New and effective EGFR-targeted fluorescence imaging technology for intraoperative rapid determination of lung cancer in freshly isolated tissue.

PURPOSE: During lung cancer surgery, it is very important to define tumor boundaries and determine the surgical margin distance. In previous research, systemically application of fluorescent probes can help medical professionals determine the boundaries of tumors and find small tumors and metastases, thereby improving the accuracy of surgical resection. However, there are very few safe and effective probes that can be applied to clinical trials up to now, which limits the clinical application of fluorescence imaging. Here we developed a new technology that can quickly identify the tumor area in the resected lung tissue during the operation and distinguish the tumor boundary and metastatic lymph nodes. EXPERIMENTAL DESIGN: For animal studies, a PDX model of lung cancer was established. The tumors, lungs, and peritumoral muscle tissues of tumor-bearing mice were surgically removed and incubated with a probe targeting epidermal growth factor receptor (EGFR) for 20 min, and then imaged by a closed-field near-infrared two-zone (NIR-II) fluorescence imaging system. For clinical samples, ten surgically removed lung tissues and 60 lymph nodes from 10 lung cancer patients undergoing radical resection were incubated with the targeting probe immediately after intraoperative resection and imaged to identify the tumor area and distinguish the tumor boundary and metastatic lymph nodes. The accuracy of fluorescence imaging was confirmed by HE staining and immunohistochemistry. RESULTS: The ex vivo animal imaging experiments showed a fluorescence enhancement of tumor tissue. For clinical samples, our results showed that this new technology yielded more than 85.7% sensitivity and 100% specificity in identifying the tumor area in the resected lung tissue. The average fluorescence tumor-to-background ratio was 2.5 ± 1.3. Furthermore, we also used this technique to image metastatic lymph nodes intraoperatively and showed that metastatic lymph nodes have brighter fluorescence than normal lymph nodes, as the average fluorescence tumor-to-background signal ratio was 2.7 ± 1.1. Calculations on the results of the fluorescence signal in relation to the number of metastatic lymph nodes yielded values of 77.8% for sensitivity and 92.1% for specificity. We expect this new technology to be a useful diagnostic tool for rapid intraoperative pathological detection and margin determination. CONCLUSIONS: By using fluorescently labeled anti-human EGFR recombinant antibody scFv fragment to incubate freshly isolated tissues during surgery, the probes can quickly accumulate in lung cancer tissues, which can be used to quickly identify tumor areas in the resected lung tissues and distinguish tumor boundaries and find metastases in lymph nodes. This technology is expected to be used for rapid intraoperative pathological detection and margin determination.

Novel multifunctional NIR-II aggregation-induced emission nanoparticles-assisted intraoperative identification and elimination of residual tumor.

Incomplete tumor resection is the direct cause of the tumor recurrence and metastasis after surgery. Intraoperative accurate detection and elimination of microscopic residual cancer improve surgery outcomes. In this study, a powerful D1-π-A-D2-R type phototheranostic based on aggregation-induced emission (AIE)-active the second near-infrared window (NIR-II) fluorophore is designed and constructed. The prepared theranostic agent, A1 nanoparticles (NPs), simultaneously shows high absolute quantum yield (1.23%), excellent photothermal conversion efficiency (55.3%), high molar absorption coefficient and moderate singlet oxygen generation performance. In vivo experiments indicate that NIR-II fluorescence imaging of A1 NPs precisely detect microscopic residual tumor (2 mm in diameter) in the tumor bed and metastatic lymph nodes. More notably, a novel integrated strategy that achieves complete tumor eradication (no local recurrence and metastasis after surgery) is proposed. In summary, A1 NPs possess superior imaging and treatment performance, and can detect and eliminate residual tumor lesions intraoperatively. This work provides a promising technique for future clinical applications achieving improved surgical outcomes.

Quantum limit of photon-counting imaging based on compressed sensing.

