF-α, and Iba-1+ microglia), prevented the M1 microglia polarization (less iNOS+Iba-1+ microglia), and restored the microglial IGF-1 expression. Conclusions: Our data highlighted the importance of maintaining spinal IGF-1 signaling in treating microglia-related neuroinflammation in PDN. This study also provides novel insights into the neuroprotective mechanisms of EGCG against neuropathic pain and neuroinflammation through IGF-1 signaling, indicating that this agent may be a promising treatment for PDN in the clinical setting.

Periodic mesoporous organosilicas with controlled pore symmetries for peptides enrichment.

Periodic mesoporous organosilicas (PMOs) with controlled structures have been synthesized by using cetyltrimethylammonium bromide (CTAB) and sodium perfluorooctanoate (PFONa) as co-templates, 1,2-bis (triethoxysilyl)ethane (BTEE) as an organosilica precursor. By increasing the weight ratio of PFONa/CTAB, a structure transformation from a cubic (Pm-3n) to a two-dimensional hexagonal (p6m) mesostructure and then to multilamellar vesicles can be observed. The cubic and hexagonal samples have similar particle size (200-750 nm), pore size (2.6 and 2.8 nm, respectively), total pore volume (approximately 0.7 cm3/g), and surface area (approximately 900 m2/g), providing ideal candidates to study the peptide enrichment performance influenced simply by pore symmetries. Matrix-assisted laser desorption ionization time-of-flight mass spectroscopy (MALDI-TOF MS) analysis indicates that PMO with a cubic (Pm-3n) structure is more effective in small molecular weight peptides enrichment compared with PMO with a hexagonal structure, showing the importance of mesostructural control for targeted applications. The phenomena can be attributed to the cage-type structure of the Pm-3n symmetry, which possesses cages with a relatively larger pore size and connectivity with a relatively smaller size. It is suggested that the pore entrances with small size are responsible for entrapping small molecular weight peptides. Our study may shed light on the designed synthesis of functional porous materials with controlled structures and enhanced performance in peptides enrichment.

[Cloning, expression and purification of silent information regulator 2 from Giardia lamblia].

OBJECTIVE: To clone and express silent information regulator 2 (Sir2) gene from Giardia lamblia. METHODS: The GlSir2 gene was amplified by PCR from genomic DNA of Giardia lamblia (Chinese strain C2 clone). PCR product was cloned into pMD-19T vector and transformed into E coli JM109. The recombinant plasmid was sequenced and then cloned into the pET28b vector. The pET28b-Gl1Sir2 recombinant plasmid was transformed into E. coli BL21(DE3), followed by expression of the protein induced by IPTG. The recombinant protein was analyzed by SDS-PAGE. Inclusion bodies were dissolved with 8 mol/L urea, and the supernatant was collected and applied to Ni2+ affinity chromatography. The purified recombinant protein was renatured by dialysis and verified by Western blotting using anti-His tag antibody. RESULTS: GlSir2 gene sequence was cloned. The GISir2 open reading frame (1 680 bp) encoded a 559-amino acid protein with Mr 62 800. The recombinant plasmid pET28b-GlSir2 expressed an inclusion body protein of GISir2 after being induced with IPTG. The protein purity reached above 80% after purification. The purified protein was renatured by dialysis. The recombinant GISir2 was recognized by anti-His tag antibody. CONCLUSION: The coding sequence of GLSir2 gene was cloned and expressed in vitro. The recombinant protein was identified by anti-His tag antibody.

Chemotherapy-induced peripheral neuropathy is promoted by enhanced spinal insulin-like growth factor-1 levels via astrocyte-dependent mechanisms.

BACKGROUND: Chemotherapy-induced peripheral neuropathy (CIPN) is a common and intractable complication in chemotherapy-receiving patients. Insulin-like growth factor-1 (IGF-1) is a popular neurotrophin with various functions, such as maintaining neuronal survival and synaptic functioning in the central nervous system. Therefore, we hypothesized that the IGF-1 signaling pathway could be a candidate target for treating CIPN. METHODS: We established the CIPN model by injecting mice intraperitoneally with oxaliplatin and assessed IGF-1 protein expression, its receptor IGF1R, phospho-IGF1R (p-IGF1R), interleukin-17A (IL-17A), tumor necrosis factor-α (TNF-α), and calcitonin gene-related peptide (CGRP) in the lumbar spinal cord with Western blot and immunofluorescence. To examine the effect of IGF-1 signaling on CIPN, we injected mice intrathecally or intraperitoneally with mouse recombinant IGF-1 (rIGF-1). RESULTS: IGF-1 protein expression decreased significantly in the spinal cord on D3 and D10 (the 3rd and 10th days after beginning oxaliplatin chemotherapy) and was co-localized with astrocytes primarily in the lumbar spinal cord, whereas IGF1R was predominantly expressed on neurons. Both intrathecally- and intraperitoneally-administered rIGF-1 relieved the chemotherapy-induced pain-like behavior and reduced IL-17A, TNF-α, and CGRP protein expressions in the spinal cord. CONCLUSION: Our results indicate a vital role for IGF-1 signaling in CIPN. Targeting IGF-1 signaling could be a potent therapeutic strategy for treating CIPN in clinical settings.

