Enhanced extracellular production of alpha-lactalbumin from Bacillus subtilis through signal peptide and promoter screening

Alpha-lactalbumin(α-LA)is a major whey protein found in breast milk and plays a crucial role in the growth and development of infants.In this study,Bacillus subtilis RIK1285 harboring AprE signal peptide(SP)was selected as the original strain for the production ofα-LA.It was found thatα-LA was identified in the pellet after ultrasonic disruption and centrifugation instead of in the fermentation supernatant.The original strain most likely only producedα-LA intracellular,but not extracellular.To improve the expression and secretion ofα-LA in RIK1285,a library of 173 homologous SPs from the B.subtilis 168 genome was fused with target LALBA gene in the pBE-S vector and expressed extracellularly in RIK1285.SP YjcN was determined to be the best signal peptide.Bands in supernatant were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and purified by nickel column to calculate the highest yield signal peptide.In addition,different promoters(P_(aprE),P_(43),and P_(glv))were compared and applied.The results indicated that the strain RIK1285-pBE-P_(glv)-YjcN-LALBA had the highestα-LA yield,reaching 122.04μg/mL.This study demonstrates successful expression and secretion of humanα-LA in B.subtilis and establishes a foundation for simulating breast milk for infant formulas and developing bioengineered milk.

Low RBC counts predict high on-treatment platelet reactivity in patients undergoing percutaneous coronary intervention and treated with clopidogrel

Dear Editor,Cardiovascular disease is the leading cause of deaths worldwide,with coronary artery disease(CAD)accounting for approximately 50%of its mortality.Dual antiplatelet therapy,including aspirin and a P2Y12 inhibitor,is the most important treatment for CAD patients undergoing percutaneous coronary intervention(PCI)to prevent recurrent ischemic events and cardiac death.Clopidogrel is one of the commonly used P2Y12 inhibitors.However,up to 30%of patients treated with a standard dose of clopidogrel present with high on-treatment platelet reactivity(HOPR),which is associated with the increased ischemic risks[1].The causes of HOPR are multifactorial and complex.

Diagnostic and prognostic value of minor elevated cardiac troponin levels for percutaneous coronary intervention-related myocardial injury:a prospective,single-center and double-blind study

Cardiac troponin-I （cTnI） and -T （cTnT） are sensitive and specific markers of myocardial injury. However, the role of increased cTnI and cTnT in percutaneous coronary intervention （PCI）-related myocardial injury remains controversial. In this prospective, single-center and double-blind study, we aimed to determine the diagnostic and prognostic value of cTnI as well as cTnT （cTns） in PCI-related myocardial injury in a Chinese population. A total of 1,008 patients with stable angina pectoris and non-ST-segment elevation acute coronary syndrome were recruited. The levels of cTnI and cTnT were examined before and after PCI. All patients were followed up for 26± 9 months to observe the incidence of major adverse cardiac events （MACEs）. Our results showed that post- PCI cTnI and/or cTnT levels were increased to more than the 99^th percentile upper reference limit （URL） in 133 （13.2%） patients, among which 22 （2.2%） were more than 5 × 99^th percentile URL. By univariate analysis, an elevation in cTns after PCI was not an independent predictor of increased MACEs, HR 1.35 （P = 0.33, 95% CI： 0.74-2.46）. In conclusion, our data demonstrate that the incidence of PCI-related myocardial injury is not common in a Chinese population and minor elevated cTns levels may not be a sensitive prognostic marker for MACEs.

Root growth and spatio-temporal distribution of three common annual halophytes in a saline desert, northern Xinjiang

Root growth and spatial and temporal distribution in the 0-100 cm soil profiles of three common annual halophytes Salsola subcrassa, Suaeda acuminate and Petrosimonia sibirica distributed in a saline desert in north- ern Xinjiang, China were studied in 2009 and 2010. The results showed that the root systems of the three halo- phytes were of the taproot type, vertically distributed in the 90-cm soil profile, and were deepest in late July. Their taproots reached maximum depth rapidly, early in the growth period, but with rare lateral roots. They were then dug out in an orderly way, from bottom to top, exhibiting vertical development first and then horizontal development. The distribution of specific root length, which reflects the characteristics of the feeder root, was gradually increased from top to bottom, whereas root weight displayed an opposite distribution pattern. The root length distribution of the three halophytes was concentrated （62% to 76%） in the middle soil profile （20-60 cm）, with less distribution in the surface （0-20 cm） and bottom （60-90 cm） soil profiles. The results indicated that the roots of the three annual halophytes grew rapidly into the deeper soil layer after germination, which ensured the plant survival and uptake of water and nutrition, and thus built up a strong tolerance to an arid, high-salt environment.