We experimentally demonstrate that the sensitivity of photon-counting imaging can be improved by 2 orders of magnitude with the compressed sensing (CS) theory. The maximum sensitivity of CS imaging under the quantum limit, which is approximately 1 photon in each pixel during one measurement, is quantitatively obtained through theoretical derivation and proved experimentally. The influences of dark noise and shot noise on photon-counting imaging are also studied to confirm the fundamental constrains on the imaging sensitivity of different imaging methods, which can guide the effort for further enhancing the ultra-weak light imaging ability.

Modified calanolides incorporated with furan-2-nitro mimics against Mycobacterium tuberculosis.

(+)-Calanolide A was identified as an active agent against both HIV-1 and Mycobacterium tuberculosis (Mtb). We modified Ring D of calanolide compounds with furan-2-nitro mimics and obtained 15 compounds. After in vitro tests, two compounds were shown to be active against replicating (R) Mtb, but not against non-replicating (NR) Mtb. Further optimization for potent candidates against both R Mtb and NR Mtb will result from this research.

Cloning, expression and characterization of protein disulfide isomerase of Schistosoma japonicum.

The excretory/secretory (ES) proteins of schistosomes play important roles in modulating host immune systems and are regarded as potential vaccine candidates and drug targets. Protein disulfide isomerase (PDI) is an essential enzyme that is involved in disulfide bond formation and rearrangement. In the present study, SjPDI, a 52.8 kDa protein previously identified in a proteomics analysis as one of the ES proteins of Schistosoma japonicum, was cloned and characterized. Western blot analysis showed that recombinant SjPDI (rSjPDI) was recognized by serum from rabbits vaccinated with schistosome worm antigen. Worm protein extracts and ES protein extracts from S. japonicum could react with anti-rSjPDI mouse serum. Real-time PCR analysis indicated that SjPDI was expressed at all developmental stages tested, and a high expression level was detected in 42-day-old male worms. Immunofluorescence analysis revealed that SjPDI was mainly distributed on the tegument and parenchyma of S. japonicum worms. An enzyme-linked immunosorbent assay (ELISA) demonstrated that rSjPDI could induce a high level of rSjPDI-specific IgG antibodies. The biological activity of purified rSjPDI was confirmed by isomerization and antioxidative activity assays. The 35.32%, 26.19% reduction in the worm burden and 33.17%, 31.7% lower liver egg count were obtained in mice vaccinated with rSjPDI compared with the blank control group in two independent trials. Our preliminary results suggest that rSjPDI plays an important role in the development of the schistosome and is a potential vaccine candidate for schistosomiasis.

Highly stereoselective synthesis of (Z)- and (E)-chloro-substituted-α-methylene-γ-butyrolactone by possibly controlling cis- and trans-chloropalladation.

A palladium-catalyzed selective synthesis of cis- or trans-chloro-substituted-α-methylene-γ-butyrolactones from single substrate (propiolic acid) has been realized by controlling cis- or trans-chloropalladation. This method is highly effective for building C-Cl, C-O, and C-C bonds into a one-pot procedure because of its good atom and step efficiency.

NIR-II fluorescence-guided liver cancer surgery by a small molecular HDAC6 targeting probe.

