A basal-level activity of ATR links replication fork surveillance and stress response.

Mammalian cells use diverse pathways to prevent deleterious consequences during DNA replication, yet the mechanism by which cells survey individual replisomes to detect spontaneous replication impediments at the basal level, and their accumulation during replication stress, remain undefined. Here, we used single-molecule localization microscopy coupled with high-order-correlation image-mining algorithms to quantify the composition of individual replisomes in single cells during unperturbed replication and under replicative stress. We identified a basal-level activity of ATR that monitors and regulates the amounts of RPA at forks during normal replication. Replication-stress amplifies the basal activity through the increased volume of ATR-RPA interaction and diffusion-driven enrichment of ATR at forks. This localized crowding of ATR enhances its collision probability, stimulating the activation of its replication-stress response. Finally, we provide a computational model describing how the basal activity of ATR is amplified to produce its canonical replication stress response.

Response of serum LDL cholesterol to oatmeal consumption depends on CYP7A1_rs3808607 genotype in Chinese.

BACKGROUND AND OBJECTIVES: Notable inter-individual differences in cholesterol-lowering effects following oatmeal consumption have been previously reported. Genetic variations may among the reasons for the heterogeneous response to lipid modulations. And to determine whether SNP of cytochrome P450 family 7 subfamily A member 1 gene rs3808607 and isoforms of apolipoprotein E are associated with the inter-individual variations in cholesterol-lowering effects of oatmeal consumption, we did this study. METHODS AND STUDY DESIGN: Data in this study were extracted from a parallel, controlled trial, in which 62 medication-naive hypercholesterolemic patients provided with staple food substitute of either 80 g/d oatmeal (n=31) or 80 g/d refined white rice (n=31) for 45 days. Fasting blood samples were collected at baseline and endpoint of the study for lipid profiling, glycemic testing, and genotyping. RESULTS: Totally, 56 of 62 participants completed the study and were thus included. Genotype- diet interactions were observed between oatmeal consumption and SNP in the cytochrome P450 family 7 subfamily A member 1 gene rs3808607 in regulating LDL cholesterol (p=0.04); rs3808607-TT homozygotes exhibited significantly higher responsiveness to oatmeal (reduction in LDL cholesterol) than G allele carriers (GG/GT) (p=0.02). However, obvious genotype-diet interactions were not observed between oatmeal consumption and apolipoprotein E isoforms in cholesterol and glycemic modulation (p>0.05). CONCLUSIONS: SNP in cytochrome P450 family 7 subfamily A member 1 gene rs3808607 was associated with the extent of LDL cholesterol reduction following oatmeal consumption. Trials with larger sample sizes are required to confirm the findings.

An Algorithm Based Wavelet Entropy for Shadowing Effect of Human Detection Using Ultra-Wideband Bio-Radar.

Ultra-wide band (UWB) radar for short-range human target detection is widely used to find and locate survivors in some rescue missions after a disaster. The results of the application of bistatic UWB radar for detecting multi-stationary human targets have shown that human targets close to the radar antennas are very often visible, while those farther from radar antennas are detected with less reliability. In this paper, on account of the significant difference of frequency content between the echo signal of the human target and that of noise in the shadowing region, an algorithm based on wavelet entropy is proposed to detect multiple targets. Our findings indicate that the entropy value of human targets was much lower than that of noise. Compared with the method of adaptive filtering and the energy spectrum, wavelet entropy can accurately detect the person farther from the radar antennas, and it can be employed as a useful tool in detecting multiple targets by bistatic UWB radar.

Detection and Identification of Multiple Stationary Human Targets Via Bio-Radar Based on the Cross-Correlation Method.

