A "Ferroptosis-Amplifier" Hydrogel for Eliminating Refractory Cancer Stem Cells Post-lumpectomy.

The unique "Iron Addiction" feature of cancer stem cells (CSCs) with tumorigenicity and plasticity generally contributes to the tumor recurrence and metastasis after a lumpectomy. Herein, a novel "Ferroptosis Amplification" strategy is developed based on integrating gallic acid-modified FeOOH (GFP) and gallocyanine into Pluronic F-127 (F127) and carboxylated chitosan (CC)-based hydrogel for CSCs eradication. This "Ferroptosis Amplifier" hydrogel is thermally sensitive and achieves rapid gelation at the postsurgical wound in a breast tumor model. Specifically, gallocyanine, as the Dickkopf-1 (DKK1) inhibitor, can decrease the expression of SLC7A11 and GPX4 and synergistically induce ferroptosis of CSCs with GFP. Encouragingly, it is found that this combination suppresses the migratory and invasive capability of cancer cells via the downregulation of matrix metalloproteinase 7 (MMP7). The in vivo results further confirm that this "Ferroptosis Amplification" strategy is efficient in preventing tumor relapse and lung metastasis, manifesting an effective and promising postsurgical treatment for breast cancer.

HtrAs are essential for the survival of the haloarchaeon Natrinema gari J7-2 in response to heat, high salinity, and toxic substances.

Bacterial and eukaryotic HtrAs can act as an extracytoplasmic protein quality control (PQC) system to help cells survive in stress conditions, but the functions of archaeal HtrAs remain unknown. Particularly, haloarchaea route most secretory proteins to the Tat pathway, enabling them to fold properly in well-controlled cytoplasm with cytosolic PQC systems before secretion. It is unclear whether HtrAs are required for haloarchaeal survival and stress response. The haloarchaeon Natrinema gari J7-2 encodes three Tat signal peptide-bearing HtrAs (NgHtrA, NgHtrB, and NgHtrC), and the signal peptides of NgHtrA and NgHtrC contain a lipobox. Here, the in vitro analysis reveals that the three HtrAs show different profiles of temperature-, salinity-, and metal ion-dependent proteolytic activities and could exhibit chaperone-like activities to prevent the aggregation of reduced lysozyme when their proteolytic activities are inhibited at low temperatures or the active site is disrupted. The gene deletion and complementation assays reveal that NgHtrA and NgHtrC are essential for the survival of strain J7-2 at elevated temperature and/or high salinity and contribute to the resistance of this haloarchaeon to zinc and inhibitory substances generated from tryptone. Mutational analysis shows that the lipobox mediates membrane anchoring of NgHtrA or NgHtrC, and both the membrane-anchored and free extracellular forms of the two enzymes are involved in the stress resistance of strain J7-2, depending on the stress conditions. Deletion of the gene encoding NgHtrB in strain J7-2 causes no obvious growth defect, but NgHtrB can functionally substitute for NgHtrA or NgHtrC under some conditions.IMPORTANCEHtrA-mediated protein quality control plays an important role in the removal of aberrant proteins in the extracytoplasmic space of living cells, and the action mechanisms of HtrAs have been extensively studied in bacteria and eukaryotes; however, information about the function of archaeal HtrAs is scarce. Our results demonstrate that three HtrAs of the haloarchaeon Natrinema gari J7-2 possess both proteolytic and chaperone-like activities, confirming that the bifunctional nature of HtrAs is conserved across all three domains of life. Moreover, we found that NgHtrA and NgHtrC are essential for the survival of strain J7-2 under stress conditions, while NgHtrB can serve as a substitute for the other two HtrAs under certain circumstances. This study provides the first biochemical and genetic evidence of the importance of HtrAs for the survival of haloarchaea in response to stresses.

Probiotic Compounds Enhanced Recovery after Surgery for Patients with Distal Gastric Cancer: A Prospective, Controlled Clinical Trial.