BACKGROUND: Hepatocellular carcinoma (HCC) is the sixth most common malignancy globally and ranks third in terms of both mortality and incidence rates. Surgical resection holds potential as a curative approach for HCC. However, the residual disease contributes to a high 5-year recurrence rate of 70%. Due to their excellent specificity and optical properties, fluorescence-targeted probes are deemed effective auxiliary tools for addressing residual lesions, enabling precise surgical diagnosis and treatment. Research indicates histone deacetylase 6 (HDAC6) overexpression in HCC cells, making it a potential imaging biomarker. This study designed a targeted small-molecule fluorescent probe, SeCF3-IRDye800cw (SeCF3-IRD800), operating within the Second near-infrared window (NIR-II, 1000-1700 nm). The study confirms the biocompatibility of SeCF3-IRD800 and proceeds to demonstrate its applications in imaging in vivo, fluorescence-guided surgery (FGS) for liver cancer, liver fibrosis imaging, and clinical samples incubation, thereby preliminarily validating its utility in liver cancer. METHODS: SeCF3-IRD800 was synthesized by combining the near-infrared fluorescent dye IRDye800cw-NHS with an improved HDAC6 inhibitor. Initially, a HepG2-Luc subcutaneous tumor model (n = 12) was constructed to investigate the metabolic differences between SeCF3-IRD800 and ICG in vivo. Subsequently, HepG2-Luc (n = 12) and HCCLM3-Luc (n = 6) subcutaneous xenograft mouse models were used to assess in vivo targeting by SeCF3-IRD800. The HepG2-Luc orthotopic liver cancer model (n = 6) was employed to showcase the application of SeCF3-IRD800 in FGS. Liver fibrosis (n = 6) and HepG2-Luc orthotopic (n = 6) model imaging results were used to evaluate the impact of different pathological backgrounds on SeCF3-IRD800 imaging. Three groups of fresh HCC and normal liver samples from patients with liver cancer were utilized for SeCF3-IRD800 incubation ex vivo, while preclinical experiments illustrated its potential for clinical application. FINDINGS: The HDAC6 inhibitor 6 (SeCF3) modified with trifluoromethyl was labeled with IRDy800CW-NHS to synthesize the small-molecule targeted probe SeCF3-IRD800, with NIR-II fluorescence signals. SeCF3-IRD800 was rapidly metabolized by the kidneys and exhibited excellent biocompatibility. In vivo validation demonstrated that SeCF3-IRD800 achieved optimal imaging within 8 h, displaying high tumor fluorescence intensity (7658.41 ± 933.34) and high tumor-to-background ratio (5.20 ± 1.04). Imaging experiments with various expression levels revealed its capacity for HDAC6-specific targeting across multiple HCC tumor models, suitable for NIR-II intraoperative imaging. Fluorescence-guided surgery experiments were found feasible and capable of detecting sub-visible 2 mm tumor lesions under white light, aiding surgical decision-making. Further imaging of liver fibrosis mice showed that SeCF3-IRD800's imaging efficacy remained unaffected by liver pathological conditions. Correlations were observed between HDAC6 expression levels and corresponding fluorescence intensity (R2 = 0.8124) among normal liver, liver fibrosis, and HCC tissues. SeCF3-IRD800 identified HDAC6-positive samples from patients with HCC, holding advantages for perspective intraoperative identification in liver cancer. Thus, the rapidly metabolized HDAC6-targeted small-molecule NIR-II fluorescence probe SeCF3-IRD800 holds significant clinical translational value. INTERPRETATION: The successful application of NIR-II fluorescence-guided surgery in liver cancer indicates that SeCF3-IRD800 has great potential to improve the clinical diagnosis and treatment of liver cancer, and could be used as an auxiliary tool for surgical treatment of liver cancer without being affected by liver pathology. FUNDING: This paper is supported by the National Natural Science Foundation of China (NSFC) (92,059,207, 62,027,901, 81,930,053, 81,227,901, 82,272,105, U21A20386 and 81,971,773), CAS Youth Interdisciplinary Team (JCTD-2021-08), the Zhuhai High-level Health Personnel Team Project (Zhuhai HLHPTP201703), and Guangdong Basic and Applied Basic Research Foundation under Grant No. 2022A1515011244.

NIR-II fluorescence imaging-guided colorectal cancer surgery targeting CEACAM5 by a nanobody.

