Laccase is a multitasking protein for synthetic gene circuits in the yeast Saccharomycescerevisiae.

Laccase is a multicopper oxidase enzyme that oxidizes a variety of substrates, including polyphenols and polycyclic aromatic hydrocarbons (PAHs). It catalyzes the four-electron reduction of molecular oxygen that results in the production of water as a by-product. Thus, laccase can play an important role in environmental care. Previously, we have successfully expressed Trametes trogii laccase (TtLcc1) in the yeast Saccharomyces cerevisiae. In this work, we have expressed in yeast another laccase, LacA from Trametes sp. AH28-2, and tested its function on PAHs. Yeast cells engineered to produce the two laccases performed efficient PAH degradation. Both TtLcc1 and LacA led to the construction of spatiotemporal fluorescence-pulse generators when combined with a benzoate/salicylate yeast biosensor in a two-population system. Moreover, laccases returned a visual output signal in yeast synthetic circuits-upon reacting with ABTS (2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)). Thus, in S. cerevisiae, laccases are a powerful alternative to fluorescent reporter proteins.

Detection and diagnosis of automated breast ultrasound in patients with BI-RADS category 4 microcalcifications: a retrospective study.

BACKGROUND: Automated Breast Ultrasound (AB US) has shown good application value and prospects in breast disease screening and diagnosis. The aim of the study was to explore the ability of AB US to detect and diagnose mammographically Breast Imaging Reporting and Data System (BI-RADS) category 4 microcalcifications. METHODS: 575 pathologically confirmed mammographically BI-RADS category 4 microcalcifications from January 2017 to June 2021 were included. All patients also completed AB US examinations. Based on the final pathological results, analyzed and summarized the AB US image features, and compared the evaluation results with mammography, to explore the detection and diagnostic ability of AB US for these suspicious microcalcifications. RESULTS: 250 were finally confirmed as malignant and 325 were benign. Mammographic findings including microcalcifications morphology (61/80 with amorphous, coarse heterogeneous and fine pleomorphic, 13/14 with fine-linear or branching), calcification distribution (189/346 with grouped, 40/67 with linear and segmental), associated features (70/96 with asymmetric shadow), higher BI-RADS category with 4B (88/120) and 4 C (73/38) showed higher incidence in malignant lesions, and were the independent factors associated with malignant microcalcifications. 477 (477/575, 83.0%) microcalcifications were detected by AB US, including 223 malignant and 254 benign, with a significantly higher detection rate for malignant lesions (x2 = 12.20, P < 0.001). Logistic regression analysis showed microcalcifications with architectural distortion (odds ratio [OR] = 0.30, P = 0.014), with amorphous, coarse heterogeneous and fine pleomorphic morphology (OR = 3.15, P = 0.037), grouped (OR = 1.90, P = 0.017), liner and segmental distribution (OR = 8.93, P = 0.004) were the independent factors which could affect the detectability of AB US for microcalcifications. In AB US, malignant calcification was more frequent in a mass (104/154) or intraductal (20/32), and with ductal changes (30/41) or architectural distortion (58/68), especially with the both (12/12). BI-RADS category results also showed that AB US had higher sensitivity to malignant calcification than mammography (64.8% vs. 46.8%). CONCLUSIONS: AB US has good detectability for mammographically BI-RADS category 4 microcalcifications, especially for malignant lesions. Malignant calcification is more common in a mass and intraductal in AB US, and tend to associated with architectural distortion or duct changes. Also, AB US has higher sensitivity than mammography to malignant microcalcification, which is expected to become an effective supplementary examination method for breast microcalcifications, especially in dense breasts.

dCas12a:Pre-crRNA: A New Tool to Induce mRNA Degradation in Saccharomyces cerevisiae Synthetic Gene Circuits.