Ultra-wideband (UWB) radar has been widely used for detecting human physiological signals (respiration, movement, etc.) in the fields of rescue, security, and medicine owing to its high penetrability and range resolution. In these applications, especially in rescue after disaster (earthquake, collapse, mine accident, etc.), the presence, number, and location of the trapped victims to be detected and rescued are the key issues of concern. Ample research has been done on the first issue, whereas the identification and localization of multi-targets remains a challenge. False positive and negative identification results are two common problems associated with the detection of multiple stationary human targets. This is mainly because the energy of the signal reflected from the target close to the receiving antenna is considerably stronger than those of the targets at further range, often leading to missing or false recognition if the identification method is based on the energy of the respiratory signal. Therefore, a novel method based on cross-correlation is proposed in this paper that is based on the relativity and periodicity of the signals, rather than on the energy. The validity of this method is confirmed through experiments using different scenarios; the results indicate a discernible improvement in the detection precision and identification of the multiple stationary targets.

A Novel Method for Speech Acquisition and Enhancement by 94 GHz Millimeter-Wave Sensor.

In order to improve the speech acquisition ability of a non-contact method, a 94 GHz millimeter wave (MMW) radar sensor was employed to detect speech signals. This novel non-contact speech acquisition method was shown to have high directional sensitivity, and to be immune to strong acoustical disturbance. However, MMW radar speech is often degraded by combined sources of noise, which mainly include harmonic, electrical circuit and channel noise. In this paper, an algorithm combining empirical mode decomposition (EMD) and mutual information entropy (MIE) was proposed for enhancing the perceptibility and intelligibility of radar speech. Firstly, the radar speech signal was adaptively decomposed into oscillatory components called intrinsic mode functions (IMFs) by EMD. Secondly, MIE was used to determine the number of reconstructive components, and then an adaptive threshold was employed to remove the noise from the radar speech. The experimental results show that human speech can be effectively acquired by a 94 GHz MMW radar sensor when the detection distance is 20 m. Moreover, the noise of the radar speech is greatly suppressed and the speech sounds become more pleasant to human listeners after being enhanced by the proposed algorithm, suggesting that this novel speech acquisition and enhancement method will provide a promising alternative for various applications associated with speech detection.

[Low fat milk powder containing esterified plant sterols improves the blood lipid profile of adults with hypercholesterolemia].

OBJECTIVE: To observe the impact of plant sterol esters (PSE) mixed in low fat milk powder (2.5 g of PSE/day) on plasma cholesterol levels in hypercholesterolemic subjects during a 6-week intervention period. METHODS: In this double-blind, randomized, placebo-controlled study, 59 subjects (19 males, mean age (60.28 ± 6.98) years) with primary hypercholesterolemia (fasting LDL cholesterol between 3.4-6.0 mmol/L) were randomly divided into two groups (treatment group, 2.5 g of plant sterol esters a day, n = 30) and placebo group (n = 29). Blood samples were collected at week 0, 3 and 6. The primary outcome was change in plasma LDL-cholesterol (LDL-C). Secondary outcomes were changes in total cholesterol (TC), HDL cholesterol (HDL-C), triglycerides (TG), anthropometry and blood biochemistry. RESULTS: LDL-C significantly reduction from baseline (4.18 ± 0.54) mmol/L to (3.44 ± 0.61) mmol/L (-17.7%, P < 0.05) at week 3 and (3.35 ± 0.39) mmol/L (-19.9%, P < 0.05) at week 6 in the treatment group, whereas in placebo group from (4.11 ± 0.54) mmol/L at baseline to (3.47 ± 0.60) mmol/L (-15.57%, P < 0.05) and (3.61 ± 0.39) mmol/L (-12.17%, P < 0.05) at week 3 and week 6, respectively. TC was reduced from (6.30 ± 0.86) mmol/L at baseline to (5.92 ± 0.75) mmol/L (-6.03%, P > 0.05) at week 3 and (5.43 ± 0.77) mmol/L (-13.8%, P < 0.05) at week 6 in treatment group, from (6.20 ± 0.76) mmol/L at week 0 to (5.70 ± 0.76) mmol/L (-8.06%, P < 0.05) at week 3 and (5.84 ± 0.75) mmol/L (-5.81%, P < 0.05) at week 6 in placebo group. PSE-enriched milk did not affect plasma HDL-C level and TG level at both week 3 and week 6. After normalization to the placebo group, the treatment group showed significant reduction in LDL-C and total cholesteron after 6 weeks. The observed difference of reduction was 7.69% (-0.33 mmol/L, P < 0.05) for LDL-C and 8.00% (-0.51 mmol/L, P < 0.05) for TC between the two groups. There were no significant changes in safety parameters, including blood biochemistry tests during the study period. CONCLUSION: Plant sterol ester enriched milk powder is effective in reducing LDL-C among Chinese hypercholesterolemic subjects at a dosage recommended by EFSA.