BACKGROUND: Surgery is the cornerstone of colorectal cancer (CRC) treatment, yet complete removal of the tumour remains a challenge. The second near-infrared window (NIR-II, 1000-1700 nm) fluorescent molecular imaging is a novel technique, which has broad application prospects in tumour surgical navigation. We aimed to evaluate the ability of CEACAM5-targeted probe for CRC recognition and the value of NIR-II imaging-guided CRC resection. METHODS: We constructed the probe 2D5-IRDye800CW by conjugated anti-CEACAM5 nanobody (2D5) with near-infrared fluorescent dye IRDye800CW. The performance and benefits of 2D5-IRDye800CW at NIR-II were confirmed by imaging experiments in mouse vascular and capillary phantom. Then mouse colorectal cancer subcutaneous tumour model (n = 15), orthotopic model (n = 15), and peritoneal metastasis model (n = 10) were constructed to investigate biodistribution of probe and imaging differences between NIR-I and NIR-II in vivo, and then tumour resection was guided by NIR-II fluorescence. Fresh human colorectal cancer specimens were incubated with 2D5-IRDye800CW to verify its specific targeting ability. FINDINGS: 2D5-IRDye800CW had an NIR-II fluorescence signal extending to 1600 nm and bound specifically to CEACAM5 with an affinity of 2.29 nM. In vivo imaging, 2D5-IRDye800CW accumulated rapidly in tumour (15 min) and could specifically identify orthotopic colorectal cancer and peritoneal metastases. All tumours were resected under NIR-II fluorescence guidance, even smaller than 2 mm tumours were detected, and NIR-II had a higher tumour-to-background ratio than NIR-I (2.55 ± 0.38, 1.94 ± 0.20, respectively). 2D5-IRDye800CW could precisely identify CEACAM5-positive human colorectal cancer tissue. INTERPRETATION: 2D5-IRDye800CW combined with NIR-II fluorescence has translational potential as an aid to improve R0 surgery of colorectal cancer. FUNDINGS: This study was supported by Beijing Natural Science Foundation (JQ19027), the National Key Research and Development Program of China (2017YFA0205200), National Natural Science Foundation of China (NSFC) (61971442, 62027901, 81930053, 92059207, 81227901, 82102236), Beijing Natural Science Foundation (L222054), CAS Youth Interdisciplinary Team (JCTD-2021-08), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16021200), the Zhuhai High-level Health Personnel Team Project (Zhuhai HLHPTP201703), the Fundamental Research Funds for the Central Universities (JKF-YG-22-B005) and Capital Clinical Characteristic Application Research (Z181100001718178). The authors would like to acknowledge the instrumental and technical support of the multi-modal biomedical imaging experimental platform, Institute of Automation, Chinese Academy of Sciences.

Identification of ITGAX and CCR1 as potential biomarkers of atherosclerosis via Gene Set Enrichment Analysis.

OBJECTIVE: Atherosclerosis (AS) is a life-threatening disease in aging populations worldwide. However, the molecular and gene regulation mechanisms of AS are still unclear. This study aimed to identify gene expression differences between atheroma plaques and normal tissues in humans. METHODS: The expression profiling dataset GSE43292 was obtained from the Gene Expression Omnibus (GEO) dataset. The differentially expressed genes (DEGs) were identified between the atheroma plaques and normal tissues via GEO2R, and functional annotation of the DEGs was performed by GSEA. STRING and MCODE plug-in of Cytoscape were used to construct a protein-protein interaction (PPI) network and analyze hub genes. Finally, quantitative polymerase chain reaction (qPCR) was performed to verify the hub genes. RESULTS: Overall, 134 DEGs were screened. Functional annotation demonstrated that these DEGs were mainly enriched in sphingolipid metabolism, apoptosis, lysosome, and more. Six hub genes were identified from the PPI network: ITGAX, CCR1, IL1RN, CXCL10, CD163, and MMP9. qPCR analysis suggested that the relative expression levels of the six hub genes were significantly higher in AS samples. CONCLUSIONS: We used bioinformatics to identify six hub genes: ITGAX, CCR1, IL1RN, CXCL10, CD163, and MMP9. These hub genes are potential promising diagnostic and therapeutic targets for AS.

Interaction effect of obesity and thyroid autoimmunity on the prevalence of hyperthyrotropinaemia.