We describe a new way to trigger mRNA degradation in Saccharomyces cerevisiae synthetic gene circuits. Our method demands to modify either the 5'- or the 3'-UTR that flanks a target gene with elements from the pre-crRNA of type V Cas12a proteins and expresses a DNase-deficient Cas12a (dCas12a). dCas12a recognizes and cleaves the pre-crRNA motifs on mRNA sequences. Our tool does not require complex engineering operations and permits an efficient control of protein expression via mRNA degradation.

Clinical Characteristics of Chlamydia psittaci Infection Diagnosed by Metagenomic Next-Generation Sequencing: A Retrospective Multi-Center Study in Fujian, China.

Objective: This study aimed to describe and compare the epidemiological, demographic, clinical, laboratory and radiological characteristics as well as the complications, treatments, and outcomes of these patients. Methods: We retrospectively investigated clinical data of patients with C. psittaci infection (psittacosis) in eight Grade IIIA hospitals of Fujian. Metagenomic next-generation sequencing (mNGS) was used identify C. psittaci in clinical samples of all included patients. Results: A total of 74 patients (39 severe/35 non-severe) was diagnosed with psittacosis, 25 (33.8%) of whom had history of poultry exposure. Common symptoms included high fever (98% [37/74]), fatigue (52.7% [39/74]), and dyspnea (51.4% [38/74]). Common manifestations in imaging included consolidation (89.2%), pleural effusion (77.0%), and air bronchogram (66.2%). Common complications included acute respiratory distress syndrome (55.4% [41/74]), type I respiratory failure (52.7% [39/74]), acute liver injury (41.9% [31/74]), and secondary infection (27.0% [20/74]). The in-hospital mortality rate was 8.11% (6/74). Conclusion: C. psittaci infection is represents an underestimated cause of CAP. For SCAP patients with poultry and bird contact history, specimens were encouraged to be sended for mNGS test in time. C. psittaci infection can lead to severe, multiple system involvement, and several complications. mNGS facilitate timely diagnosis of C. psittaci infection.

Scaffold RNA engineering in type V CRISPR-Cas systems: a potent way to enhance gene expression in the yeast Saccharomyces cerevisiae.

New, orthogonal transcription factors in eukaryotic cells have been realized by engineering nuclease-deficient CRISPR-associated proteins and/or their guide RNAs. In this work, we present a new kind of orthogonal transcriptional activators, in Saccharomyces cerevisiae, made by turning type V CRISPR RNA into a scaffold RNA (ScRNA) able to recruit a variable number of VP64 activation domains. The activator arises from the complex between the synthetic ScRNA and DNase-deficient type V Cas proteins: dCas12e and denAsCas12a. The transcription activation achieved via the newly engineered dCas:ScRNA system is up to 4.7-fold higher than that obtained with the direct fusion of VP64 to Cas proteins. The new transcription factors have been proven to be functional in circuits such as Boolean gates, converters, multiplex-gene and metabolic-pathway activation. Our results extend the CRISPR-Cas-based technology with a new effective tool that only demands RNA engineering and improves the current design of transcription factors based on type V Cas proteins.

Structure-directed identification of pyridine-2-methylamine derivatives as MmpL3 inhibitors for use as antitubercular agents.

Mycobacterial membrane protein Large 3 (MmpL3), an inner membrane protein, plays a crucial role in the transport of mycolic acids that are essential for the viability of M. tuberculosis and has been a promising therapeutic target for new anti-TB agents. Herein, we report the discovery of pyridine-2-methylamine antitubercular compounds using a structure-based drug design strategy. Compound 62 stands out as the most potent compound with high activity against M. tb strain H37Rv (MIC = 0.016 µg/mL) as well as the clinically isolated strains of MDR/XDR-TB (MIC = 0.0039-0.0625 µg/mL), low Vero cell toxicity (IC50 ≥ 16 µg/mL), and moderate liver microsomal stability (CLint = 28 µL/min/mg). Furthermore, the resistant mutant of S288T due to single nucleotide polymorphism in mmpL3 was resistant to pyridine-2-methylamine 62, demonstrating compound 62 is likely target to MmpL3.

