Highly efficient expression of Rasamsonia emersonii lipase in Pichia pastoris: characterization and gastrointestinal simulated digestion in vitro.

BACKGROUND: Acidic lipases with high catalytic activities under acidic conditions have important application values in the food, feed and pharmaceutical industries. However, the availability of acidic lipases is still the main obstacle to their industrial applications. Although a novel acidic lipase Rasamsonia emersonii (LIPR) was heterologously expressed in Escherichia coli, the expression level was unsatisfactory. RESULTS: To achieve the high-efficiency expression and secretion of LIPR in Pichia pastoris GS115, the combinatorial optimization strategy was adopted including gene codon preference, signal peptide, molecular chaperone co-expression and disruption of vacuolar sorting receptor VPS10. The activity of the combinatorial optimization engineered strain in a shake flask reached 1480 U mL-1, which was 8.13 times greater than the P. pastoris GS115 parental strain. After high-density fermentation in a 5-L bioreactor, the highest enzyme activity reached as high as 11 820 U mL-1. LIPR showed the highest activity at 40 °C and pH 4.0 in the presence of Ca2+ ion. LIPR exhibited strong tolerance to methanol, indicating its potential application in biodiesel biosynthesis. Moreover, the gastrointestinal digestion simulation results demonstrated that LIPR was tolerant to pepsin and trypsin, but its activity was inhibited by sodium taurodeoxycholate. CONCLUSION: This study provided an effective approach for the high expression of acidic lipase LIPR. LIPR was more appropriate for lipid digestion in the stomach than in intestine according to the gastrointestinal digestion simulation results. © 2024 Society of Chemical Industry.

A refined method of quantifying deceleration capacity index for heart rate variability analysis.

BACKGROUND: Phase-rectified signal averaging (PRSA) was often applied to assess the cardiac vagal modulation. Despite its broad use, this method suffers from the confounding effects of anomalous variants of sinus rhythm. This study aimed to improve the original PRSA method in deceleration capacity (DC) quantification. METHODS: The refined deceleration capacity (DCref) was calculated by excluding from non-vagally mediated abnormal variants of sinus rhythms. Holter recordings from 202 healthy subjects and 51 patients with end-stage renal disease (ESRD) have been used for validity. The DCref was compared to original DC (DCorg) by the area under receiver operating characteristic curve. RESULTS: Experimental results demonstrate that the original and refined DCs calculated from 24-h, 2-h, and 30-min Holter recordings are significantly lower in patients with ESRD than those in the healthy group. In receiver operating characteristic curve analysis, the DCref provides better performance than the DCorg in distinguishing between the patients with ESRD and healthy control subjects. Furthermore, the refined PRSA technique enhances the low frequency and attenuates high frequency components for spectral analysis in ESRD patients. CONCLUSIONS: The DCref appears to reduce the influence of non-vagally mediated abnormal variants of sinus rhythm and highlighting the pathological influence. DCref, especially assessed from short-term electrocardiography recordings, may be complementary to existing autonomic function assessment, risk stratification, and efficacy prediction strategies.

[Variation of SpO2 during a Step-wise Paced Breathing Procedure].

Rhythmic respiratory movement in a deep and slow pattern can be beneficial to cardiovascular system, this paper investigates the effect of step-wise paced breathing procedure on blood oxygen saturation (SpO2). Experiment objects were divided into two groups, the normobaric hypoxia (simulated altitude of 4 500 meters hypoxia environment, 8 persons), normoxia and normobaric group (laboratory environment, 49 persons). The respiratory movements were performed by a high-to-low progressive change in two groups respectively. During the experiment, each object's blood oxygen saturation and heart rate were recorded. Results showed that progressive guided breathing could significantly increase the subjects' blood oxygen saturation level from 90% to 95% under the hypoxic condition. Even under the normobaric and normoxic condition, progressive guided breathing with stable blood oxygen saturation level can also enhance the blood oxygen saturation level. In both groups, mean heart rate declined in the progressive guided breathing. The research showed that the step-wise paced breathing technique could regulate the blood oxygen saturation and effectively improve the level of blood oxygen saturation by adjusting the respiratory motion in the low oxygen environment.

[Study on the Optimum Order of Autoregressive Models for Heart Rate Variability Analysis].