PURPOSE: The role of thyroid autoimmunity in the association between obesity and hyperthyrotropinaemia remains unclear. We aimed to assess the relationship between obesity, autoimmunity, and hyperthyrotropinaemia. METHODS: In this population-based cross-sectional study, 12531 Chinese individuals (18-80 years) with thyroid function test were categorized into three groups by body mass index (BMI) and were categorized into three layers by thyroid autoantibodies. Multivariate logistic regression was employed to assess the correlation and interaction effect. RESULTS: There was no significant difference in prevalence of hyperthyrotropinaemia (P = 0.637) among three BMI groups. After stratification, the difference of serum thyrotropin (P < 0.01) and prevalence of hyperthyrotropinaemia (P < 0.01) between the three groups have significant linear trends at the positive levels of thyroid peroxidase antibody (TPOAb) or/and thyroglobulin antibody (TgAb). When TPOAb and TgAb were positive, the risk of hyperthyrotropinaemia increased 1.857-fold in overweight group and 2.201-fold in obese group compared with normal group. Compared with negative TPOAb and TgAb, the risk of hyperthyrotropinaemia for individuals with two positive antibodies increased 3.310-fold, 4.969-fold, and 5.122-fold in the three BMI groups. The adjusted OR (95% CI) for interaction were 1.033 (0.752-1.419) for overweight and one positive antibodies, 1.935 (1.252-2.990) for overweight and two positive antibodies, 1.435 (0.978-2.105) for obesity and one positive antibodies and 2.191 (1.252-3.832) for obesity and two positive antibodies. CONCLUSION: Overweight and obesity were associated with hyperthyrotropinaemia only in presence of thyroid autoimmunity, and obesity might aggravate the pathogenic effect of autoimmunity on hyperthyrotropinaemia. There was an interaction effect between obesity and autoimmunity on the prevalence of hyperthyrotropinaemia.

[Data analysis of laser desorption/ionization mass spectrum of individual particle using adaptive resonance theory based neural network].

On-line measurement of size and chemical composition of single particle using an aerosol laser time-of-flight mass spectrometer (ALTOFMS) was designed in our lab. Each particle's aerodynamic diameter is determined by measuring the delay time between two continuous-wave lasers operating at 650 nm. A Nd : YAG laser desorbs and ionizes molecules from the particle, and the time-of-flight mass spectrometer collects a mass spectrum of the generated ions. Then the composition of single particle is obtained. ALTOFMS generates large amount of data during the process period. How to process these data quickly and extract valuable information is one of the key problems for the ALTOFMS. In the present paper, an adaptive resonance theory-based neural network, ART-2a algorithm, was used to classify mixed mass spectra of aerosol particles of NaCl, CaCl2, dioctylphthalate (DOP), and 2,5-dihydroxybenzoic acid (DHB). Compared with the traditional methods, ART-2a can recognize input patterns self-organically, self-adaptively and self-steadily without considering the complexity and the number of the patterns, so it is more favorable for the analysis of the mass spectra data. Experimental results show that when vigilance parameter is 0.40, learning rate is 0.05 and iteration number is 6, ART-2a algorithm can successfully reveal these four particle categories. The weight vectors for these four particle classes were obtained, which can represent the characters of these four particle classes remarkably.

[Trace detection of ammonia at 1.531 microm].

A compact instrument based on the off-axis integrated-cavity output spectroscopy (ICOS) technology was developed for sensitive measurements of gas mixing ratios (ammonia in air) at room temperature by using fiber-coupled distributed feedback (DFB) diode laser operating at 1.531 microm. The absorption line of ammonia at 6 528.764 cm(-1) was chosen for trace detection. The mirrors' effective reflectivity R2 of 0.996 9 was first calibrated by carbon dioxide under this condition, and the cavity 35.8 cm in length as an absorption cell could yield an optical path of presumably 115.46 m. As a result, a minimum detectable concentration of approximately 2.66 ppmv (S/N-3) at the total pressure of 100 torr was obtained. Then the lock-in amplifier was added in the system to acquire the second harmonic signal by combination of wavelength modulation technology, which could better suppress background noise and improve the signal-to-noise ratio, and a detection limit of 0.293 ppmv (S/N-3) was achieved eventually. This work demonstrated the potential of the system for a range of atmospheric species sensing in the future.