Abnormal Insulin-like Growth Factor 1 Signaling Regulates Neuropathic Pain by Mediating the Mechanistic Target of Rapamycin-Related Autophagy and Neuroinflammation in Mice.

Neuropathic pain is a chronic condition with little specific treatment. Insulin-like growth factor 1 (IGF1), interacting with its receptor, IGF1R, serves a vital role in neuronal and brain functions such as autophagy and neuroinflammation. Yet, the function of spinal IGF1/IGF1R in neuropathic pain is unclear. Here, we examined whether and how spinal IGF1 signaling affects pain-like behaviors in mice with chronic constriction injury (CCI) of the sciatic nerve. To corroborate the role of IGF1, we injected intrathecally IGF1R inhibitor (nvp-aew541) or anti-IGF1 neutralizing antibodies. We found that IGF1 (derived from astrocytes) in the lumbar cord increased along with the neuropathic pain induced by CCI. IGF1R was predominantly expressed on neurons. IGF1R antagonism or IGF1 neutralization attenuated pain behaviors induced by CCI, relieved mTOR-related suppression of autophagy, and mitigated neuroinflammation in the spinal cord. These findings reveal that the abnormal IGF1/IGF1R signaling contributes to neuropathic pain by exacerbating autophagy dysfunction and neuroinflammation.

Anti-HIV activities of the compounds isolated from Polygonum cuspidatum and Polygonum multiflorum.

The 70 % EtOH extract of Polygonum cuspidatum showed inhibitory action against HIV-1-induced syncytium formation at non-cytotoxic concentrations in vitro with a 50 % effective concentration (EC(50)) of 13.94 +/- 3.41 microg/mL. Through bioactivity-guided fractionation, 20 phenolic compounds, including eight stilbenoids, were isolated from the roots of Polygonum cuspidatum, and their anti-HIV-1 activities were evaluated. Results showed that compounds 1, 13, 14, and 16 demonstrated fairly strong antiviral activity against HIV-1-induced cytopathic effects in C8166 lymphocytes at non-cytotoxic concentrations, with EC (50) values of 4.37 +/- 1.96 microg/mL, 19.97 +/- 5.09, 14.4 +/- 1.34 microg/mL, and 11.29 +/- 6.26 microg/mL and therapeutic index (TI) values of 8.12, > 10.02, > 13.89, and > 17.71, respectively. Other compounds showed either weak or no effects. Compound 6 also showed weak inhibition (153.42 +/- 19.25 microg/mL); however, it possesses very good water solubility and showed almost no cytotoxicity (> 2000 microg/mL), therefore achieving a fairly good TI (13.04). The activities of the two compounds (3 and 18) from Polygonum multiflorum were also assayed. The relationship between molecular structures and their bioactivities was also discussed.

The anti-HIV activity of three 2-alkylsulfanyl-6-benzyl-3, 4-dihydropyrimidin-4 (3H)-one derivatives acting as non-nucleoside reverse transcriptase inhibitor in vitro.

It was recently shown that several new synthetic 2-alkylsulfanyl-6-benzyl-3, 4-dihydropyrimidin-4(3H)-one (S-DABO) derivatives demonstrated anti-HIV-1 activity. Three of the derivatives namely RZK-4, RZK-5 and RZK-6 were used in this study to explore their inhibitory effects on a variety of HIV strains. These compounds at a concentration of 200 microg mL(-1) almost completely inhibited the activity of recombinant HIV-1 reverse transcriptase. All of the three compounds reduced replication of HIV-1 laboratory-derived strains, low-passage clinical isolated strain, and the drug resistant strain. In particular RZK-6 showed potent activity against the HIV-1 drug resistant strain. In general, the antiviral activities are similar in magnitude to nevirapine (NVP), which is a non-nucleoside reverse transcriptase inhibitor approved by FDA. The therapeutic indexes of these compounds were remarkable, ranging from 3704 to 38462 indicating extremely low cytotoxicity. These results suggest that the three S-DABO derivatives in this study have good potential for further development in anti-HIV-1 therapy. It may be particularly useful to target at the non-nucleoside reverse transcriptase inhibitors resistant HIV-1 strain.

Sifuvirtide, a potent HIV fusion inhibitor peptide.