Efficiency and impact factors of anatomical intelligence for breast and hand-held ultrasound in lesion detection.

PURPOSE: To investigate the efficiency and impact factors of anatomical intelligence for breast (AI-Breast) and hand-held ultrasound (HHUS) in lesion detection. METHODS: A total of 172 outpatient women were randomly selected, underwent AI-Breast ultrasound (Group AI) once and HHUS twice. HHUS was performed by breast imaging radiologists (Group A) and general radiologists (Group B). For the AI-Breast examination, a trained technician performed the whole-breast scan and data acquisition, while other general radiologists performed image interpretation. The examination time and lesion detection rate were recorded. The impact factors for breast lesion detection, including breast cup size, number of lesions, and benign or malignant lesions were analyzed. RESULTS: The detection rates of Group AI, A, and B were 92.8 ± 17.0%, 95.0 ± 13.6%, and 85.0 ± 22.9%, respectively. Comparable lesion detection rates were observed in Group AI and Group A (P > 0.05), but a significantly lower lesion detection rate was observed in Group B compared to the other two (both P < 0.05). Regarding missed diagnosis rates of malignant lesions, comparable performance was observed in Group AI, Group A, and Group B (8% vs. 4% vs. 14%, all P > 0.05). Scan times of Groups AI, A, and B were 262.15 ± 40.4 s, 237.5 ± 110.3 s, 281.2 ± 86.1 s, respectively. The scan time of Group AI was significantly higher than Group A (P < 0.01), but was slightly lower than Group B (P > 0.05). We found a strong linear correlation between scan time and cup size in Group AI (r = 0.745). No impacts of cup size and number of lesions were found on the lesion detection rate in Group AI (P > 0.05). CONCLUSIONS: With the assist of AI-Breast system, the lesion detection rate of AI-Breast ultrasound was comparable to that of a breast imaging radiologist and superior to that of the general radiologist. AI-Breast ultrasound may be used as a potential approach for breast lesions surveillance.

Synthesis and Biological Evaluation of 3-Amino-4,4-Dimethyl Lithocholic Acid Derivatives as Novel, Selective, and Cellularly Active Allosteric SHP1 Activators.

Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP1), a non-receptor member of the protein tyrosine phosphatase (PTP) family, negatively regulates several signaling pathways that are responsible for pathological cell processes in cancers. In this study, we report a series of 3-amino-4,4-dimethyl lithocholic acid derivatives as SHP1 activators. The most potent compounds, 5az-ba, showed low micromolar activating effects (EC50: 1.54-2.10 µM) for SHP1, with 7.63-8.79-fold maximum activation and significant selectivity over the closest homologue Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP2) (>32-fold). 5az-ba showed potent anti-tumor effects with IC50 values of 1.65-5.51 µM against leukemia and lung cancer cells. A new allosteric mechanism of SHP1 activation, whereby small molecules bind to a central allosteric pocket and stabilize the active conformation of SHP1, was proposed. The activation mechanism was consistent with the structure-activity relationship (SAR) data. This study demonstrates that 3-amino-4,4-dimethyl lithocholic acid derivatives can be selective SHP1 activators with potent cellular efficacy.

CRISPR-associated type V proteins as a tool for controlling mRNA stability in S. cerevisiae synthetic gene circuits.

Type V-A CRISPR-(d)Cas system has been used in multiplex genome editing and transcription regulation in both eukaryotes and prokaryotes. However, mRNA degradation through the endonuclease activity of Cas12a has never been studied. In this work, we present an efficient and powerful tool to induce mRNA degradation in the yeast Saccharomyces cerevisiae via the catalytic activity of (d)Cas12a on pre-crRNA structure. Our results point out that dFnCas12a, (d)LbCas12a, denAsCas12a and two variants (which carry either NLSs or NESs) perform significant mRNA degradation upon insertion of pre-crRNA fragments into the 5'- or 3' UTR of the target mRNA. The tool worked well with two more Cas12 proteins-(d)MbCas12a and Casϕ2-whereas failed by using type VI LwaCas13a, which further highlights the great potential of type V-A Cas proteins in yeast. We applied our tool to the construction of Boolean NOT, NAND, and IMPLY gates, whose logic operations are fully based on the control of the degradation of the mRNA encoding for a reporter protein. Compared to other methods for the regulation of mRNA stability in yeast synthetic gene circuits (such as RNAi and riboswitches/ribozymes), our system is far easier to engineer and ensure very high performance.