[Data analysis of laser desorption/ionization mass spectrum of atmospheric aerosol particles using fuzzy clustering algorithms].

On-line measurement of size and composition of single particle using an aerosol time-of-flight Laser mass spectrometry (ATOFLMS) had been designed in our lab. Each particle's aerodynamic diameter is determined by measuring the delay time between two continuous-wave lasers, A Nd : YAG laser desorbs and ionizes molecules from the particle, and the time-of-flight mass spectrometer collects a mass spectrum of the generated ions. Then the composition of single particle is obtained. ATOFLMS generates large amount of data during the process period. How to process these data and extract valuable information is one of the key problems for the ATOFLMS. In this paper, the fuzzy clustering used to classify large numbers of mass spectral of air indoor by an ATOFLMS. Each revised spectrum is converted to a normalized 300-point vector, each point representing one mass unit. Then the positive ion mass spectra of a single particle are described as 300-dimensional data vectors using the ion masses as dimensions and the ion signal peak areas as values. The data vectors of all particles measured are written into a classification matrix. Each spectrum's data was stored as one row in this matrix. The Fuzzy c-means algorithm is an iterative method starting the calculation with random class centers to find a substructure in the data. The procedure works in such a way that finally similar objects (particle spectra) have a minimum distance between their corresponding data vectors, on the one hand, and to the center of a cluster, on the other hand. So the aim of the iteration is to find local minima in the N-dimensional space where N is the number of evaluated peak masses. The particle data used in this study were collected over a period one day in Hefei. During the campaign, inorganic salts, mineral particles, and carbonaceous particles, with varying degrees of secondary components, were identified. The detection results of particle size exhibit that aerosol is predominanantly in the form of fine particles, and the particles whose diameter larger than 1 microm are scare. The particles whose diameter less than 1 microm are make up of 95% of the total particles, and these particles are major distributed in 0.4-0.8 microm.

[Single particle measurement of suspended soil dust using laser desorption/ionization time-of-flight mass spectrometry].

Real-time measurement of size and composition of single soil dust particles using an aerosol time-of-flight laser mas spectrometry (ATOFLMS) was designed in our lab. Each particle's aerodynamic diameter was determined by measuring the delay time between two continuous-wave lasers, a Nd:YAG laser with a 266 nm pulsed output was used to desorb and ionize aerosol particles, and ions formed in the laser desorption/ionization process were accelerated into the time-of-flight drift region where they separated by mass-to-charge ratio, then the composition of single particle was obtained. In the present paper, soil samples were collected from four different areas in China. After the pretreatment and suspension, the particle sample was then transferred to ATOFLMS through a plastic transfer line. During the campaign, a large number of size and mass spectra of single par ticles were obtained. The presence of crustal elements was observed in the mass spectra of individual particles. Iron, potassium aluminum and calcium constitute the two most commonly detected cations. Other common cations observed in the mass spectra o soil particles include magnesium, and sodium. The detection results exhibit that the coarse particles with size of 1-2 microm are dominate in the detected particles. Experimental results show the ATOFLMS have important practical value for researching and monitoring of atmospheric aerosol environment.

Comparison of apoptosis between adult worms of Schistosoma japonicum from susceptible (BALB/c mice) and less-susceptible (Wistar rats) hosts.