BACKGROUND: Enhanced recovery after surgery (ERAS) for radical distal gastrectomy needs to be improved urgently. We investigated the effects of probiotic compounds (including Lactobacillus plantarum, L. rhamnosus, L. acidophilus, and Bifidobacterium animalis subsp.lactis) on enhance recovery after gastrectomy. METHODS: The patients in this prospective study were divided into probiotic group (PG group, n = 36) and placebo group (CG group, n = 38), taking corresponding capsule according to the protocol during the perioperative period. We compared the trends in perioperative hematologic findings and the postoperative outcomes. Patients' feces were collected for bacterial 16S rRNA sequencing. Patients were followed up at 1 month postoperatively. RESULTS: After the application of probiotics, the patients' postoperative inflammatory response level was reduced, and the trend of postoperative NLR decrease was significantly faster in the patients of the PG group than in the CG group (P = 0.047, partial η2 = 0.054). The trend of postoperative increase in serum albumin concentration in the patients of the PG group was significantly better than that in the CG group (P = 0.016, partial η2 = 0.078). In addition, patients in the PG group met discharge criteria earlier postoperatively and had fewer medical expenses. The quality of life of PG group was improved postoperatively. Postoperative inflammation-related markers, including the ratio of Firmicutes/Bacteroidetes, were increasing in untreated patients. In addition, the postoperative microbial diversity and abundance in the PG group remained stable. CONCLUSIONS: Probiotic compounds can reduce the inflammatory response after gastrectomy and enhance the recovery of the DGC patients by maintaining the stability of the gut microbiota.

GM-lncLoc: LncRNAs subcellular localization prediction based on graph neural network with meta-learning.

In recent years, a large number of studies have shown that the subcellular localization of long non-coding RNAs (lncRNAs) can bring crucial information to the recognition of lncRNAs function. Therefore, it is of great significance to establish a computational method to accurately predict the subcellular localization of lncRNA. Previous prediction models are based on low-level sequences information and are troubled by the few samples problem. In this study, we propose a new prediction model, GM-lncLoc, which is based on the initial information extracted from the lncRNA sequence, and also combines the graph structure information to extract high level features of lncRNA. In addition, the training mode of meta-learning is introduced to obtain meta-parameters by training a series of tasks. With the meta-parameters, the final parameters of other similar tasks can be learned quickly, so as to solve the problem of few samples in lncRNA subcellular localization. Compared with the previous methods, GM-lncLoc achieved the best results with an accuracy of 93.4 and 94.2% in the benchmark datasets of 5 and 4 subcellular compartments, respectively. Furthermore, the prediction performance of GM-lncLoc was also better on the independent dataset. It shows the effectiveness and great potential of our proposed method for lncRNA subcellular localization prediction. The datasets and source code are freely available at https://github.com/JunzheCai/GM-lncLoc .

Research on the Security of IPv6 Communication Based on Petri Net under IoT.

The distribution of wireless network systems challenges the communication security of Internet of Things (IoT), and the IPv6 protocol is gradually becoming the main communication protocol under the IoT. The Neighbor Discovery Protocol (NDP), as the base protocol of IPv6, includes address resolution, DAD, route redirection and other functions. The NDP protocol faces many attacks, such as DDoS attacks, MITM attacks, etc. In this paper, we focus on the communication-addressing problem between nodes in the Internet of Things (IoT). We propose a Petri-Net-based NS flooding attack model for the flooding attack problem of address resolution protocols under the NDP protocol. Through a fine-grained analysis of the Petri Net model and attacking techniques, we propose another Petri-Net-based defense model under the SDN architecture, achieving security for communications. We further simulate the normal communication between nodes in the EVE-NG simulation environment. We implement a DDoS attack on the communication protocol by an attacker who obtains the attack data through the THC-IPv6 tool. In this paper, the SVM algorithm, random forest algorithm (RF) and Bayesian algorithm (NBC) are used to process the attack data. The NBC algorithm is proven to exhibit high accuracy in classifying and identifying data through experiments. Further, the abnormal data are discarded through the abnormal data processing rules issued by the controller in the SDN architecture, to ensure the security of communications between nodes.

Negative Design Margin Realization through Deep Path Activity Detection Combined with Dynamic Voltage Scaling in a 55 nm Near-Threshold 32-Bit Microcontroller.

