Multiple Regulatory Mechanisms Synergistically Control the Soluble Expression of CsCE for Enhanced Enzymatic Productivity of Lactulose in E. coli.

The limited expression of cellobiose 2-epimerase poses a significant constraint on the industrial enzymatic production of lactulose. Extensive modifications to the expression cassette offer a means to enhance the yield of recombinant proteins. In this study, an integrated strategy, combining individual and collaborative approaches, is proposed to fine-tune each stage of the CsCE overexpression program. This strategy involves the multidimensional integration of standardized genetic elements at various levels, including transcription, translation, folding, and three-dimensional structure. The volumetric activity of the final recombinant strain was markedly increased by 12-fold compared to the wild-type strain, reaching 2260.62 U/L. The protein expression in the newly developed high-yield recombinant strain exhibited a significant enhancement, with a higher proportion of soluble protein compared to that of inclusion bodies. Our findings offer insights into the multifaceted synergistic regulation of protein expression processes, holding promising implications for the production of heterologous recombinant proteins.

Next-generation photodynamic antimicrobial materials made by direct synthesis of functional bacterial cellulose.

Bacterial cellulose (BC) regularly uses chemical or physical modifications to produce antimicrobial wound dressings. However, there is a risk of loss of functional components during application. Moreover, a significant hurdle lies in successfully integrating durable and highly effective bactericidal entities with BC. Herein, we successfully synthesized a photodynamic antibacterial cellulose through direct in situ microbial fermentation, incorporating the photosensitizer protoporphyrin IX-modified glucosamine (PPIX-GlcN) into cellulose to form PIXX-BC biopolymers. Excitingly, the PPIX-BC membrane exhibited robust and uniform red fluorescence, which is crucial for monitoring the bacterial fermentation process. Our results demonstrated that the biocompatibility PPIX-BC membrane possessed potent light-triggered photodynamic bactericidal activity, effectively suppressing the growth of E. coli and S. aureus while also promoting skin wounds repair. Consequently, this research validated the possibility of leveraging microorganisms to bio-functionalize BC, conferring it with photocatalytic antibacterial properties. Furthermore, successfully modification of the microorganisms' glucose carbon source offers valuable insights into biosynthesis of other living materials through microbial metabolism.

[Recent advances in metabolic engineering of microorganisms for production of tyrosol and its derivatives].

Tyrosol is a natural phenolic compound with antioxidant, anti-inflammatory and other biological activities, serving as an important precursor of high-value products such as hydroxytyrosol and salidroside. Therefore, the green and efficient biosynthesis of tyrosol and its derivatives has become a research hotspot in recent years. Building cell factories by metabolic engineering of microorganisms is a potential industrial production way, which has low costs and environmental friendliness. This paper introduces the biosynthesis pathway of tyrosol and presents the key regulated nodes in the de novo synthesis of tyrosol in Escherichia coli and Saccharomyces cerevisiae. In addition, this paper reviews the recent advances in metabolic engineering for the production of hydroxytyrosol and salidroside. This review can provide a reference for engineering the strains for the high-yield production of tyrosol and its derivatives.

Inverse metabolic engineering based on metabonomics for efficient production of hydroxytyrosol by Saccharomyces cerevisiae.

Metabolic engineering provides a powerful approach to efficiently produce valuable compounds, with the aid of emerging gene editing tools and diverse metabolic regulation strategies. However, apart from the current known biochemical pathway information, a variety of unclear constraints commonly limited the optimization space of cell phenotype. Hydroxytyrosol is an important phenolic compound that serves various industries with prominent health-beneficial properties. In this study, the inverse metabolic engineering based on metabolome analysis was customized and implemented to disclose the hidden rate-limiting steps and thus to improve hydroxytyrosol production in Saccharomyces cerevisiae (S. cerevisiae). The potential rate-limiting steps involved three modules that were eliminated individually via reinforcing and balancing metabolic flow, optimizing cofactor supply, and weakening the competitive pathways. Ultimately, a 118.53 % improvement in hydroxytyrosol production (639.84 mg/L) was achieved by inverse metabolic engineering.

Molecular insights into FucR transcription factor to control the metabolism of L-fucose in Bifidobacterium longum subsp. infantis.