Characteristics of mineral elements in shoots of three annual halophytes in a saline desert, Northern Xinjiang

Halophytes are valuable salt-, alkali- and drought-resistant germplasm resources. However, the char- acteristics of mineral elements in halophytes have not been investigated as intensively as those in crops. This study attempted to investigate the characteristics of mineral elements for annual halophytes during their growth period to reveal their possible physiological mechanisms of salt resistance. By using three native annual halophytes （Salsola subcrassa, Suaeda acuminate and Petrosimonia sibirica） distributed in the desert in Northern Xinjiang of China, the dynamic changes in the mineral element contents of annual halophytes were analyzed through field sampling and laboratory analyses. The results demonstrated that the annual halophytes were able to absorb water and mineral nutrients selectively. In the interaction between the annual halophytes and saline soil, the adaptability of the annual halophytes was manifested as the accumulation of S, Na and CI during the growth period and maintenance of water and salt balance in the plant, thus ensuring their selective absorption of N, P, K, Ca, Mg and other mineral nutrients according to their growth demand. By utilizing this property, halophyte planting and mowing （before the wilting and death periods） could bioremediate heavy saline-alkali soil.

Simultaneous determination of clopidogrel,2-oxo-clopidogrel,and the thiol metabolite of clopidogrel in human plasma by LC-MS/MS

Clopidogrel is a pro-drug which needs two-step metabolism to produce the active thiol metabolite.This study aimed to explore an efficient method to simultaneously determine the plasma clopidogrel,2-oxo-clopidogrel(2-Oxo-CLP),and the clopidogrel active metabolite(CAM).A high-throughput liquid chromatography tandem mass spectrometry(LC-MS/MS)was therefore developed.The analytes were extracted from plasma by using methyl tert-butyl ether(MTBE).Chromatographic separation was performed on a C18 column under an isocratic elution,accompanied with acetonitrile and deionized water containing 0.1%formic acid.After optimizing the condition of LC-MS/MS,a stable linearity was observed in the standard curves over the concentration ranges of 0.05 to 50.0 ng/mL for clopidogrel,0.5 to 50.0 ng/mL for 2-Oxo-CLP,and 0.5 to 100 ng/mL for clopidogrel active metabolite derivative(CAMD).The retention time was 4.78 minutes,3.79 minutes,3.59 minutes,and 4.82 minutes for clopidogrel,2-Oxo-CLP,CAMD,and internal standard,respectively.Both the relative standard deviation and the relative error were within the requirement of operating criteria.No significant degradation of clopidogrel,2-Oxo-CLP,and CAMD occurred under different storage conditions.This method was successfully validated in 3 patients with coronary artery disease.The results showed that the current LC-MS/MS method was efficient for simultaneously detecting clopidogrel,2-Oxo-CLP,and CAM with fine linearity,accuracy,precision,and stability.

Comparison of N uptake and internal use efficiency in two tobacco varieties

To explain the observation in field experiments that tobacco variety CB-1 was more nitrogen(N)-efficient than K326, the influence of two N levels on growth, N uptake and N flow within plants of the two tobacco varieties was studied. Xylem sap from the upper and lower leaves of both tobacco varieties cultured in quartz sand was collected by application of pressure to the root system. CB-1 took up more N with smaller roots at both high(HN, 10 mmol L-1) and low(LN, 1 mmol L-1) N levels, and built up more new tissues in upper leaves especially at LN level,than K326. Both varieties showed luxury N uptake, and CB-1 accumulated significantly less NO-3in new tissues than K326, when grown at the HN level. At both N levels, the amount of xylem-transported N and phloem-cycled N from shoot to root in K326 was greater than those in CB-1, indicating higher N use efficiency in CB-1 shoots than in K326 shoots. The major nitrogenous compound in the xylem sap was NO-3irrespective of N level and variety. Low N supply did not cause more NO-3reduction in the root. The results indicated that the N-efficient tobacco variety CB-1 was more efficient in both N uptake by smaller roots and N utilization in shoots, especially when grown at the LN level.