This paper presents an innovative approach for predicting timing errors tailored to near-/sub-threshold operations, addressing the energy-efficient requirements of digital circuits in applications, such as IoT devices and wearables. The method involves assessing deep path activity within an adjustable window prior to the root clock's rising edge. By dynamically adapting the prediction window and supply voltage based on error detection outcomes, the approach effectively mitigates false predictions-an essential concern in low-voltage prediction techniques. The efficacy of this strategy is demonstrated through its implementation in a near-/sub-threshold 32-bit microprocessor system. The approach incurs only a modest 6.84% area overhead attributed to well-engineered lightweight design methodologies. Furthermore, with the integration of clock gating, the system functions seamlessly across a voltage range of 0.4 V-1.2 V (5-100 MHz), effectively catering to adaptive energy efficiency. Empirical results highlight the potential of the proposed strategy, achieving a significant 46.95% energy reduction at the Minimum Energy Point (MEP, 15 MHz) compared to signoff margins. Additionally, a 19.75% energy decrease is observed compared to the zero-margin operation, demonstrating successful realization of negative margins.

[Application of copy number variation sequencing in patients with intellectual disability/developmental delay and autistic spectrum disorder].

OBJECTIVE: To assess the value of copy number variation sequencing (CNV-seq) for the diagnosis of children with intellectual disability (ID), developmental delay (DD), and autistic spectrum disorder (ASD). METHODS: Forty patients with ID/DD/ASD referred to Nanshan Maternity and Child Health Care Hospital from September 2018 to January 2022 were enrolled. G-banded karyotyping analysis was carried out for the patients. Genomic DNA was extracted from peripheral blood samples and subjected to CNV-Seq analysis to detect chromosome copy number variations (CNVs) in such patients. ClinVar, DECIPHER, OMIM and other database were searched for data annotation. RESULTS: Among the 40 patients (including 30 males and 10 females), 16, 15 and 6 were diagnosed with ID, DD and ASD, respectively. One patient had combined symptoms of ID and DD, whilst the remaining two had combined ID and ASD. Four patients were found with abnormal karyotypes, including 47,XY,+mar, 46,XY,inv(8)(p11.2q21.2), 46,XX,del(5)(p14) and 46,XX[76]/46,X,dup(X)(p21.1q12). Chromosome polymorphism was also found in two other patients. CNV-seq analysis has detected 32 CNVs in 20 patients (50.0%, 20/40). Pathogenic CNVs were found in 10 patients (25.0%), 15 CNVs of uncertain clinical significance were found in 12 patients (30.0%), and 7 likely benign CNVs were found in 4 patients (10.0%). CONCLUSION: Chromosome CNVs play an important role in the pathogenesis of ID/DD/ASD. CNV-seq can detect chromosomal abnormalities including microdeletions and microduplications, which could provide a powerful tool for revealing the genetic etiology of ID/DD/ASD patients.

Sec-Dependent Secretion of Subtilase SptE in Haloarchaea Facilitates Its Proper Folding and Heterocatalytic Processing by Halolysin SptA Extracellularly.

In response to high-salt conditions, haloarchaea export most secretory proteins through the Tat pathway in folded states; however, it is unclear why some haloarchaeal proteins are still routed to the Sec pathway. SptE is an extracellular subtilase of Natrinema sp. strain J7-2. Here, we found that SptE precursor comprises a Sec signal peptide, an N-terminal propeptide, a catalytic domain, and a long C-terminal extension (CTE) containing seven domains (C1 to C7). SptE is produced extracellularly as a mature form (M180) in strain J7-2 and a proform (ΔS) in the ΔsptA mutant strain, indicating that halolysin SptA mediates the conversion of the secreted proform into M180. The proper folding of ΔS is more efficient in the presence of NaCl than KCl. ΔS requires SptA for cleavage of the N-terminal propeptide and C-terminal C6 and C7 domains to generate M180, accompanied by the appearance of autoprocessing product M120 lacking C5. At lower salinities or elevated temperatures, M180 and M120 could be autoprocessed into M90, which comprises the catalytic and C1 domains and has a higher activity than M180. When produced in Haloferax volcanii, SptE could be secreted as a properly folded proform, but its variant (TSptE) with a Tat signal peptide does not fold properly and suffers from severe proteolysis extracellularly; meanwhile, TSptE is more inclined to aggregate intracellularly than SptE. Systematic domain deletion analysis reveals that the long CTE is an important determinant for secretion of SptE via the Sec rather than Tat pathway to prevent enzyme aggregation before secretion. IMPORTANCE While Tat-dependent haloarchaeal subtilases (halolysins) have been extensively studied, the information about Sec-dependent subtilases of haloarchaea is limited. Our results demonstrate that proper maturation of Sec-dependent subtilase SptE of Natrinema sp. strain J7-2 depends on the action of halolysin SptA from the same strain, yielding multiple hetero- and autocatalytic mature forms. Moreover, we found that the different extra- and intracellular salt types (NaCl versus KCl) of haloarchaea and the long CTE are extrinsic and intrinsic factors crucial for routing SptE to the Sec rather than Tat pathway. This study provides new clues about the secretion and adaptation mechanisms of Sec substrates in haloarchaea.