Heart rate variability (HRV) analysis technology based on an autoregressive (AR) model is widely used in the assessment of autonomic nervous system function. The order of AR models has important influence on the accuracy of HRV analysis. This article presents a method to determine the optimum order of AR models. After acquiring the ECG signal of 46 healthy adults in their natural breathing state and extracting the beat-to-beat intervals (RRI) in the ECG, we used two criteria, i. e. final prediction error (FPE) criterion to estimate the optimum model order for AR models, and prediction error whiteness test to decide the reliability of the model. We compared the frequency domain parameters including total power, power in high frequency (HF), power in low frequency (LF), LF power in normalized units and ratio of LF/HF of our HRV analysis to the results of Kubios-HRV. The results showed that the correlation coefficients of the five parameters between our methods and Kubios-HRV were greater than 0.95, and the Bland-Altman plot of the parameters was in the consistent band. The results indicate that the optimization algorithm of HRV analysis based on AR models proposed in this paper can obtain accurate results, and the results of this algorithm has good coherence with those of the Kubios-HRV software in HRV analysis.

[The Study of the Measurement of Heart Rate Variability Using ECG and Photoplethysmographic Signal].

In comparison with the measurement of heart rate variability from ECG and from photoplethysmographic signal from 46 healthy adults in their spontaneous breathing state. The beat-to-beat intervals in ECG and pulse-to-pulse intervals in photoplethysmographic signal are extracted, and then the parameters of heart rate variability are calculated. Three kinds of algorithms are chosen to get the pulse-to-pulse intervals, which are the intervals of maximum of second derivative, the maximum of PPG signal and the tangent intersection. The results show that the correlation coefficients of the HRV parameters in the two calculation methods are highly correlated. The Bland-Altman scattered plots show the relative bias results from the algorithm of the maximum of PPG signal are smallest and singular points that deviate from the consistent limits are the least compared with the other two algorithms.

[Change of pulse transit time during stepwise paced breathing].

To investigate the effect of stepwise paced breathing (PB) on pulse transit time (PTT), we collected physiological signals of electrocardiogram (ECG), respiration and arterial pulse wave during a procedure of stepwise PB, which consists of 6 different breathing rates changing in a protocol of 14.0-12.5-11.0-9.5-8.0-7.0 breath per minute (BPM), with each breathing rate lasting 3 minutes. Twenty two healthy adults involved in this experiment and the change of PTT was analyzed during the stepwise PB procedure. In our study, the PTT was measured by calculating the time interval from the R-spike of the ECG to the peaks of the second derivative of the arterial pulse wave. Ensemble empirical mode decomposition (EEMD) was applied to PTT to decompose the signal into different intrinsic mode function, and respiratory oscillation and trend component (baseline) in PTT were further extracted. It was found that the respiratory oscillations in the PTT increased with decreasing of the PB rate, and many of the subjects (14 out of 22) showed the phenomena of PTT baseline increasing during the stepwise PB procedure. The results indicated that the stepwise PB procedure induced a high level of cardiovascular oscillation and produced an accumulative effect of PTT baseline increase. As PTT is capable of predicting changes in BP over a short period of time, increase of PTT baseline indicates the decrease of blood pressure. The experiments showed that the stepwise PB procedure could reduce blood pressure for most subjects. For future work, it is necessary to develop certain indices differentiating the effectiveness of the stepwise PB procedure on the PTT baseline change, and to test the effectiveness of this stepwise PB procedure on blood pressure reduction for patients with essential hypertension.

[Application of three kinds of method for extracting nonlinear trend in biomedical signal analysis].

Biomedical signal analysis often needs to separate the trend component from the non-trend component to achieve different purposes and applications in signal analysis. This article introduces three kinds of detrending nonlinear component methods used in the process of biomedical signal analysis: wavelet analysis, empirical mode decomposition, smoothness priors approach as well as the application in the separation of the actual biomedical data. The different separation methods should be selected according to different research goals as well as the feature of the signal.

[Comprehensive testing system for cardiorespiratory interaction research].