Schistosomiasis remains a serious public health concern in China. BALB/c mice are susceptible to Schistosoma japonicum infection, whereas the Wistar rats are less susceptible. Apoptosis phenomenon was observed in 42d adult worms of S. japonicum from both rats and mice at the morphologic, DNA, cellular, and gene levels by transmission electron microscopy (TEM), fluorometric terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analysis, fluorescein isothiocyanate-annexin-V/propidium iodide staining flow cytometry (FCM) analysis, and real-time PCR. The results showed that the apoptotic state in worms from two different susceptible hosts was diverse. Several classical hallmarks of apoptosis, including cell shrinkage, chromatin condensation and lunate marginalization, splitting of the nucleoli, nuclear shrinkage and apoptotic body formation were observed by TEM. TUNEL analysis showed that there were much more apoptosis spots in adult worms from rats than those from mice. Statistical analysis revealed that the degree of apoptosis and percentage of necrotic cells in adult worms from Wistar rats were significantly greater (P<0.01) than those from BALB/c mice by flow cytometry. A total of 15 apoptosis-associated genes including the major components of an intrinsic cell-death pathway were identified from S. japonicum in this study, suggested that a similar apoptosis pathway might occur in S. japonicum. Real-time PCR analyses revealed that the expression levels of most of the tested apoptosis-associated genes, except CASP7, were significantly higher or at the similar level in adult worms from Wistar rats, as compared to those from BALB/c mice. The results obtained in this study collectively demonstrated that differential development of adult S. japonicum in less-susceptible rats and susceptible mice was significantly associated with apoptosis in the worm, and provided valuable information to guide further investigations of the mechanisms governing apoptosis and host interactions in schistosome infection.

Benzimidazole-based compounds kill Mycobacterium tuberculosis.

Tuberculosis remains one of the deadliest infectious diseases, killing 1.4 million people annually and showing a rapid increase in cases resistant to multiple drugs. New antibiotics against tuberculosis are urgently needed. Here we describe the design, synthesis and structure-activity relationships of a series of benzimidazole-based compounds with activity against Mycobacterium tuberculosis (Mtb) in a replicating state, a physiologically-induced non-replicating state, or both. Compounds 49, 67, 68, 69, 70, and 72, which shared a 5-nitrofuranyl moiety, exhibited high potency and acceptable selectivity indices (SI). As illustrated by compound 70 (MIC90 < 0.049 µg/mL, SI > 512), the 5-nitrofuranyl group was compatible with minimal cytotoxicity and good intra-macrophage killing, although it lacked non-replicating activity when assessed by CFU assays. Compound 70 had low mutagenic potential by SOS Chromotest assay, making this class of compounds good candidates for further evaluation and target identification.

Synthetic calanolides with bactericidal activity against replicating and nonreplicating Mycobacterium tuberculosis.

It is urgent to introduce new drugs for tuberculosis to shorten the prolonged course of treatment and control drug-resistant Mycobacterium tuberculosis (Mtb). One strategy toward this goal is to develop antibiotics that eradicate both replicating (R) and nonreplicating (NR) Mtb. Naturally occurring (+)-calanolide A was active against R-Mtb. The present report details the design, synthesis, antimycobacterial activities, and structure-activity relationships of synthetic calanolides. We identified potent dual-active nitro-containing calanolides with minimal in vitro toxicity that were cidal to axenic Mtb and Mtb in human macrophages, while sparing Gram-positive and -negative bacteria and yeast. Two of the nitrobenzofuran-containing lead compounds were found to be genotoxic to mammalian cells. Although genotoxicity precluded clinical progression, the profound, selective mycobactericidal activity of these calanolides will be useful in identifying pathways for killing both R- and NR-Mtb, as well as in further structure-based design of more effective and drug-like antimycobacterial agents.

Induced apoptosis of osteoblasts proliferating on polyhydroxyalkanoates.