Antimicrobial activity against Staphylococcus aureus and genome features of Lactiplantibacillus plantarum LR-14 from Sichuan pickles.

The persistence of Staphylococcus aureus within biofilm can lead to contamination of medical devices and life-threatening infections. Luckily, lactic acid bacteria (LAB) have an inhibitory effect on the growth of these bacteria. This study aims to select LAB strains from fermented vegetables, and analyze their potential inhibition activities against S. aureus. In total, 45 isolates of LAB were successfully isolated from Sichuan pickles, and the CFS of Lactiplantibacillus plantarum LR-14 exerted the strongest inhibitory effect against S. aureus. Moreover, S. aureus cells in planktonic and biofilm states both wrinkled and damaged when treated with the CFS of L. plantarum LR-14. In addition, whole genome sequencing analysis indicates that L. plantarum LR-14 contains various functional genes, including predicted extracellular polysaccharides (EPS) biosynthesis genes, and genes participating in the synthesis and metabolism of fatty acid, implying that L. plantarum LR-14 has the potential to be used as a probiotic with multiple functions.

A self-assessment tool for predicting discomfort and tolerance in Chinese patients undergoing esophagogastroduodenoscopy.

BACKGROUND: For patients taking esophagogastroduodenoscopy (EGD), sedation should ideally be used individually based on patients' comfort and tolerance level. However, currently there is no valid predictive tool. We undertook this study to develop and temporally validate a self-assessment tool for predicting discomfort and tolerance in Chinese patients undergoing EGD. METHODS: We recruited 1522 patients undergoing routine diagnostic EGD without sedation. We collected candidate predictor variables before endoscopy and evaluated discomfort and tolerance with a 5-point visual analogue scale after the procedure. We developed logistic regression predictive models based on the first 2/3 of participants, and evaluated the calibration and discrimination of the models in the later 1/3 of patients. RESULTS: 30.2% and 23.0% participants reported severe discomfort or poor tolerance to EGD respectively. The predictive factors in the model for discomfort included sex, education, expected level of discomfort, and anxiety before endoscopy. The model for tolerance included income, expected level of discomfort, and anxiety before endoscopy. In the validation population, the established models showed a moderate discriminative ability with a c-index of 0.74 for discomfort and 0.78 for tolerance. Hosmer-Lemeshow test suggested the models had fine calibration ability (discomfort: P = 0.37, tolerance: P = 0.41). CONCLUSIONS: Equations for predicting discomfort and tolerance in Chinese patients undergoing EGD demonstrated moderate discrimination and variable calibration. Further studies are still required to validate these tools in other population. TRIAL REGISTRATION: Chinese Clinical Trial Registry (ChiCTR1800020236).

Peatland Loss in Southeast Asia Contributing to U.S. Biofuel's Greenhouse Gas Emissions.

Land use change (LUC) induced by biofuel production could lead to greenhouse gas (GHG) emissions, which potentially increase biofuel's carbon intensity. Among the sources of LUC-related emissions for soy biodiesel, the contribution from peatland loss to agricultural plantations in Southeast Asia remains uncertain. Here, we analyzed LUC in Malaysia and Indonesia and modeled its impacts on the GHG emissions of soy biodiesel produced in the United States. It shows that oil palm plantations have more than doubled over 2001-2016 and the area of palm-on-peatlands (PoP) has expanded 3.7 times. Over new palm plantations, the share of PoP is about 19% regardless of time and location and the emission factor (EF) for peatland-to-palm conversion is estimated to be 41.5 Mg CO2 ha-1 yr-1. With these updates on PoP and EF, the contribution of peatland loss (0.7-5.1 g CO2e MJ-1) to biodiesel emissions is only 40-65% of previous estimates, which reduces discrepancies among model simulations used by different agencies. Based on emerging evidence on LUC and related carbon changes, our analysis reexamines regional peatland loss and its impacts on LUC emissions modeling and provides new insights into the estimation of LUC impacts on biofuels' carbon intensity.