To investigate the modulation effects of breathing movement on cardiovascular system and to study the physiological coupling relationship between respiration and cardiovascular system, we designed a comprehensive testing system for cardiorespiratory interaction research. This system, comprising three parts, i. e. physiological signal conditioning unit, data acquisition and USB medical isolation unit, and a PC based program, can acquire multiple physiological data such as respiratory flow, rib cage and abdomen movement, electrocardiograph, artery pulse wave, cardiac sounds, skin temperature, and electromyography simultaneously under certain experimental protocols. Furthermore this system can be used in research on short-term cardiovascular variability by paced breathing. Preliminary experiments showed that this system could accurately record rib cage and abdomen movement under very low breathing rate, using respiratory inductive plethysmography to acquire respiration signal in direct-current coupling mode. After calibration, this system can be used to estimate ventilation non-intrusively and correctly. The PC based program can generate audio and visual biofeedback signal, and guide the volunteers to perform a slow and regular breathing. An experiment on healthy volunteers showed that this system was able to guide the volunteers to do slow breathing effectively and simultaneously record multiple physiological data during the experiments. Signal processing techniques were used for off-line data analysis, such as non-invasive ventilation calibration, QRS complex wave detection, and respiratory sinus arrhythmia and pulse wave transit time calculation. The experiment result showed that the modulation effect on RR interval, respiratory sinus arrhythmia (RSA), pulse wave transit time (PWTT) by respiration would get stronger with the going of the slow and regular breathing.

Highly efficient expression and secretion of human lysozyme using multiple strategies in Pichia pastoris.

BACKGROUND: Human lysozyme (hLYZ), an emerging antibacterial agent, has extensive application in the food and pharmaceutical industries. However, the source of hLYZ is particularly limited. RESULTS: To achieve highly efficient expression and secretion of hLYZ in Pichia pastoris, multiple strategies including G418 sulfate screening, signal sequence optimization, vacuolar sorting receptor VPS10 disruption, and chaperones/transcription factors co-expression were applied. The maximal enzyme activity of extracellular hLYZ in a shaking flask was 81,600 ± 5230 U mL-1 , which was about five times of original strain. To further reduce the cost, the optimal medium RDMY was developed and the highest hLYZ activity reached 352,000 ± 16,696.5 U mL-1 in a 5 L fermenter. CONCLUSION: This research provides a very useful and cost-effective approach for the hLYZ production in P. pastoris and can also be applied to the production of other recombinant proteins.

Active Ingredients of Reduning Injection Maintain High Potency against SARS-CoV-2 Variants.

OBJECTIVE: To investigate the anti-coronavirus potential and the corresponding mechanisms of the two ingredients of Reduning Injection: quercetin and luteolin. METHODS: A pseudovirus system was designed to test the efficacy of quercetin and luteolin to inhibit SARS-CoV-2 infection and the corresponding cellular toxicity. Luteolin was tested for its activities against the pseudoviruses of SARS-CoV-2 and its variants. Virtual screening was performed to predict the binding sites by Autodock Vina 1.1.230 and PyMol. To validate docking results, surface plasmon resonance (SPR) was used to measure the binding affinity of the compounds with various proteins of the coronaviruses. Quercetin and luteolin were further tested for their inhibitory effects on other coronaviruses by indirect immunofluorescence assay on rhabdomyosarcoma cells infected with HCoV-OC43. RESULTS: The inhibition of SARS-CoV-2 pseudovirus by luteolin and quercetin were strongly dose-dependent, with concentration for 50% of maximal effect (EC50) of 8.817 and 52.98 µmol/L, respectively. Their cytotoxicity to BHK21-hACE2 were 177.6 and 405.1 µmol/L, respectively. In addition, luetolin significantly blocked the entry of 4 pseudoviruses of SARS-CoV-2 variants, with EC50 lower than 7 µmol/L. Virtual screening and SPR confirmed that luteolin binds to the S-proteins and quercetin binds to the active center of the 3CLpro, PLpro, and helicase proteins. Quercetin and luteolin showed over 99% inhibition against HCoV-OC43. CONCLUSIONS: The mechanisms were revealed of quercetin and luteolin inhibiting the infection of SARS-CoV-2 and its variants. Reduning Injection is a promising drug for COVID-19.

Development of a respiratory inductive plethysmography module supporting multiple sensors for wearable systems.