A 94-GHz millimeter-wave sensor for speech signal acquisition.

High frequency millimeter-wave (MMW) radar-like sensors enable the detection of speech signals. This novel non-acoustic speech detection method has some special advantages not offered by traditional microphones, such as preventing strong-acoustic interference, high directional sensitivity with penetration, and long detection distance. A 94-GHz MMW radar sensor was employed in this study to test its speech acquisition ability. A 34-GHz zero intermediate frequency radar, a 34-GHz superheterodyne radar, and a microphone were also used for comparison purposes. A short-time phase-spectrum-compensation algorithm was used to enhance the detected speech. The results reveal that the 94-GHz radar sensor showed the highest sensitivity and obtained the highest speech quality subjective measurement score. This result suggests that the MMW radar sensor has better performance than a traditional microphone in terms of speech detection for detection distances longer than 1 m. As a substitute for the traditional speech acquisition method, this novel speech acquisition method demonstrates a large potential for many speech related applications.

A two-step mechanism governing PARP1-DNA retention by PARP inhibitors.

PARP inhibitors (PARPi) have emerged as promising cancer therapeutics capable of targeting specific DNA repair pathways, but their mechanism of action with respect to PARP1-DNA retention remains unclear. Here, we developed single-molecule assays to directly monitor the retention of PARP1 on DNA lesions in real time. Our study reveals a two-step mechanism by which PARPi modulate the retention of PARP1 on DNA lesions, consisting of a primary step of catalytic inhibition via binding competition with NAD+ followed by an allosteric modulation of bound PARPi. While clinically relevant PARPi exhibit distinct allosteric modulation activities that can either increase retention of PARP1 on DNA or induce its release, their retention potencies are predominantly determined by their ability to outcompete NAD+ binding. These findings provide a mechanistic basis for improved PARPi selection according to their characteristic activities and enable further development of more potent inhibitors.

Contactless multiscale measurement of cardiac motion using biomedical radar sensor.

Introduction: A contactless multiscale cardiac motion measurement method is proposed using impulse radio ultra-wideband (IR-UWB) radar at a center frequency of 7.29 GHz. Motivation: Electrocardiograph (ECG), heart sound, and ultrasound are traditional state-of-the-art heartbeat signal measurement methods. These methods suffer from defects in contact and the existence of a blind information segment during the cardiogram measurement. Methods: Experiments and analyses were conducted using coarse-to-fine scale. Anteroposterior and along-the-arc measurements were taken from five healthy male subjects (aged 25-43) when lying down or prone. In every measurement, 10 seconds of breath-holding data were recorded with a radar 55 cm away from the body surface, while the ECG was monitored simultaneously as a reference. Results: Cardiac motion detection from the front was superior to that from the back in amplitude. In terms of radar detection angles, the best cardiac motion information was observed at a detection angle of 120°. Finally, in terms of cardiac motion cycles, all the ECG information, as well as short segments of cardiac motion details named blind ECGs segments, were detected. Significance: A contactless and multiscale cardiac motion detection method is proposed with no blind detection of segments during the entire cardiac cycle. This paves the way for a potentially significant method of fast and accurate cardiac disease assessment and diagnosis that exhibits promising application prospects in contactless online cardiac monitoring and in-home healthcare.

[Laser Raman and infrared spectrum analysis of low-density lipoproteins purified from hen egg yolk].

During the experiment, diversified proteins were separated from hen egg yolk by ammonium sulphate rapid fractionation, and pure LDL was obtained after filtrating through Sephadex G-200 chromatography. After the qualitative detection of SDS-PAGE, the authors discovered that LDL consists of five major apoprotein. The Raman and infrared spectrum showed CH2 asymmetric stretching and symmetric stretching mode. However, the authors found C==O stretching vibrations of protein peptide bonds and N+ (CH3)3 asymmetric stretching vibration from the choline group in phospholipids. Laser Raman and infrared spectrum analysis of LDL provided useful information for studying their structure.