Modeling Analysis of SM2 Construction Attacks in the Open Secure Sockets Layer Based on Petri Net.

The detection and defense of malicious attacks are critical to the proper functioning of network security. Due to the diversity and rapid updates of the attack methods used by attackers, traditional defense mechanisms have been challenged. In this context, a more effective method to predict vulnerabilities in network systems is considered an urgent need to protect network security. In this paper, we propose a formal modeling and analysis approach based on Petri net vulnerability exploitation. We used the Common Vulnerabilities and Exposures (CVE)-2021-3711 vulnerability source code to build a model. A patch model was built to address the problems of this model. Finally, the time injected by the actual attacker and the time simulated by the software were calculated separately. The results showed that the simulation time was shorter than the actual attack time, and ultra-real-time simulation could be achieved. By modeling the network system with this method, the model can be found to arrive at an illegitimate state according to the structure of Petri nets themselves and thus discover unknown vulnerabilities. This method provides a reference method for exploring unknown vulnerabilities.

An ultrahigh-throughput screening platform based on flow cytometric droplet sorting for mining novel enzymes from metagenomic libraries.

Uncultivable microbial communities provide enormous reservoirs of enzymes, but their experimental identification by functional metagenomics is challenging, mainly due to the difficulty of screening enormous metagenomic libraries. Here, we propose a reliable and convenient ultrahigh-throughput screening platform based on flow cytometric droplet sorting (FCDS). The FCDS platform employs water-in-oil-in-water double emulsion droplets serving as single-cell enzymatic micro-reactors and a commercially available flow cytometer, and it can efficiently isolate novel biocatalysts from metagenomic libraries by processing single cells as many as 108 per day. We demonstrated the power of this platform by screening a metagenomic library constructed from domestic running water samples. The FCDS assay screened 30 million micro-reactors in only 1 h, yielding a collection of esterase genes. Among these positive hits, Est WY was identified as a novel esterase with high catalytic efficiency and distinct evolutionary origin from other lipolytic enzymes. Our study manifests that the FCDS platform is a robust tool for functional metagenomics, with the potential to significantly improve the efficiency of exploring novel enzymes from nature.

Highly Efficient InGaN Nanorods Photoelectrode by Constructing Z-scheme Charge Transfer System for Unbiased Water Splitting.

Unbiased photoelectrochemical water splitting for the promising InGaN nanorods photoelectrode is highly desirable, but it is practically hindered by the serious recombination of charge carrier in bulk and surface of InGaN nanorods. Herein, an unbiased Z-scheme InGaN nanorods/Cu2 O nanoparticles heterostructured system with boosted interfacial charge transfer is constructed for the first time. The introduced Cu2 O nanoparticles pose double-sided effect on photoelectrochemical (PEC) performance of InGaN nanorods, which enables a robust hybrid structure and induces weakened light absorption capability simultaneously. As a result, the optimized InGaN/Cu2 O-1.5C photoelectrode with the uniform morphology exhibits an enhanced photocurrent density of ≈170 µA cm-2 at 0 V versus Pt, with 8.5-fold enhancement compared with pure InGaN nanorods. Comprehensive investigations into experimental results and theoretical calculations reveal that the electrons accumulation and holes depletion of Cu2 O facilitate to form a typical Z-scheme band alignment, thus providing a large photovoltage to drive unbiased water splitting and enhancing the stability of Cu2 O. This work provides a novel and facile strategy to achieve InGaN nanorods and other catalyst-based PEC water splitting without external bias, and to relieve the bottlenecks of charge transfer dynamics at the electrode bulk and electrode/electrolyte interface by constructing Z-scheme heterostructure.

[Analysis of results of concurrent hearing and deafness genetic screening and follow up of 33 911 newborns].