In this paper, we present an RIP module with the features of supporting multiple inductive sensors, no variable frequency LC oscillator, low power consumption, and automatic gain adjustment for each channel. Based on the method of inductance measurement without using a variable frequency LC oscillator, we further integrate pulse amplitude modulation and time division multiplexing scheme into a module to support multiple RIP sensors. All inductive sensors are excited by a high-frequency electric current periodically and momentarily, and the inductance of each sensor is measured during the time when the electric current is fed to it. To improve the amplitude response of the RIP sensors, we optimize the sensing unit with a matching capacitor parallel with each RIP sensor forming a frequency selection filter. Performance tests on the linearity of the output with cross-sectional area and the accuracy of respiratory volume estimation demonstrate good linearity and accurate lung volume estimation. Power consumption of this new RIP module with two sensors is very low. The performance of respiration measurement during movement is also evaluated. This RIP module is especially desirable for wearable systems with multiple RIP sensors for long-term respiration monitoring.

[Research on the respiratory sinus arrhythmia in the process of guided breathing].

To investigate the effects of gradually guided breathing on respiratory sinus arrhythmia (RSA), 15 healthy men's simultaneous ECG and respiratory signals during the guided breathing process (14 beats/min-12.5 beats/ min--1 beats/min--9.5 beats/min--8 beats/minute--7 beats/min) were acquired. Based on the frequency domain and time domain methods, the effects of respiratory rate on RSA were studied. A general tendency increase features the three parameters of RSA as respiratory rate decreased. RSA represents the reflection control of respiratory system to cardiovascular system. The results suggested that the function of the reflection control of respiratory system to cardiovascular system increaseed with guided breathing decreasing. The conclusion indicates that the cardiovascular system could be improved by regulating the mode of respiration.

[The development of heart rate variability analysis system].

A heart rate variability (HRV)analysis system is presented. The change of the activity of Sympathetic and vagus nerve is evaluated by detecting the parameters of heart rate variability of the tested in different states using the system. The system provides a tool for the study of heart rate variability.

[Design of hand-held heart rate variability acquisition and analysis system].

A design of handheld heart rate variability acquisition and analysis system is proposed. The system collects and stores the patient's ECG every five minutes through both hands touching on the electrodes, and then -uploads data to a PC through USB port. The system uses software written in LabVIEW to analyze heart rate variability parameters, The parameters calculated function is programmed and generated to components in Matlab.

[Study of analysis of the singularity of R-wave by using wavelet transform].

Singularity is a basic feature of biological signals. Based on the variations of wavelet transform modulus maxima in multi-scales, we studied the basic theorem for analyzing the singularity and proposed an algorithm for calculating lipschitz exponent. Then we applied the algorithm to calculate the singularity of R-wave in ECG. Our study found that the level of singularity of R-waves in ECG between the 10 arrhythm patients randomly chosen and the healthy persons was significantly different, with the level of singularity of R-wave of normal persons remarkably higher than that of arrhythmia patients.

[Research progress of quantitative analysis for respiratory sinus arrhythmia].

Respiratory sinus arrhythmia (RSA) is known as fluctuations of heart rate associated with breathing. It has been increasingly used as a noninvasive index of cardiac vagal tone in psychophysiological research recently. Its analysis is often influenced or distorted by respiratory parameters, posture and action, etc. This paper reviews five methods of quantification, including the root mean square of successive differences (RMSSD), peak valley RSA (pvRSA), cosinor fitting, spectral analysis, and joint timing-frequency analysis (JTFA). Paced breathing, analysis of covariance, residua method and msRSA per liter tidal volume are adjustment strategies of measurement and analysis of RSA in this article as well. At last, some prospects of solutions of the problems of RSA research are given.

R-Wave Singularity: A New Morphological Approach to the Analysis of Cardiac Electrical Dyssynchrony.