Oatmeal induced gut microbiota alteration and its relationship with improved lipid profiles: a secondary analysis of a randomized clinical trial.

BACKGROUND: In vitro and animal experiments reported a microbiota-regulating ability of oatmeal, however, related in vivo evidences remained limited. Thus, we conducted this study aiming to investigate the oatmeal-induced alteration of gut microbiota and its potential relationship with the improvements of lipid profiles. METHODS AND STUDY DESIGN: Data of anthropometric measurements and biochemical parameters were extracted from a randomized, controlled clinical trial, in which 62 hypercholesterolemic men and women (18-65 years old) were provided with either treatment of 80 g/day oatmeal or 80 g/day refined white rice for 45 days. Fasting blood samples and fecal samples were collected both at baseline and endpoint of the study for lipid profiling and microbiota 16S rRNA amplicon sequencing, respectively. RESULTS: Totally 28 participants (56 fecal samples) qualified with the new criteria and were thus included in this secondary analysis. The results of microbiota analysis showed that no significant difference was observed in the alteration of its overall α or ß diversity between two groups throughout the study. Nor did any notable between-group difference was found in the relative abundance changes of microorganism at different taxonomies. However, results from linear discriminant analysis effect size in the oatmeal group indicated a significant positive response of Firmicutes phylum following oatmeal consumption. Further Procrustes analysis suggested a concordance trend between microorganism alteration and alleviation of hypercholesterolemia phenotypes throughout the study (P = 0.05). The results of within-group comparison from Spearman's correlation in the oatmeal group demonstrated a significant association between the enrichment of Blautia genus and the reduction of serum total cholesterol (P < 0.05), low-density lipoprotein cholesterol (P < 0.01), and apolipoprotein B (P < 0.05). CONCLUSIONS: Positive response of Firmicutes phylum might be a critical characteristic of oatmeal-induced alteration of microbiota, whereas, one of the underlying cholesterol-lowering mechanism of oatmeal consumption might be its microbiota-manipulating ability, in which the enrichment of Blautia genus played a potentially significant role. Current results should be taken cautiously and more studies were needed for further verification.Trial registration: ChiCTR, ChiCTR180001864. Registered 30 September 2018, http://www.chictr.org.cn/showproj.aspx?proj=31469.

Activity of andrographolide and its derivatives against influenza virus in vivo and in vitro.

Infections with influenza A viruses are still a major threat to humans and several animal species. The occurrence of highly pathogenic avian influenza viruses capable of infecting and killing humans highlights the urgency for a new and efficient strategy for the treatment of diseases caused by the virus. Andrographolide and its derivatives have been widely used for treating respiratory infections in China for decades. We have recently synthesized new andrographolide derivatives and found that some of the compounds including 14-alpha-lipoyl andrographolide (AL-1) have significant activity against bacterial infections with an unique mechanism of action. We report here the antiviral activity of AL-1 and other andrographolide drugs. AL-1 showed significant activity against influenza A viruses including the H5N1 avian influenza virus. The administration of AL-1 by oral gavage to mice infected with avian influenza A/Chicken/Guangdong /96 (H9N2), A/Duck/Guangdong/99 (H5N1), and human influenza A/PR/8/34 (H1N1) viruses greatly reduced the death rate, prolonged life, inhibited lung consolidation, and reduced viral titers in the lung. The most effective dosage of AL-1 in these studies ranged from 100 to 200 mg/kg/d, when administered twice daily for 7 d beginning 24 h before viral exposure. The LD(50) of AL-1 was 1243 mg/kg/d. AL-1 was effective against avian influenza A (H9N2 and H5N1) and human influenza A H1N1 viruses in vitro, with the 50% effective concentrations ranging from 7.2 to 15.2 microM and the selective indexes ranging from 51 to 109. Significant inhibition of viral adsorption onto red blood cells with minimum inhibitory concentrations ranging from 5.3 to 16.8 mM suggested that AL-1 was capable of directly interfering with viral hemagglutinin to block binding to cellular receptors. With potent antiviral activity and a potentially new mechanism of action, AL-1 may warrant further evaluation as a possible therapy for influenza.