OBJECTIVE: To analyze the results of concurrent hearing and deafness genetic screening and follow up of newborns. METHODS: In total 33 911 babies born to 5 designated hospitals in Nanshan District of Shenzhen city from October 2017 to December 2019 were included. All subjects underwent concurrent hearing and deafness genetic screening covering 21 variants of 4 genes including GJB2, SLC26A4, GJB3 and Mt12SrRNA. For those with positive results, Sanger sequencing was carried out for confirmation. RESULTS: 93.32% subjects passed the first-round hearing screening, and 87.01% passed the recheck testing. The overall detection rate was 4.18%. The detection rates for GJB2, SLC26A4, GJB3 and Mt12srRNA variants were 1.98%, 1.58%, 0.37% and 0.25%, respectively. 126 and 84 subjects were found with high risk for delayed-onset and drug-induced hearing loss, respectively. In addition, 4 and 5 subjects were found to harbor homozygous/compound heterozygous variants of the GJB2 and SLC26A4 genes, respectively. Concurrent screening showed that subjects (with heterozygous variants) who did not passed the two round hearing test were as follows: GJB2 with 6.75% in the first round and 2.61% in the second round testing, SLC26A4 (3.3%/1.2%), GJB3 (0.72%/0.14%) and 12SrRNA (0.36%/Nil), respectively. Moreover, the No-pass rate in the subjects with homozygous or compound variants in single gene, heterozygous variant in single gene, heterozygous variant in multiple genes, and homozygous variant in GJB3 gene were significantly higher than the subjects with negative results of genetic screening. CONCLUSION: Concurrent newborn genetic screening can enhance the effectiveness of hearing screening and enable earlier identification and intervention for children with hearing impairment. Follow-up can improve the diagnostic rate for children who are positive for the concurrent screening. Nevertheless, genetic and hearing screening cannot replace the diagnostic testing. It is necessary to conduct comprehensive analysis for the results of genetic and hearing screening and radiological examinations. Sanger sequencing and next-generation sequencing are critical for ascertain the diagnosis.

Remaining Vigilant to Paradoxical Air Embolism in Patients Undergoing Hysteroscopic Surgery: A Case Report and Review of the Literature.

The purpose of this case study and review was to understand the perianesthestic care of paradoxical air embolism (PAE) in patients undergoing hysteroscopic surgery. The perianesthestic management record of a patient undergoing hysteroscopic surgery was analyzed to study the characteristics of PAE, and the literature describing the perianesthestic care for PAE was reviewed. The first symptom of PAE in hysteroscopic surgery is often a decrease in end-tidal carbon dioxide (ETCO2), and the complications include embolism of the pulmonary artery, coronary artery, and cerebral artery. The best monitoring method is continuous ETCO2 monitoring, and intraoperative echocardiography is an excellent method to diagnose and guide the treatment of PAE. PAE is a rare but serious complication of hysteroscopic surgery that is associated with organ ischemia and hypoxia. Prevention and treatment of PAE requires the vigilance and cooperation of not only perianesthesia nurses and anesthesiologists but also the surgeons and operating room nurses. Early prevention, proper monitoring, and effective treatment are the keys to successful rescue for PAE.

Activation of Aryl Carboxylic Acids by Diboron Reagents towards Nickel-Catalyzed Direct Decarbonylative Borylation.

The Ni-catalyzed decarbonylative borylation of (hetero)aryl carboxylic acids with B2 cat2 has been achieved without recourse to any additives. This Ni-catalyzed method exhibits a broad substrate scope covering poorly reactive non-ortho-substituted (hetero)aryl carboxylic acids, and tolerates diverse functional groups including some of the groups active to Ni0 catalysts. The key to achieve this decarbonylative borylation reaction is the choice of B2 cat2 as a coupling partner that not only acts as a borylating reagent, but also chemoselectively activates aryl carboxylic acids towards oxidative addition of their C(acyl)-O bond to Ni0 catalyst via the formation of acyloxyboron compounds. A combination of experimental and computational studies reveals a detailed plausible mechanism for this reaction system, which involves a hitherto unknown concerted decarbonylation and reductive elimination step that generates the aryl boronic ester product. This mode of boron-promoted carboxylic acid activation is also applicable to other types of reactions.

Intramuscular injection of tetracycline decreased gut microbial diversity in mouse.