R-wave singularity (RWS) measures the intermittence or discontinuousness of R waves. It has been broadly used in QRS (QRS complex of electrocardiogram) detection, electrocardiogram (ECG) beats classification, etc. In this article, we novelly developed RWS to the analysis of QRS morphology as the measurement of ventricular dyssynchrony and tested the hypothesis that RWS could enhance the discrimination between control and acute myocardial infarction (AMI) patients. Holter ECG recordings were obtained from the Telemetric and Holter ECG Warehouse database, among which database Normal was extracted as normal controls (n = 202) and database AMI (n = 93) as typical subjects of autonomic nervous system dysfunction and cardiac electrical dyssynchrony with high risk for cardiac arrhythmias and sudden cardiac death. Experimental results demonstrate that RWS measured by Lipschitz exponent calculated from 5-min Holter recordings was significantly less negative in early AMI and late AMI than that in Normal subjects for overall, elderly, and elderly male groups, which suggested the heterogeneous depolarization of the ventricular myocardium during AMI. Receiver operating characteristic curve analyses show that combined with heart rate variability parameters, Lipschitz exponent provides higher accuracy in distinguishing between the patients with AMI and healthy control subjects for overall, elderly, elderly male, and elderly female groups. In summary, our study demonstrates the significance of using RWS to probe the cardiac electrical dyssynchrony for AMI. Lipschitz exponent may be valuable and complementary for existing cardiac resynchronization therapy and autonomic nervous system assessment.

Comparative genome analysis and mining of secondary metabolites of Paenibacillus polymyxa.

Paenibacillus polymyxa is a well-known Gram-positive biocontrol bacterium. It has been reported that many P. polymyxa strains can inhibit bacteria, fungi and other plant pathogens. Paenibacillus polymyxa employs a variety of mechanisms to promote plant growth, so it is necessary to understand the biocontrol ability of bacteria at the genome level. In the present study, thanks to the widespread availability of Paenibacillus genome data and the development of bioinformatics tools, we were able to analyze and mine the genomes of 43 P. polymyxa strains. The strain NCTC4744 was determined not to be P. polymyxa according to digital DNA-DNA hybridization and average nucleotide identity. By analysis of the pan-genome and the core genome, we found that the pan-genome of P. polymyxa was open and that there were 3,192 core genes. In a gene cluster analysis of secondary metabolites, 797 secondary metabolite gene clusters were found, of which 343 are not similar to known clusters and are expected to reveal a large number of new secondary metabolites. We also analyzed the plant growth-promoting genes that were mined and found, surpisingly, that these genes are highly conserved. The results of the present study not only reveal a large number of unknown potential secondary metabolite gene clusters in P. polymyxa, but also suggest that plant growth promotion characteristics are evolutionary adaptations of P. polymyxa to plant-related habitats.

Genomics and LC-MS Reveal Diverse Active Secondary Metabolites in Bacillus amyloliquefaciens WS-8.

Bacillus amyloliquefaciens is an important plant disease-preventing and growth-promoting microorganism. B. amyloliquefaciens WS-8 can stimulate plant growth and has strong antifungal properties. In this study, we sequenced the complete genome of B. amyloliquefaciens WS-8 by Pacific Biosciences RSII (PacBio) Single Molecule Real-Time (SMRT) sequencing. The genome consists of one chromosome (3,929,787 bp) and no additional plasmids. The main bacteriostatic substances were determined by genome, transcriptome, and mass spectrometry data. We thereby laid a theoretical foundation for the utilization of the strain. By genomic analysis, we identified 19 putative biosynthetic gene clusters for secondary metabolites, most of which are potentially involved in the biosynthesis of numerous bioactive metabolites, including difficidin, fengycin, and surfactin. Furthermore, a potential class II lanthipeptide biosynthetic gene cluster and genes that are involved in auxin biosynthesis were found. Through the analysis of transcriptome data, we found that the key bacteriostatic genes, as predicted in the genome, exhibited different levels of mRNA expression. Through metabolite isolation, purification, and exposure experiments, we found that a variety of metabolites of WS-8 exert an inhibitory effect on the necrotrophic fungus Botrytis cinerea, which causes gray mold; by mass spectrometry, we found that the main substances are mainly iturins and fengycins. Therefore, this strain has the potential to be utilized as an antifungal agent in agriculture.

[Interactive guided breathing technology].

OBJECT: Scientific guided breathing technology is to be studied based on the cardiopulmonary interaction. METHODS: Heart rate variability was used as the target function to study the smoothly respiratory relaxation procedure in order to acquire the common pattern of regular and slow breathing. RESULTS: Music based on the acquired common pattern was created and a musical pattern temporally-related to the breathing movement monitored by a sensor could be chosen to guide the breathing interactively.