Bifidobacterium longum subsp. infantis commonly colonizes the human gut and is capable of metabolizing L-fucose, which is abundant in the gut. Multiple studies have focused on the mechanisms of L-fucose utilization by B. longum subsp. infantis, but the regulatory pathways governing the expression of these catabolic processes are still unclear. In this study, we have conducted a structural and functional analysis of L-fucose metabolism transcription factor FucR derived from B. longum subsp. infantis Bi-26. Our results indicated that FucR is a L-fucose-sensitive repressor with more α-helices, fewer ß-sheets, and ß-turns. Transcriptional analysis revealed that FucR displays weak negative self-regulation, which is counteracted in the presence of L-fucose. Isothermal titration calorimetry indicated that FucR has a 2:1 stoichiometry with L-fucose. The key amino acid residues for FucR binding L-fucose are Asp280 and Arg331, with mutation of Asp280 to Ala resulting in a decrease in the affinity between FucR and L-fucose with the Kd value from 2.58 to 11.68 µM, and mutation of Arg331 to Ala abolishes the binding ability of FucR towards L-fucose. FucR specifically recognized and bound to a 20-bp incomplete palindrome sequence (5'-ACCCCAATTACGAAAATTTTT-3'), and the affinity of the L-fucose-loaded FucR for the DNA fragment was lower than apo-FucR. The results provided new insights into the regulating L-fucose metabolism by B. longum subsp. infantis.

Curative effect analysis of robot-assisted drainage surgery in treatment of spontaneous hypertensive brainstem hemorrhage.

Objective: Spontaneous hypertensive brainstem hemorrhage (HBSH) is characterized by sudden onset, rapid progression and poor prognosis. There has been a growing tendency of surgical treatment for HBSH. This study aimed to investigate outcomes and potential factors associated with the prognosis of robot-assisted drainage surgery for HBSH treatment. Methods: Patients with HBSH from July 2016 to March 2023 at a single neurosurgery center were included and divided into conservative group and surgical groups. Baseline and clinical data, radiographic characteristics, complications, and outcome evaluations were recorded and analyzed. Results: A total of 125 patients, with 74 in the conservative group and 51 in the surgical group, were enrolled in the study. Mortality at 6 months was 59/74 (79.7%) in the conservative group and 9/51 (17.6%) in the surgical group. Twenty-four patients (47.1%) achieved favorable outcomes in the surgical group, whereas this rate in the conservative group was only 5.4% (4/74). There was a significant difference in NIHSS, GCS, and mRS at 6 months between surviving patients in the conservative and surgical groups. In prognostic analysis in the surgical subgroup, initial GCS score [5 (IQR 4-7) vs. 3 (IQR 3-4), p < 0.001], NIHSS [36 (IQR 32-38) vs. 40 (IQR 38-40), p < 0.001], smoking history [45.8% (11/24) vs. 74.1% (20/27), p = 0.039], hematoma volume [6.9 (IQR 6.2-7.6) vs. 9.6 (IQR 7.3-11.4), p = 0.001], and hemorrhage location (p = 0.001) were potential risk factors for poor 6-month prognosis after robot-assisted surgery for HBSH. Conclusion: Based on the results of this study, robot-assisted minimally invasive drainage of brain stem hematoma may significantly reduce mortality and improve prognosis. Surgery should be conducted for selected patients.

A nomogram for predicting the possibility of effusion deterioration in patients with traumatic subdural effusion.

BACKGROUND: Traumatic subdural effusion (TSDE) may increase progressively or evolve into chronic subdural hematoma. These events, defined as deterioration of the effusion, often require close observation or even surgical treatment. The aim of our study was to develop and validate a nomogram for predicting the possibility of an effusion deteriorating in patients with TSDE based on the available clinical characteristics. METHODS: Clinical data from 78 patients with TSDE were retrospectively analyzed. All patients were admitted from January 2019 to May 2022. Logistic regression was applied to the data to screen for independent predictors of effusion deterioration within six months; then, a predictive nomogram model was established in R language. The consistency, predictive accuracy and clinical utility of the model were evaluated with the C-index, calibration plots, ROC curves and decision curve analysis (DCA). Furthermore, we performed internal validation using a bootstrap approach to assess the effectiveness of the model. RESULTS: Time of effusion after trauma, maximum thickness of the effusion, CT value of the effusion as well as the use of atorvastatin were identified as predictors in the nomogram. The predictive model was well calibrated and demonstrated good discrimination (C-index: 0.893). The AUC of the model was 0.893 (95% CI: 0.824-0.962), and the modified C-index (0.865) indicated excellent performance in the internal validation. In addition, DCA revealed that the nomogram had clinical value. CONCLUSIONS: This predictive model can effectively assess the risk of effusion deterioration in TSDE patients within six months and identify high-risk patients early.