The mechanism study on behaviors of cells influenced by biomaterial surface properties can provide profound guidances for functional tissue engineering scaffolds design. In this study, regulation of integrin-mediated cell-substrate interactions using rat osteoblasts incubated on PHA films was investigated. Compared with tissue culture plate (TCP), poly-3-hydroxybutyrate (PHB), copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV) and copolymer of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx), osteoblasts inoculated on a terpolymer of 3-hydroxybutyrate, 3-hydroxyvalerate and 3-hydroxyhexanoate (PHBVHHx) were found to have higher apoptosis rates. Several integrin subunits in osteoblasts grown on PHBVHHx showed altered expressions. Simultaneously, extracellular matrics (ECM) were also remodeled on the material surface. Osteoblasts showed a higher expression of integrin subunit ß3 and αv on PHBVHHx films compared with that on TCP. On the other hand, less vitronectin, osteopontin and fibronectin, the main ligands for integrin ß3 were expressed and deposited in ECM. The unligated integrin ß3 could recruit caspase-8 to the membrane and activate its downstream signaling which was proven by the caspase-8 activation assay. It was therefore concluded that the induced apoptosis of osteoblasts on PHBVHHx was regulated by recruitment of caspase-8 to the unligated integrin ß3.

Quinoxalin-2(1H)-one derivatives as inhibitors against hepatitis C virus.

Hepatitis C virus (HCV) infection is a serious problem worldwide, but no effective drugs are currently available. Through screening of our privileged structure library, quinoxalin-2(1H)-one derivative N-(7-(cyclohexyl(methyl)amino)-3-oxo-3,4-dihydroquinoxalin-6-ylcarbamothioyl)benzamide (compound 1) was identified as potent HCV inhibitor in vitro. Subsequently, a structure-activity relationship analysis was carried out that showed N-(7-(cyclohexyl(methyl)amino)-3-oxo-3,4-dihydroquinoxalin-6-ylcarbamothioyl)furan-2-carboxamide (compound 11, EC(50)=1.8 µM, SI=9.6), 6-(cyclohexyl(methyl)amino)-7-(4-phenylthiazol-2-ylamino)quinoxalin-2(1H)-one (compound 33, EC(50)=1.67 µM, SI=37.4), 2-(cyclohexyl(methyl)amino)-3-(4-phenylthiazol-2-ylamino)-7,8,9,10-tetrahydro-5H-pyrido[1,2-a]quinoxalin-6(6aH)-one (compound 60, EC(50)=1.19 µM, SI=9.27), 8-(cyclohexyl(methyl)amino)-7-(4-phenylthiazol-2-ylamino)pyrrolo[1,2-a]quinoxalin-4(5H)-one (compound 65, EC(50)=1.82 µM, SI=9.9), and 6-(diethylamino)-7-(4-phenylthiazol-2-ylamino)quinoxalin-2(1H)-one (compound 78, EC(50)=1.27 µM, SI=17.9) acted against HCV. The data from the structure-activity relationship study suggests that quinoxalin-2(1H)-one derivatives exhibited potent activity against HCV.

An assessment of the risks of carcinogenicity associated with polyhydroxyalkanoates through an analysis of DNA aneuploid and telomerase activity.

Polyhydroxyalkanoates (PHA) are aliphatic polyesters synthesized by many bacteria. Because of their flexible mechanical strengths, superior elastic property, biodegradability and biocompatibility, PHA have been developed for applications as medical implants, drug delivery matrices, and devices to support cell growth. Lots of studies showed that PHA matrices improved cell proliferation and tissue regeneration. However, the possibility of whether rapid cell proliferation on PHA matrices will induce tumor formation is unclear. Here we confirmed that proliferating rat osteoblasts grown on films of various PHA including PHB, PHBV, P3HB4HB, PHBHHx and PHBVHHx did not lead to cancer induction at least for p8th. Cell proliferation was evaluated by the incorporation of 5-bromodeoxyuridine (BrdU), the transcript expression of cancer related genes Ki67, p53 and c-Fos was monitored by quantitative Real-time PCR, the results showed the cells proliferating on the PHA films were under normal cell cycle regulation. Moreover, DNA aneuploid and telomerase activity were only detected in the positive control UMR-108 cells; compared with cells grown on films, UMR-108 cells had longer telomeres, further demonstrated the normal status of cells proliferating on the PHA films. It indicated that the above PHA family members could be used to support cell growth without indication of susceptibility to tumor induction. These results will be important for promoting the application of PHA as new members of biomaterials.