Antibiotics contribute a lot to human beings and can kill bacteria effectively. However, more and more studies show that antibiotics can disturb the intestinal microbial community. It has been widely reported that oral antibiotics can reduce the diversity of intestinal microflora, but the effect of intramuscular injection on intestinal microflora is less studied. In this study, we sequenced the intestinal microflora of mice treated with tetracycline by 16SrRNA method, and found that intramuscular injection of tetracycline (TET) can also reduce the intestinal microbial richness of mice. In addition, the results showed that within a certain range (3 mg), with the increase of TET injection concentration, the wind of intestinal microflora in mice decreased significantly. When the injection concentration reached saturation, although the amount of TET injection was increased, the degree of intestinal flora affected was not increased. The results showed that the degree of diversity decrease was in direct proportion to the amount of tetracycline injection in the saturated concentration, but not positively related to the high amount of TET injection after exceeding the saturated concentration.

Structure-guided reshaping of the acyl binding pocket of 'TesA thioesterase enhances octanoic acid production in E. coli.

Medium-chain fatty acids (C6-C10) have attracted much attention recently for their unique properties compared to their long-chain counterparts, including low melting points and relatively higher carbon conversion yield. Thioesterase enzymes, which can catalyze the hydrolysis of acyl-ACP (acyl carrier protein) to release free fatty acids (FAs), regulate both overall FA yields and acyl chain length distributions in bacterial and yeast fermentation cultures. These enzymes typically prefer longer chain substrates. Herein, seeking to increase bacterial production of MCFAs, we conducted structure-guided mutational screening of multiple residues in the substrate-binding pocket of the E. coli thioesterase enzyme 'TesA. Confirming our hypothesis that enhancing substrate selectivity for medium-chain acyl substrates would promote overall MCFA production, we found that replacement of residues lining the bottom of the pocket with more hydrophobic residues strongly promoted the C8 substrate selectivity of 'TesA. Specifically, two rounds of saturation mutagenesis led to the identification of the 'TesARD-2 variant that exhibited a 133-fold increase in selectivity for the C8-ACP substrate as compared to C16-ACP substrate. Moreover, the recombinant expression of this variant in an E. coli strain with a blocked ß-oxidation pathway led to a 1030% increase in the in vivo octanoic acid (C8) production titer. When this strain was fermented in a 5-L fed-batch bioreactor, it produced 2.7 g/L of free C8 (45%, molar fraction) and 7.9 g/L of total free FAs, which is the highest-to-date free C8 titer to date reported using the E. coli type II fatty acid synthetic pathway. Thus, reshaping the substrate binding pocket of a bacterial thioesterase enzyme by manipulating the hydrophobicity of multiple residues altered the substrate selectivity and therefore fatty acid product distributions in cells. Our study demonstrates the relevance of this strategy for increasing titers of industrially attractive MCFAs as fermentation products.

Improved thermostability of creatinase from Alcaligenes Faecalis through non-biased phylogenetic consensus-guided mutagenesis.

BACKGROUND: Enzymatic quantification of creatinine has become an essential method for clinical evaluation of renal function. Although creatinase (CR) is frequently used for this purpose, its poor thermostability severely limits industrial applications. Herein, we report a novel creatinase from Alcaligenes faecalis (afCR) with higher catalytic activity and lower KM value, than currently used creatinases. Furthermore, we developed a non-biased phylogenetic consensus method to improve the thermostability of afCR. RESULTS: We applied a non-biased phylogenetic consensus method to identify 59 candidate consensus residues from 24 creatinase family homologs for screening afCR mutants with improved thermostability. Twenty-one amino acids of afCR were selected to mutagenesis and 11 of them exhibited improved thermostability compared to the parent enzyme (afCR-M0). Combination of single-site mutations in sequential screens resulted in a quadruple mutant D17V/T199S/L6P/T251C (M4-2) which showed ~ 1700-fold enhanced half-life at 57 °C and a 4.2 °C higher T5015 than that of afCR-M0. The mutant retained catalytic activity equivalent to afCR-M0, and thus showed strong promise for application in creatinine detection. Structural homology modeling revealed a wide range of potential molecular interactions associated with individual mutations that contributed to improving afCR thermostability. CONCLUSIONS: Results of this study clearly demonstrated that the non-biased-phylogenetic consensus design for improvement of thermostability in afCR is effective and promising in improving the thermostability of more enzymes.