Brain region changes following a spinal cord injury.

Brain-related complications are common in clinical practice after spinal cord injury (SCI); however, the molecular mechanisms of these complications are still unclear. Here, we reviewed the changes in the brain regions caused by SCI from three perspectives: imaging, molecular analysis, and electrophysiology. Imaging studies revealed abnormal functional connectivity, gray matter volume atrophy, and metabolic abnormalities in brain regions after SCI, leading to changes in the structure and function of brain regions. At the molecular level, chemokines, inflammatory factors, and damage-associated molecular patterns produced in the injured area were retrogradely transmitted through the corticospinal tract, cerebrospinal fluid, or blood circulation to the specific brain area to cause pathologic changes. Electrophysiologic recordings also suggested abnormal changes in brain electrical activity after SCI. Transcranial magnetic stimulation, transcranial direct current stimulation, and deep brain stimulation alleviated pain and improved motor function in patients with SCI; therefore, transcranial therapy may be a new strategy for the treatment of patients with SCI.

Acid-induced conformation regulation of peanut polysaccharide and its effect on stability and digestibility of oil-in-water emulsion.

BACKGROUND: Developing the stable and healthy emulsion-based food is in accord with the needs of people for health. In the present study, acidification at pH 3.0 of peanut polysaccharide (APPSI) was employed to regulate its conformation and further improve its advantages in preparing oil-in-water emulsion. RESULTS: The results indicated that acidification induced conversion of PPSI aggregates into linear chains. Increasing concentration promoted formation of cross-linked network structure shown in transmission electron microscopy images. Consequently, the viscosity, yield stress, storage modulus and flow activation energy significantly increased, further fabricating gel structure. Moreover, aggregation behavior suggested that more exposed proteins were involved in gel structure, thereby forming many hydrophobic cores as verified by fluorescence spectroscopy of pyrene. Afterwards, emulsion characteristics indicated that APPSI produced strong and thick steric hindrance around oil droplets and the coil-like interweaved chains locked the continuous phase, bringing strong elasticity and resistance to stress and creaming. Meanwhile, the lower fatty acid in APPSI-emulsion was released after simulated gastrointestinal digestion, mainly as a result of the high retention ratio of emulsion droplets. Furthermore, the elastic and viscous Lissajous curves suggested that the structure strength of APPSI-emulsion was similar to that of the salad dressing within the strain 53.22%. CONCLUSION: The conformation of PPSI after acidification at pH 3.0 was suitable for preparing the stable emulsion. The obtained emulsion could resist digestion and maintain a strong structure, comprising a cholesterol-free and low-fat salad dressing substitute. © 2023 Society of Chemical Industry.

Highly efficient production and simultaneous purification of d-tagatose through one-pot extraction-assisted isomerization of d-galactose.

A one-pot extraction-assisted d-galactose-to-d-tagatose isomerization strategy was proposed based on the selective extraction of d-tagatose by phenylborate anions. 4-Vinylphenylboronic acid was selected with high extraction efficiency and selectivity towards d-tagatose. The extracted sugars could be desorbed through a two-staged stripping process with the purity of d-tagatose significantly increased. In-situ extraction-assisted d-galactose-to-d-tagatose isomerization was implemented for the first time ever reported, and the effect of boron-to-sugar ratio (boron: sugar) was investigated. The conversion yield of d-tagatose at 60 °C increased from âˆ¼ 39 % (boron: sugar = 0.5) to âˆ¼ 56 % (boron: sugar = 1) but then decreased to âˆ¼ 44 % (boron: sugar = 1.5). With temperature increased to 70 °C, the conversion yield of d-tagatose was further improved to âˆ¼ 61 % (boron: sugar = 1.5), with the minimized formation of byproducts. Moreover, high purity (∼83 %) and concentrated d-tagatose solution (∼40 g/L) was obtained after sequential desorption. The proposed extraction-assisted isomerization strategy achieved improving the yield and purity of d-tagatose, proving its feasibility in industrial applications.