Therapeutic potential of coumestan Pks13 inhibitors for tuberculosis.

Polyketide synthase 13 (Pks13) is an important enzyme found in Mycobacterium tuberculosis (M. tuberculosis) that condenses two fatty acyl chains to produce α-alkyl ß-ketoesters, which in turn serve as the precursors for the synthesis of mycolic acids that are essential building blocks for maintaining the cell wall integrity of M. tuberculosis Coumestan derivatives have recently been identified in our group as a new chemotype that exert their antitubercular effects via targeting of Pks13. These compounds were active on both drug-susceptible and drug-resistant strains of M. tuberculosis as well as showing low cytotoxicity to healthy cells and a promising selectivity profile. No cross-resistance was found between the coumestan derivatives and first-line TB drugs. Here we report that treatment of M. tuberculosis bacilli with 15 times the MIC of compound 1, an optimized lead coumestan compound, resulted in a colony forming unit (CFU) reduction from 6.0 log10 units to below the limit of detection (1.0 log10 units) per mL culture, demonstrating a bactericidal mechanism of action. Single dose (10 mg/kg) pharmacokinetic studies revealed favorable parameters with a relative bioavailability of 19.4%. In a mouse infection and chemotherapy model, treatment with 1 showed dose-dependent mono-therapeutic activity, whereas treatment with 1 in combination with rifampin showed clear synergistic effects. Together these data suggest that coumestan derivatives are promising agents for further TB drug development.

Extracellular polymeric substances trigger microbial immigration from partial denitrification (PD) to anammox biofilms in a long-term operated PD/anammox process in low-strength wastewater.

The immigration of microbial communities in a synergistic partial denitrification/anammox (SPDA) system was investigated in a moving bed biofilm reactor (MBBR) inoculated with partial denitrification (PD) and anaerobic ammonium oxidation (anammox) biofilms. The SPDA system was operated at 25 ± 1 °C over 260 days. The total nitrogen (TN) of the effluent was only 3.71 ± 0.92 mg·L-1 in the stable phase with a TN removal efficiency of 95.23%. The anammox process was the dominant nitrogen removal pathway with an average contribution of 74.31% to TN removal. The results of the in situ activity and key enzymatic activity revealed that the nitrate-reducing bacteria tended to immigrate to anammox biofilms. Correspondingly, the abundance of the genus Thauera, the second most dominant bacteria in anammox biofilms, quickly increased from 0.78 to 10.69% on day 50 and eventually to 16.45% on day 221 according to the Illumina MiSeq sequencing data. The microbial immigration might be caused by different extracellular polymeric substance (EPS)-mediated mechanisms in PD and anammox biofilms. For fast-growing denitrifiers, PD biofilms tend to increase the ability of mass transfer by excreting more polysaccharides to form loosely-bound EPS at the expense of the ability to harbor the nitrate-reducing bacteria. However, for the slow-growing anaerobic ammonium oxidizing bacteria (AnAOB), the anammox biofilms tend to increase the retention of AnAOB by excreting more proteins to form enhanced tightly-bound EPS at the expense of the mass transfer ability, thereby causing the detached nitrate-reducing bacteria to immigrate into anammox biofilms.

Value of multimodal imaging in the diagnosis of breast sclerosing adenosis associated with malignant lesions.