Enfuvirtide (ENF) is currently the only FDA approved HIV fusion inhibitor in clinical use. Searching for more drugs in this category with higher efficacy and lower toxicity seems to be a logical next step. In line with this objective, a synthetic peptide with 36 amino acid residues, called Sifuvirtide (SFT), was designed based on the crystal structure of gp41. In this study, we show that SFT is a potent anti-HIV agent with relatively low cytotoxicity. SFT was found to inhibit replication of all tested HIV strains. The effective concentrations that inhibited 50% viral replication (EC(50)), as determined in all tested strains, were either comparable or lower than benchmark values derived from well-known anti-HIV drugs like ENF or AZT, while the cytotoxic concentrations causing 50% cell death (CC(50)) were relatively high, rendering it an ideal anti-HIV agent. A GST-pull down assay was performed to confirm that SFT is a fusion inhibitor. Furthermore, the activity of SFT on other targets in the HIV life cycle was also investigated, and all assays showed negative results. To further understand the mechanism of action of HIV peptide inhibitors, resistant variants of HIV-1(IIIB) were derived by serial virus passage in the presence of increasing doses of SFT or ENF. The results showed that there was cross-resistance between SFT and ENF. In conclusion, SFT is an ideal anti-HIV agent with high potency and low cytotoxicity, but may exhibit a certain extent of cross-resistance with ENF.

Determination of the chemical components and phospholipids of velvet antler using UPLC/QTOF-MS coupled with UNIFI software.

Velvet antler, which exhibits immune and growth enhancing effects, is commonly used in a variety of Asian health care products, but its complex components remain unknown. The current study analyzed extracts using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry in the MSE mode. Automated detection and data filtering were performed using UNIFI software and peaks were compared with a proprietary scientific library (Traditional Medicine Library; TML). The results obtained using different data processing parameters (including 3D peak detection, target by mass and fragment identification) were evaluated against 87 compounds comprising 1 lignan, 30 terpenoids (including 20 triterpenes), 39 steroids, 8 alkaloids, 4 organic acids and 5 esters in the TML. Using a screening method with a mass accuracy cutoff of ±2 mDa, a retention time cutoff of ±0.2 min, a minimum response threshold of 1,000 counts and an average of 10 false detects per sample analysis, 16 phospholipids were identified in the extracts of velvet antler, three of which were quantified. The results demonstrated that there was 1.07±0.02 µg/g of 1-myristoyl-sn-glycero-3-phosphocholine, 7.05±0.52 ng/g of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 18.81±0.55 ng/g of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine in velvet antler. The current study successfully identified certain components of velvet antler. Furthermore, the results may provide an experimental basis for further pharmacological and clinical study.

Efficient synthesis and utilization of phenyl-substituted heteroaromatic carboxylic acids as aryl diketo acid isosteres in the design of novel HIV-1 integrase inhibitors.

Three new types of aryl diketo acid (ADK) isosteres were designed by conversion of the biologically labile 1,3-diketo unit into heteroaromatic motif such as isoxazole, isothiazole, or 1H-pyrazole to improve the physicochemical property of ADK-based HIV-1 integrase (IN) inhibitors. The synthesis of the heteroaromatic carboxylic acids was established by employing phenyl beta-diketoester or benzaldehyde as the starting material and 1,3-dipolar cycloaddition as the key reaction. Of the compounds tested, the 3-benzyloxyphenyl-substituted isoxazole carboxylic acid displayed the best IN inhibitory and antiviral activities, with N-hydroxylamidation enhancing the in vitro and in vivo potency. These findings are important for further optimization of ADK-based IN inhibitors.

Anti-HIV-1 activities of compounds isolated from the medicinal plant Rhus chinensis.

AIM OF THE STUDY: Previously, we reported that the petroleum ether fraction, RC-1, and EtOAc fraction, RC-2, of the medicinal plant Rhus chinensis showed potent anti-HIV-1 activities. To address anti-HIV-1 constituents of RC-1 and RC-2, 17 compounds were isolated. Anti-HIV-1 activities and possible action mechanisms of these compounds were investigated. METHODS: The syncytial formation induced by HIV-1 was determined under the inverted microscope, cellular toxicity and protection assay were assessed by MTT method, reduction of p24 antigen expression level and RT activity were measured by ELISA, and inhibition of recombinant HIV-1 PR was monitored by the fluorescent signal. RESULTS: The compounds 1 and 13 inhibited HIV-1-induced syncytium formation potently with TI value of 42.31 and 19.07, respectively. Compounds 4, 5, 6, 9 and 10 were less potent with TI value of 8.94, 8.22, 4.14, 5.11 and 5.34, respectively. Compound 1, a benzofuranone-type compound, previously reported as a novel anti-HIV-1 agent, might target late-steps of HIV-1 life cycle. Compound 13 inhibited HIV-1 replication with EC(50) of 7.16mug/ml and might target at/before integration step. CONCLUSION: These compounds might contribute to anti-HIV-1 activities extracts of the medicinal plant Rhus chinensis.

Synthesis of analogues of flazin, in particular, flazinamide, as promising anti-HIV agents.

Flazin isolated from the fruiting bodies of Suillus granulatus was found to possess weak anti-HIV activity (EC(50)=2.36 microM, TI=12.1). To establish a SAR study, 46 flazin analogues were synthesized, and their anti-HIV activities were evaluated in vitro. Among them, flazinamide (9a) showed the most potent activity with an EC(50) value of 0.38 microM and a TI value of 312.0. The results suggested that appropriate substituents at positions 3, 1', and 5' of flazin might play a crucial role in determining their anti-HIV activities, and that flazinamide can be considered as a promising, readily available anti-HIV agent.