Exploration of the common pathogenic link between COVID-19 and diabetic foot ulcers: An in silico approach.

Background and Aims: The Coronavirus Disease-19 (COVID-19) is posing an ongoing threat to human health. Patients of diabetic foot ulcer (DFU) are susceptible to COVID-19-induced adverse outcomes. Nevertheless, investigations into their mutual molecular mechanisms have been limited to date. In the present work, we tried to uncover the shared pathogenesis and regulatory gene targets of COVID-19 and DFU. Methods: In this study, we chose GSE161281 as the COVID-19 data set, which contained severe acute respiratory syndrome coronavirus 2 infected human induced embryonic stem cell-derived peripheral neurons (n = 2) with uninfected controls (n = 2). The GSE134431 designated as the DFU data set, comprising full-thickness DFU (n = 13) and diabetic foot skin (n = 8) samples from diabetic patients. The differential expressed genes (DEGs) were identified from GSE161281 and GSE134431, and the common DEGs between COVID-19 and DFU were extracted. Multifactor regulatory network and co-expression network of the common DEGs were analyzed, along with candidate drug prediction. Results: Altogether, six common DEGs (dickkopf-related protein 1 [DKK1], serine proteinase inhibitor A3 [SERPINA3], ras homolog family member D [RHOD], myelin protein zero like 3 [MPZL3], Claudin-11 [CLDN11], and epidermal growth factor receptor pathway substrate 8-like 1 [EPS8L1]) were found between COVID-19 and DFU. Functional analyses indicated that pathways of apoptotic and Wnt signaling may contribute to progression of COVID-19. Gene co-expression network implied the shared pathways of immune regulation and cytokine response participated collectively in the development of DFU and COVID-19. A multifactor regulatory network was constructed integrating the corresponding microRNAs (miRNAs) and transcription factors. Additionally, we proposed potential drug objects for the combined therapy. Conclusion: Our study revealed the shared molecular mechanisms underlying COVID-19 and DFU. The identified pivotal targets and common pathways can provide new perspectives for further research and assist the development of management strategies in patients of DFU complicated with COVID-19.

A precise swaying map for how promiscuous cellobiose-2-epimerase operate bi-reaction.

Promiscuous enzymes play a crucial role in organism survival and new reaction mining. However, comprehensive mapping of the catalytic and regulatory mechanisms hasn't been well studied due to the characteristic complexity. The cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus (CsCE) with complex epimerization and isomerization was chosen to comprehensively investigate the promiscuous mechanisms. Here, the catalytic frame of ring-opening, cis-enediol mediated catalysis and ring-closing was firstly determined. To map the full view of promiscuous CE, the structure of CsCE complex with the isomerized product glucopyranosyl-ß1,4-fructose was determined. Combined with computational calculation, the promiscuity was proved a precise cooperation of the double subsites, loop rearrangement, and intermediate swaying. The flexible loop was like a gear, whose structural reshaping regulates the sway of the intermediates between the two subsites of H377-H188 and H377-H247, and thus regulates the catalytic directions. The different protonated states of cis-enediol intermediate catalyzed by H188 were the key point for the catalysis. The promiscuous enzyme tends to utilize all elements at hand to carry out the promiscuous functions.

Production of high-purity lactulose via an integrated one-pot boronate affinity adsorbent based adsorption-assisted isomerization and simultaneous purification.

High-purity lactulose is mandatory for its medical uses and food applications. This work developed an efficient lab-scale strategy for the synthesis of high-purity lactulose by combining lactose-to-lactulose isomerization with simultaneous recovery of lactulose, which was conducted concurrently and semi-continuously in a boronate affinity adsorbent-packed column. The first step covers the boronate affinity adsorbent-based adsorption-assisted lactose-to-lactulose isomerization. Under optimized conditions, in situ selectively binding of the newly formed lactulose onto the boronate affinity adsorbent enables a much-enhanced lactulose yield up to 80.20% with the lowest byproducts yield of 6.30%. Afterward, over 90% of the adsorbed lactulose can be recovered through sequential desorption with purity >98%. The net outcome of the applied strategy was the yield of high-purity lactulose up to 72.31%, the highest value ever reported. Moreover, the packed column displayed excellent operational stability. The encouraging results validate the high potential of this approach in the sustainable production of high-purity lactulose.