AIM: To explore the diagnostic value of multimodal imaging techniques, including automatic breast volume scanner (ABVS), mammography (MG), and magnetic resonance (MRI) in breast sclerosing adenosis (SA) associated with malignant lesions. METHODS: From January 2018 to October 2020, 76 patients (88 lesions) with pathologically confirmed as SA associated with malignant or benign lesions were retrospective analyzed. All patients completed ABVS examination, 58 patients (67 lesions) with MG and 50 patients (62 lesions) with MRI were also completed before biopsy or surgical excision, of which, six patients (eight lesions) diagnosed as Breast Imaging Reporting and Data System (BI-RADS) category 3 by all imaging examinations underwent surgical excision without biopsy, other 70 patients (80 lesions) with BI-RADS category 4 or above by any imaging examination completed biopsy, including 65 patients (75 lesions) were further surgical excised and the other five patients (five lesions) were just followed up. All lesions were retrospectively described and classified, and were divided into benign group and malignant group according to their pathological results. Image features of different examination methods between the two groups were compared and analyzed. A ROC curve was established using the sensitivity of BI-RADS categories to predict malignant lesions in different imaging techniques as the ordinate and 1-specificity as the abscissa. RESULTS: 88 lesions including 26 purely SA and 45 SA associated with benign lesions were classified as benign group, and the remaining 17 SA associated with malignant lesions were classified as malignant group. On ABVS, 40 mass lesions, their heterogeneous echo, not circumscribed margin and coronal convergence signs were statistically significant for malignant lesions (p < .05), but the remain 48 nonmass lesions lack specific sonographic features. On MG, 12 showed negative results, 55 showed with microcalcification, mass, structural distortion, and asymmetric density shadow, of which 11 lesions had the above two signs at the same time, but only microcalcification had statistical difference between the two groups. 35 mass enhanced lesions and 27 nonmass enhanced lesions on MRI, but there were no significant difference between their pathological results. Time signal intensity curves showed no differences, but ADC value <1.10 × 10-3  mm2 /s is more significant in malignant lesions (p < .05). The area under the ROC curve (AUC) of BI-RADS classification of ABVS, MG, and MRI in the diagnosis of malignant lesions were 0.611, 0.474, and 0.751, respectively, and the AUC of the combined diagnosis of the three was 0.761. CONCLUSION: Mass lesions with heterogeneous echo, not circumscribed margin and coronal convergence sign on ABVS, microcalcification on MG and the ADC value <1.10 × 10-3  mm2 /s on MRI are significant signs for SA associated with malignant lesions. The combined diagnosis of the three methods was the highest, and the following were MRI, ABVS, and MG. Therefore, be cognizant of significant characteristics in SA associated with malignancy showed in different imaging examinations can improve the preoperative evaluation of SA and better provide basis for subsequent clinical decision-making.

Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins.

Synthetic gene Boolean gates and digital circuits have a broad range of applications, from medical diagnostics to environmental care. The discovery of the CRISPR-Cas systems and their natural inhibitors-the anti-CRISPR proteins (Acrs)-provides a new tool to design and implement in vivo gene digital circuits. Here, we describe a protocol that follows the idea of the "Design-Build-Test-Learn" biological engineering cycle and makes use of dCas9/dCas12a together with their corresponding Acrs to establish small transcriptional networks, some of which behave like Boolean gates, in Saccharomyces cerevisiae. These results point out the properties of dCas9/dCas12a as transcription factors. In particular, to achieve maximal activation of gene expression, dSpCas9 needs to interact with an engineered scaffold RNA that collects multiple copies of the VP64 activation domain (AD). In contrast, dCas12a shall be fused, at the C terminus, with the strong VP64-p65-Rta (VPR) AD. Furthermore, the activity of both Cas proteins is not enhanced by increasing the amount of sgRNA/crRNA in the cell. This article also explains how to build Boolean gates based on the CRISPR-dCas-Acr interaction. The AcrIIA4 fused hormone-binding domain of the human estrogen receptor is the core of a NOT gate responsive to ß-estradiol, whereas AcrVAs synthesized by the inducible GAL1 promoter permits to mimic both YES and NOT gates with galactose as an input. In the latter circuits, AcrVA5, together with dLbCas12a, showed the best logic behavior.