Efficacy of an adjusted treatment strategy on the management and in-hospital outcome of patients with STEMI during the COVID-19 pandemic

Dear Editor,The coronavirus disease 2019(COVID-19) broke out in early December 2019 in Wuhan, China~([1]), which put tremendous pressure on the medical system. A nationwide lockdown and strict quarantine measures proved effective in reducing the spread of the pandemic. However, it might have affected the management of time-dependent diseases such as STelevation myocardial infarction(STEMI)~([2]).

Efficient biosynthesis of creatine by whole-cell catalysis from guanidinoacetic acid in Corynebacterium glutamicum

Creatine is a naturally occurring derivative of an amino acid commonly utilized in functional foods and pharmaceuticals.Nevertheless,the current industrial synthesis of creatine relies on chemical processes,which may hinder its utilization in certain applications.Therefore,a biological approach was devised that employs whole-cell biocatalysis in the bacterium Corynebacterium glutamicum,which is considered safe for use in food production,to produce safe-for-consumption creatine.The objective of this study was to identify a guanidinoacetate N-methyltransferase(GAMT)with superior catalytic activity for creatine production.Through employing whole-cell biocatalysis,a gamt gene from Mus caroli(Mcgamt)was cloned and expressed in C.glutamicum ATCC 13032,resulting in a creatine titer of 3.37 g/L.Additionally,the study employed a promoter screening strategy that utilized nine native strong promoters in C.glutamicum to enhance the expression level of GAMT.The highest titer was achieved using the P1676 promoter,reaching 4.14 g/L.The conditions of whole-cell biocatalysis were further optimized,resulting in a creatine titer of 5.42 g/L.This is the first report of successful secretory creatine expression in C.glutamicum,which provides a safer and eco-friendly approach for the industrial production of creatine.

Growth,Nitrogen Uptake and Flow in Maize Plants Affected by Root Growth Restriction

The objective of the present study was to investigate the influence of a reduced maize root-system size on root growth and nitrogen （N） uptake and flow within plants. Restriction of shoot-borne root growth caused a strong decrease in the absorption of root ： shoot dry weight ratio and a reduction in shoot growth. On the other hand, compensatory growth and an increased N uptake rate in the remaining roots were observed. Despite the limited long-distance transport pathway in the mesocotyl with restriction of shoot-borne root growth, N cycling within these plants was higher than those in control plants, implying that xylem and phloem flow velocities via the mesocotyl were considerably higher than in plants with an intact root system. The removal of the seminal roots in addition to restricting shoot-borne root development did not affect whole plant growth and N uptake, except for the stronger compensatory growth of the primary roots. Our results suggest that an adequate N supply to maize plant is maintained by compensatory growth of the remaining roots, increased N uptake rate and flow velocities within the xylem and phloem via the mesocotyl, and reduction in the shoot growth rate.

Nitrogen Under-and Over-supply Induces Distinct Protein Responses in Maize Xylem Sap

Xylem sap primarily transports water and mineral nutrients such as nitrogen （N） from roots to shoots in vascular plants. However, it remains largely unknown how nitrogenous compounds, especially proteins in xylem sap, respond to N under- or over-supply. We found that reducing N supply increased amino-N percentage of total N in maize （Zea mays L.） xylem sap. Proteomic analysis showed that 23 proteins in the xylem sap of maize plants, including 12 newly identified ones, differentially accumulated in response to various N supplies. Fifteen of these 23 proteins were primarily involved in general abiotic or biotic stress responses, whereas the other five proteins appeared to respond largely to N under- or over-supply, suggesting distinct protein responses in maize xylem upon N under- and over-supply. Furthermore, one putative xylanase inhibitor and two putative O-glycosyl hydrolases had preferential gene expression in shoots.