Highly efficient isolation and purification of high-purity tea saponins from industrial camellia oil production by porous polymeric adsorbents.

BACKGROUND: Recovery of high-purity tea saponin (TS), a promising non-ionic surfactant with well-documented properties, is one of the major challenges to broadening its industrial applications. In this study, an innovative and sustainable strategy for the highly-efficient purification of TS was developed by using well-designed highly-porous polymeric adsorbents. RESULTS: The prepared Pp-A with controllable macropores (~96 nm) and appropriate surface hydrophobic properties was found more favorable for achieving high adsorption efficiency towards TS/TS-micelles. Kinetic results showed the adsorption follows the pseudo-second-order model (R2 = 0.9800), and the Langmuir model is more qualified to explicate the adsorption isotherms with Qe-TS ~ 675 mg g-1 . Thermodynamic studies revealed the monolayer adsorption of TS was an endothermic process that was conducted spontaneously. Interestingly, ethanol-driven desorption (90% v/v ethanol) of TS was rapidly (< 30 min) complete due to the possible ethanol-mediated disassembling of TS-micelles. A possible mechanism that involves the interactions between the adsorbents and TS/TS-micelles, the formation and disassembling of TS-micelles was proposed to account for the highly efficient purification of TS. Afterwards, Pp-A-based adsorption method was developed to purify TS directly from industrial camellia oil production. Through selective adsorption, pre-washing, and ethanol-driven desorption, the applied Pp-A enabled the direct isolation of high-purity TS (~96%) with a recovery ratio > 90%. Notably, Pp-A exhibited excellent operational stability and is of high potential for long-term industrial application. CONCLUSION: Results ensured the practical feasibility of the prepared porous adsorbents in purifying TS, and the proposed methodology is a promising industrial-scale purification strategy. © 2023 Society of Chemical Industry.

Budding uninhibited by benzimidazoles 1 promotes cell proliferation, invasion, and epithelial-mesenchymal transition via the Wnt/ß-catenin signaling in glioblastoma.

The pathogenesis and progression of GBM (glioblastoma), as one of the most frequently occurring malignancies of the central nervous system, are regulated by several genes. BUB1 (budding uninhibited by benzimidazoles 1) is a mitotic checkpoint that plays an important role in chromosome segregation as well as in various tumors. However, its role in glioma is unknown. The current study discovered prominently elevated BUB1 in glioma and a significant relationship between BUB1 expression, a high World Health Organization grade, and a poor prognosis in glioma patients. Moreover, BUB1 triggered EMT (epithelial-mesenchymal transition) apart from promoting glioma cell proliferation, migration, and infiltration. Besides, BUB1 promoted EMT by activating the Wnt/ß-catenin axis. As implied by our study, BUB1 probably has the potential as a target for GBM management.

Human atlastin-3 is a constitutive ER membrane fusion catalyst.

Homotypic membrane fusion catalyzed by the atlastin (ATL) GTPase sustains the branched endoplasmic reticulum (ER) network in metazoans. Our recent discovery that two of the three human ATL paralogs (ATL1/2) are C-terminally autoinhibited implied that relief of autoinhibition would be integral to the ATL fusion mechanism. An alternative hypothesis is that the third paralog ATL3 promotes constitutive ER fusion with relief of ATL1/2 autoinhibition used conditionally. However, published studies suggest ATL3 is a weak fusogen at best. Contrary to expectations, we demonstrate here that purified human ATL3 catalyzes efficient membrane fusion in vitro and is sufficient to sustain the ER network in triple knockout cells. Strikingly, ATL3 lacks any detectable C-terminal autoinhibition, like the invertebrate Drosophila ATL ortholog. Phylogenetic analysis of ATL C-termini indicates that C-terminal autoinhibition is a recent evolutionary innovation. We suggest that ATL3 is a constitutive ER fusion catalyst and that ATL1/2 autoinhibition likely evolved in vertebrates as a means of upregulating ER fusion activity on demand.