Structure-Based Optimization of Coumestan Derivatives as Polyketide Synthase 13-Thioesterase(Pks13-TE) Inhibitors with Improved hERG Profiles for Mycobacterium tuberculosis Treatment.

Pks13 was identified as a key enzyme involved in the final step of mycolic acid biosynthesis. We previously identified antitubercular coumestans that targeted Pks13-TE, and these compounds exhibited high potency both in vitro and in vivo. However, lead compound 8 presented potential safety concerns because it inhibits the hERG potassium channel in electrophysiology patch-clamp assays (IC50 = 0.52 µM). By comparing the Pks13-TE-compound 8 complex and the ligand-binding pocket of the hERG ion channel, fluoro-substituted and oxazine-containing coumestans were designed and synthesized. Fluoro-substituted compound 23 and oxazine-containing coumestan 32 showed excellent antitubercular activity against both drug-susceptible and drug-resistant Mtb strains (MIC = 0.0039-0.0078 µg/mL) and exhibited limited hERG inhibition (IC50 ≥ 25 µM). Moreover, 32 exhibited improved metabolic stability relative to parent compound 8 while showing favorable bioavailability in mouse models via serum inhibition titration assays.

Predicting the molecular subtypes of breast cancer using nomograms based on three-dimensional ultrasonography characteristics.

Background: Molecular subtyping of breast cancer is commonly doneforindividualzed cancer management because it may determines prognosis and treatment. Therefore, preoperativelyidentifying different molecular subtypes of breast cancery can be significant in clinical practice.Thisretrospective study aimed to investigate characteristic three-dimensional ultrasonographic imaging parameters of breast cancer that are associated with the molecular subtypes and establish nomograms to predict the molecular subtypes of breast cancers. Methods: A total of 309 patients diagnosed with breast cancer between January 2017and December 2019 were enrolled. Sonographic features were compared between the different molecular subtypes. A multinomial logistic regression model was developed, and nomograms were constructed based on this model. Results: The performance of the nomograms was evaluated in terms of discrimination and calibration.Variables such as maximum diameter, irregular shape, non-parallel growth, heterogeneous internal echo, enhanced posterior echo, lymph node metastasis, retraction phenomenon, calcification, and elasticity score were entered into the multinomial model.Three nomograms were constructed to visualize the final model. The probabilities of the different molecular subtypes could be calculated based on these nomograms. Based on the receiver operating characteristic curves of the model, the macro-and micro-areaunder the curve (AUC) were0.744, and 0.787. The AUC was 0.759, 0.683, 0.747 and 0.785 for luminal A(LA), luminal B(LB), human epidermal growth factor receptor 2-positive(HER2), and triple-negative(TN), respectively.The nomograms for the LA, HER2, and TN subtypes provided good calibration. Conclusions: Sonographic features such as calcification and posterior acoustic features were significantly associated with the molecular subtype of breast cancer. The presence of the retraction phenomenon was the most important predictor for the LA subtype. Nomograms to predict the molecular subtype were established, and the calibration curves and receiver operating characteristic curves proved that the models had good performance.

Design, Synthesis and Biological Evaluation of N-phenylindole Derivatives as Pks13 Inhibitors againstMycobacterium tuberculosis.