Method for Distinguishing Humans and Animals in Vital Signs Monitoring Using IR-UWB Radar.

Radar has been widely applied in many scenarios as a critical remote sensing tool for non-contact vital sign monitoring, particularly for sleep monitoring and heart rate measurement within the home environment. For non-contact monitoring with radar, interference from house pets is an important issue that has been neglected in the past. Many animals have respiratory frequencies similar to those of humans, and they are easily mistaken for human targets in non-contact monitoring, which would trigger a false alarm because of incorrect physiological parameters from the animal. In this study, humans and common pets in families, such as dogs, cats, and rabbits, were detected using an impulse radio ultrawideband (IR-UWB) radar, and the echo signals were analyzed in the time and frequency domains. Subsequently, based on the distinct in-body structure between humans and animals, we propose a parameter, the respiratory and heartbeat energy ratio (RHER), which reflects the contribution rate of breathing and heartbeat in the detected vital signs. Combining this parameter with the energy index, we developed a novel scheme to distinguish between humans and animals. In the developed scheme, after background noise removal and direct-current component suppression, an energy indicator is used to initially identify the target. The signal is then decomposed using a variational mode decomposition algorithm, and the variational intrinsic mode functions that represent human respiration and heartbeat components are obtained and utilized to calculate the RHER parameter. Finally, the RHER index is applied to rapidly distinguish between humans and animals. Our experimental results demonstrate that the proposed approach more effectively distinguishes between humans and animals in terms of monitoring vital signs than the existing methods. Furthermore, its rapidity and need for only minimal calculation resources enable it to meet the needs of real-time monitoring.

Fine-tuning the expression of target genes using a DDI2 promoter gene switch in budding yeast.

Tuned gene expression is crucial to the proper growth and response to the environmental changes of an organism. To enable tunable gene expression as designed is desirable in both scientific research and industrial application. Here, we introduce a novel promoter switching method based on the DDI2 promoter (PDDI2) that can fine tune the expression of target genes. We constructed a recyclable cassette (PDDI2-URA3-PDDI2) and integrated it upstream of yeast target genes to replace the native promoters by DDI2 promoter without introducing any junk sequence. We found that the presence or absence of cyanamide as an inducer could turn on or off the expression of target genes. In addition, we showed that PDDI2 could act as a gene switch to linearly regulate the expression levels of target genes in vivo. We switched the original promoters of RAD18, TUP1, and CDC6 with PDDI2 as a proof-of-concept.

Visualizing the Interaction Between the Qdot-labeled Protein and Site-specifically Modified λ DNA at the Single Molecule Level.

The fluorescence microscopy has made great contributions in dissecting the mechanisms of complex biological processes at the single molecule level. In single molecule assays for studying DNA-protein interactions, there are two important factors for consideration: the DNA substrate with enough length for easy observation and labeling a protein with a suitable fluorescent probe. 48.5 kb λ DNA is a good candidate for the DNA substrate. Quantum dots (Qdots), as a class of fluorescent probes, allow long-time observation (minutes to hours) and high-quality image acquisition. In this paper, we present a protocol to study DNA-protein interactions at the single-molecule level, which includes preparing a site-specifically modified λ DNA and labeling a target protein with streptavidin-coated Qdots. For a proof of concept, we choose ORC (origin recognition complex) in budding yeast as a protein of interest and visualize its interaction with an ARS (autonomously replicating sequence) using TIRFM. Compared with other fluorescent probes, Qdots have obvious advantages in single molecule studies due to its high stability against photobleaching, but it should be noted that this property limits its application in quantitative assays.

MicroRNA-216b-3p inhibits lung adenocarcinoma cell growth via regulating PDZ binding kinase/T-LAK-cell-originated protein kinase.