A clinical case report of Balamuthia granulomatous amoebic encephalitis in a non-immunocompromised patient and literature review.

BACKGROUND: Balamuthia granulomatous amoebic encephalitis (GAE) is a peculiar parasitic infectious disease of the central nervous system, about 39% of the infected Balamuthia GAE patients were found to be immunocompromised and is extremely rare clinically. The presence of trophozoites in diseased tissue is an important basis for pathological diagnosis of GAE. Balamuthia GAE is a rare and highly fatal infection for which there is no effective treatment plan in clinical practice. CASE PRESENTATION: This paper reports clinical data from a patient with Balamuthia GAE to improve physician understanding of the disease and diagnostic accuracy of imaging and reduce misdiagnosis. A 61-year-old male poultry farmer presented with moderate swelling pain in the right frontoparietal region without obvious inducement three weeks ago. Head computed tomography(CT) and magnetic resonance imaging(MRI) revealed a space-occupying lesion in the right frontal lobe. Intially clinical imaging diagnosed it as a high-grade astrocytoma. The pathological diagnosis of the lesion was inflammatory granulomatous lesions with extensive necrosis, suggesting amoeba infection. The pathogen detected by metagenomic next-generation sequencing (mNGS) is Balamuthia mandrillaris, the final pathological diagnosis was Balamuthia GAE. CONCLUSION: When a head MRI shows irregular or annular enhancement, clinicians should not blindly diagnose common diseases such as brain tumors. Although Balamuthia GAE accounts for only a small proportion of intracranial infections, it should be considered in the differential diagnosis.

Functional bacterial cellulose nanofibrils with silver nanoparticles and its antibacterial application.

Bacterial cellulose (BC) with good biocompatibility and superior mechanical properties has broad applications. BC functionalized with silver nanoparticles (AgNPs) has been assessed as an antimicrobial membrane for wound-healing treatment. During the AgNPs synthesis, avoiding the use of toxic chemicals is very necessary for the development of environmentally friendly procedures. Herein, a Komagataeibacter xylinus-based direct biosynthetic method to fabricate D-Saccharic acid potassium salt (SA)-grafted BC (SABC) through in situ bacterial metabolism was firstly explored. Subsequently, the SABC pellicles were immersed in AgNO3 solution for ion-exchanged process, and the silver nanoparticles (AgNPs) with diameter of ∼25.2 nm were in situ synthesized on SABC nanofiber surfaces by thermal reduction instead of using a reducing agent. The morphology and microstructure of SABC/AgNPs pellicles were analyzed by field-emission scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, and X-ray photoelectron spectra. Moreover, antibacterial activity measurement performed against the Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus) by disk diffusion and plate count methods, showed high-efficiency bacteria-killing performance of SABC/AgNPs pellicles. This work proposed a new method by using microbial metabolism to prepare BC pellicles with functional groups, and antimicrobial films containing AgNPs was prepared by thermal reduction, exhibiting valuable prospects in wound healing treatment.

Precise Photothermal Treatment of Methicillin-Resistant S. aureus Infection via Phage Lysin-Cell Binding Domain-Modified Gold Nanosheets.

The increasing spread of antibiotic resistance in bacterial pathogens poses a huge threat to global human health. Precise targeting of bacterial pathogens while avoiding collateral damage to healthy tissues has become the overriding goal for bacterial infection treatment. Inspired by the host specificity of bacteriophages, a biomimetic intelligent platform was designed for highly precise photothermal treatment herein. As proof-of-concept, the lysin cell-binding domain (CBD) from a newly discovered virulent methicillin-resistant S. aureus (MRSA) phage Z was applied to the functionalization of gold nanosheets. Our results demonstrated that the bionanocomposite gold particles (Au@PEG-CBDz) could be effectively delivered directly to MRSA and kill them effectively under near infrared irradiation in vitro, while displaying good in vivo biocompatibility. This work is the first to report the combination of phage lysin navigatory function with photothermal effect-induced bactericidal activity from Au nanosheets, providing a novel therapeutic mode for the precision treatment of antibiotic-resistant bacterial infections.