Polyketide synthase 13 (Pks13), an essential enzyme for the survival of Mycobacterium tuberculosis (Mtb), is an attractive target for new anti-TB agents. In our previous work, we have identified 2-phenylindole derivatives against Mtb. The crystallography studies demonstrated that the two-position phenol was solvent-exposed in the Pks13-TE crystal structure and a crucial hydrogen bond was lost while introducing bulkier hydrophobic groups at indole N moieties. Thirty-six N-phenylindole derivatives were synthesized and evaluated for antitubercular activity using a structure-guided approach. The structure-activity relationship (SAR) studies resulted in the discovery of the potent Compounds 45 and 58 against Mtb H37Rv, with an MIC value of 0.0625 µg/mL and 0.125 µg/mL, respectively. The thermal stability analysis showed that they bind with high affinity to the Pks13-TE domain. Preliminary ADME evaluation showed that Compound 58 displayed modest human microsomal stability. This report further validates that targeting Pks13 is a valid strategy for the inhibition of Mtb and provides a novel scaffold for developing leading anti-TB compounds.

Ultrasound grayscale ratio: a reliable parameter for differentiating between papillary thyroid microcarcinoma and micronodular goiter.

BACKGROUND: The present study aimed to quantify and differentiate the echo levels of papillary thyroid microcarcinomas (PTMCs) and micronodular goiters (MNGs) using the ultrasound grayscale ratio (UGSR) and to investigate the repeatability of UGSR. METHODS: The ultrasound (US) data of 241 patients with 265 PTMCs and 141 patients with 168 MNGs confirmed by surgery and pathology were retrospectively analyzed. All patients had received outpatient ultrasonic examination and preoperative ultrasonic positioning. The RADinfo radiograph reading system was used to measure the grayscales of PTMC, MNG, and thyroid tissues at the same gain level, and the UGSR values of the PTMC, MNG, and thyroid tissue were calculated. The patients were divided into outpatient examination, preoperative positioning, and mean value groups, and the receiver operating characteristic (ROC) curves were calculated to obtain the optimal UGSR threshold to distinguish PTMC from MNG. The interclass correlation coefficient (ICC) was used to assess the consistency of UGSR measured in three groups. RESULTS: The UGSR values of the PTMC and MNG were 0.56 ± 0.14 and 0.80 ± 0.19 (t = 5.84, P < 0.001) in the outpatient examination group, 0.55 ± 0.14 and 0.80 ± 0.19 (t = 18.74, P < 0.001) in the preoperative positioning group, and 0.56 ± 0.12 and 0.80 ± 0.18 (t = 16.49, P < 0.001) in the mean value group. The areas under the ROC curves in the three groups were 0.860, 0.856, and 0.875, respectively. When the UGSR values for the outpatient examination, preoperative positioning, and mean value groups were 0.649, 0.646, and 0.657, respectively, each group obtained its largest Youden index. A reliable UGSR value was obtained between the outpatient examination and preoperative positioning groups (ICC = 0.79, P = 0.68). CONCLUSION: UGSR is a simple and repeatable method to distinguish PTMC from MNG, and hence, can be widely applicable.

Computational and Structure-Based Development of High Potent Cell-Active Covalent Inhibitor Targeting the Peptidyl-Prolyl Isomerase NIMA-Interacting-1 (Pin1).

The unique proline isomerase peptidyl-prolyl isomerase NIMA-interacting-1 (Pin1) is reported to activate numerous cancer-driving pathways simultaneously, and aberrant Pin1 activation is present in many human cancers. Here, we identified a novel hit compound, ZL-Pin01, that covalently modified Pin1 at Cys113 with an half-maximal inhibitory concentration (IC50) of 1.33 ± 0.07 µM through screening an in-house library. Crystallographic study drove the process of structure-guided optimization and led to the potent inhibitor ZL-Pin13 with an IC50 of 0.067 ± 0.03 µM. We obtained four co-crystal structures of Pin1 complexed with inhibitors that elucidated the detailed binding mode of the derivatives with Pin1. Interestingly, the co-crystal of Pin1 with ZL-Pin13 obtained by co-crystallization revealed the conformational change of Gln129 induced by the inhibitor. Furthermore, ZL-Pin13 effectively inhibited the proliferation and downregulated the Pin1 substrates in MDA-MB-231 cells. Collectively, we developed a potent covalent inhibitor of Pin1, ZL-Pin13, which could be an effective probe for studying the functional roles of Pin1.