Numerous studies have reported that microRNA (miR)-216b, as a tumor suppressor, is downregulated in a variety of cancer types. PDZ binding kinase (PBK)/T-LAK-cell-originated protein kinase (TOPK) is highly expressed in various types of human cancer, including lung cancer. The expression of miR-216b-3p and its potential roles in lung adenocarcinoma are still unclear and no research has been conducted into the association between miR-216b-3p and PBK/TOPK. Thus, the present study aimed to investigate the expression and role of miR-216b-3p in lung adenocarcinoma and to explore whether PBK/TOPK is involved in the underlying mechanisms of lung adenocarcinoma. The expression of miR-216b-3p in lung adenocarcinoma cell lines was detected. PBK/TOPK protein expression levels were also determined within lung adenocarcinoma cell lines. To investigate the association between miR-216b-3p and PBK/TOPK, TargetScan analysis was performed; PBK was predicted to be a potential target gene of miR-216b-3p, and a dual luciferase reporter assay was applied to confirm this prediction. To investigate the role of miR-216b-3p in lung adenocarcinoma, a lung adenocarcinoma cell line (GLC-82) was transfected with miR-216b-3p mimic or its negative control. An MTT assay was applied to detect cell proliferation, and cell apoptosis was analyzed by flow cytometry. Western blot analysis was performed to determine the protein expression levels of associated proteins. The results of the present study suggested that miR-216b-3p was downregulated in lung adenocarcinoma cell lines and PBK/TOPK was highly expressed in lung adenocarcinoma cells. miR-216b-3p directly targets PBK and negatively regulates its expression. miR-216b-3p overexpression may inhibit GLC-82 cell proliferation and induce cell apoptosis. In addition, miR-216b-3p overexpression may increase p53 and p21 expression, and prevent p38 MAPK activation. These effects on GLC-82 cells caused by miR-216b-3p overexpression may be eliminated by PBK/TOPK overexpression. In conclusion, miR-216b-3p was downregulated in lung adenocarcinoma and may function as a tumor suppressor by inhibiting cell growth via regulating PBK/TOPK expression.

Utilizing Biotinylated Proteins Expressed in Yeast to Visualize DNA-Protein Interactions at the Single-Molecule Level.

Much of our knowledge in conventional biochemistry has derived from bulk assays. However, many stochastic processes and transient intermediates are hidden when averaged over the ensemble. The powerful technique of single-molecule fluorescence microscopy has made great contributions to the understanding of life processes that are inaccessible when using traditional approaches. In single-molecule studies, quantum dots (Qdots) have several unique advantages over other fluorescent probes, such as high brightness, extremely high photostability, and large Stokes shift, thus allowing long-time observation and improved signal-to-noise ratios. So far, however, there is no convenient way to label proteins purified from budding yeast with Qdots. Based on BirA-Avi and biotin-streptavidin systems, we have established a simple method to acquire a Qdot-labeled protein and visualize its interaction with DNA using total internal reflection fluorescence microscopy. For proof-of-concept, we chose replication protein A (RPA) and origin recognition complex (ORC) as the proteins of interest. Proteins were purified from budding yeast with high biotinylation efficiency and rapidly labeled with streptavidin-coated Qdots. Interactions between proteins and DNA were observed successfully at the single-molecule level.

A method for labeling proteins with tags at the native genomic loci in budding yeast.

Fluorescent proteins and epitope tags are often used as protein fusion tags to study target proteins. One prevailing technique in the budding yeast Saccharomyces cerevisiae is to fuse these tags to a target gene at the precise chromosomal location via homologous recombination. However, several limitations hamper the application of this technique, such as the selectable markers not being reusable, tagging of only the C-terminal being possible, and a "scar" sequence being left in the genome. Here, we describe a strategy to solve these problems by tagging target genes based on a pop-in/pop-out and counter-selection system. Three fluorescent protein tag (mCherry, sfGFP, and mKikGR) and two epitope tag (HA and 3×FLAG) constructs were developed and utilized to tag HHT1, UBC13 or RAD5 at the chromosomal locus as proof-of-concept.