Design, synthesis, and biological evaluation of indole-based hydroxamic acid derivatives as histone deacetylase inhibitors.

The equilibrium between histone acetylation and deacetylation plays an important role in cancer initiation and progression. The histone deacetylases (HDACs) are a class of key regulators of gene expression that enzymatically remove an acetyl moiety from acetylated lysine ε-amino groups on histone tails. Therefore, HDAC inhibitors have recently emerged as a promising strategy for cancer therapy and several pan-HDAC inhibitors have globally been approved for clinical use. In the present study, we designed and synthesized a series of substituted indole-based hydroxamic acid derivatives that exhibited potent anti-proliferative activities in various tumor cell lines. Among the compounds tested, compound 4o, was found to be among the most potent in the inhibition of HDAC1 (half maximal inhibitory concentration, IC50 = 1.16 nM) and HDAC6 (IC50 = 2.30 nM). It also exhibited excellent in vitro anti-tumor proliferation activity. Additionally, compound 4o effectively increased the acetylation of histone H3 in a dose-dependent manner and inhibited cell proliferation by inducing cell cycle arrest and apoptosis. Moreover, compound 4o remarkably blocked colony formation in HCT116 cancer cells. Based on its favorable in vitro profile, compound 4o was further evaluated in an HCT116 xenograft mouse model, in which it demonstrated better in vivo efficacy than the clinically used HDAC inhibitor, suberanilohydroxamic acid. Interestingly, compound 4k was found to have a preference for the inhibition of HDAC6, with IC50 values of 115.20 and 5.29 nM against HDAC1 and HDAC6, respectively.

Synthesis and antitumour evaluation of indole-2-carboxamides against paediatric brain cancer cells.

Paediatric glioblastomas are rapidly growing, devastating brain neoplasms with an invasive phenotype. Radiotherapy and chemotherapy, which are the current therapeutic adjuvant to surgical resection, are still associated with various toxicity profiles and only marginally improve the course of the disease and life expectancy. A considerable body of evidence supports the antitumour and apoptotic effects of certain cannabinoids, such as WIN55,212-2, against a wide spectrum of cancer cells, including gliomas. In fact, we previously highlighted the potent cytotoxic activity of the cannabinoid ligand 5 against glioblastoma KNS42 cells. Taken together, in this study, we designed, synthesised, and evaluated several indoles and indole bioisosteres for their antitumour activities. Compounds 8a, 8c, 8f, 12c, and 24d demonstrated significant inhibitory activities against the viability (IC50 = 2.34-9.06 µM) and proliferation (IC50 = 2.88-9.85 µM) of paediatric glioblastoma KNS42 cells. All five compounds further retained their antitumour activities against two atypical teratoid/rhabdoid tumour (AT/RT) cell lines. When tested against a medulloblastoma DAOY cell line, only 8c, 8f, 12c, and 24d maintained their viability inhibitory activities. The viability assay against non-neoplastic human fibroblast HFF1 cells suggested that compounds 8a, 8c, 8f, and 12c act selectively towards the panel of paediatric brain tumour cells. In contrast, compound 24d and WIN55,212-2 were highly toxic toward HFF1 cells. Due to their structural resemblance to known cannabimimetics, the most potent compounds were tested in cannabinoid 1 and 2 receptor (CB1R and CB2R) functional assays. Compounds 8a, 8c, and 12c failed to activate or antagonise both CB1R and CB2R, whereas compounds 8f and 24d antagonised CB1R and CB2R, respectively. We also performed a transcriptional analysis on KNS42 cells treated with our prototype compound 8a and highlighted a set of seven genes that were significantly downregulated. The expression levels of these genes were previously shown to be positively correlated with tumour growth and progression, indicating their implication in the antitumour activity of 8a. Overall, the drug-like and selective antitumour profiles of indole-2-carboxamides 8a, 8c, 8f, and 12c substantiate the versatility of the indole scaffold in cancer drug discovery.