Enhancing the expression of chondroitin 4-O-sulfotransferase for one-pot enzymatic synthesis of chondroitin sulfate A.

Chondroitin sulfate (CS) stands as a pivotal compound in dietary supplements for osteoarthritis treatment, propelling significant interest in the biotechnological pursuit of environmentally friendly and safe CS production. Enzymatic synthesis of CS for instance CSA has been considered as one of the most promising methods. However, the bottleneck consistently encountered is the active expression of chondroitin 4-O-sulfotransferase (C4ST) during CSA biosynthesis. This study meticulously delved into optimizing C4ST expression through systematic enhancements in transcription, translation, and secretion mechanisms via modifications in the 5' untranslated region, the N-terminal encoding sequence, and the Komagataella phaffii chassis. Ultimately, the active C4ST expression escalated to 2713.1 U/L, representing a striking 43.7-fold increase. By applying the culture broth supernatant of C4ST and integrating the 3'-phosphoadenosine-5'-phosphosulfate (PAPS) biosynthesis module, we constructed a one-pot enzymatic system for CSA biosynthesis, achieving a remarkable sulfonation degree of up to 97.0 %. The substantial enhancement in C4ST expression and the development of an engineered one-pot enzymatic synthesis system promises to expedite large-scale CSA biosynthesis with customizable sulfonation degrees.

Coordinated optimization of the polymerization and transportation processes to enhance the yield of exopolysaccharide heparosan.

Heparosan as the precursor for heparin biosynthesis has attracted intensive attention while Escherichia coli Nissle 1917 (EcN) has been applied as a chassis for heparosan biosynthesis. Here, after uncovering the pivotal role of KfiB in heparosan biosynthesis, we further demonstrate KfiB is involved in facilitating KpsT to translocate the nascent heparosan polysaccharide chain. As a result, an artificial expression cassette KfiACB was constructed with optimized RBS elements, resulting in 0.77 g/L heparosan in shake flask culture. Moreover, in view of the intracellular accumulation of heparosan, we further investigated the effects of overexpression of the ABC transport system proteins on heparosan biosynthesis. By co-overexpressing KfiACB with KpsTME, the heparosan production in flask cultures was increased to 1.03 g/L with an extracellular concentration of 0.96 g/L. Eventually, the engineered strain EcN/pET-kfiACB3-galU-kfiD-glmM/pCDF-kpsTME produced 12.2 g/L heparosan in 5-L fed-batch cultures while the extracellular heparosan was about 11.2 g/L. The results demonstrate the high-efficiency of the strategy for co-optimizing the polymerization and transportation for heparosan biosynthesis. Moreover, this strategy should be also available for enhancing the production of other polysaccharides.

Construction of 5-Aminolevulinic Acid Microbial Cell Factories through Identification of Novel Synthases and Metabolic Pathway Screens and Transporters.

5-Aminolevulinic acid (5-ALA) plays a pivotal role in the biosynthesis of heme and chlorophyll and has garnered great attention for its agricultural applications. This study explores the multifaceted construction of 5-ALA microbial cell factories. Evolutionary analysis-guided screening identified a novel 5-ALA synthase from Sphingobium amiense as the best synthase. An sRNA library facilitated global gene screening that demonstrated that trpC and ilvA repression enhanced 5-ALA production by 74.3% and 102%, respectively. Subsequently, efflux of 5-ALA by the transporter Gdx increased 5-ALA biosynthesis by 25.7%. To mitigate oxidative toxicity, DNA-binding proteins from starved cells were employed, enhancing cell density and 5-ALA titer by 21.1 and 4.1%, respectively. Combining these strategies resulted in an Escherichia coli strain that produced 5-ALA to 1.51 g·L-1 in shake flask experiments and 6.19 g·L-1 through fed-batch fermentation. This study broadens the repertoire of available 5-ALA synthases and transporters and provides a new platform for optimizing 5-ALA bioproduction.

Enzymatic construction of a library of even- and odd-numbered heparosan oligosaccharides and their N-sulfonated derivatives.

Low-molecular-weight heparins (LMWHs), especially the specific-sized heparin oligosaccharides, are attractive for the therapeutic applications, while their synthesis remains challenging. In the present study, unsaturated even-numbered heparosan oligosaccharides were firstly prepared by cleaving high-molecular-weight heparosan using recombinant heparinase III (HepIII). The conversion rates of the unsaturated disaccharides, tetrasaccharides, hexasaccharides, octasaccharides, and decasaccharides were 33.9 %, 47.9 %, 78.7 %, 71.8 %, and 53.4 %, respectively. After processing the aforementioned heparosan oligosaccharides with the Δ4,5 unsaturated glycuronidase, saturated odd-numbered heparosan trisaccharides, pentasaccharides, heptasaccharides, and nonasaccharides were produced. It was observed that among them, the pentasaccharides were the smallest units of saturated odd-numbered oligosaccharides recognized by HepIII. These oligosaccharides were further catalyzed with bifunctional heparan sulfate N-deacetylase/N-sulfotransferase (NDST) under optimized reaction conditions. It was found that the tetrasaccharide was defined as the smallest recognition unit for NDST, obtaining the N-sulfonated heparosan tetrasaccharides, pentasaccharides, and hexasaccharides with a single sulfonate group, as well as N-sulfonated heparosan heptasaccharides, octasaccharides, and nonasaccharides with multiple sulfonate groups. These results provide an easy pathway for constructing a library of specific-sized N-sulfonated heparosan oligosaccharides that can be used as the substrates for the enzymatic synthesis of LMWHs and heparin oligosaccharides, shedding new light on the substrate preference of NDST.

Regulatory RNAs in Bacillus subtilis: A review on regulatory mechanism and applications in synthetic biology.

Bacteria exhibit a rich repertoire of RNA molecules that intricately regulate gene expression at multiple hierarchical levels, including small RNAs (sRNAs), riboswitches, and antisense RNAs. Notably, the majority of these regulatory RNAs lack or have limited protein-coding capacity but play pivotal roles in orchestrating gene expression by modulating transcription, post-transcription or translation processes. Leveraging and redesigning these regulatory RNA elements have emerged as pivotal strategies in the domains of metabolic engineering and synthetic biology. While previous investigations predominantly focused on delineating the roles of regulatory RNA in Gram-negative bacterial models such as Escherichia coli and Salmonella enterica, this review aims to summarize the mechanisms and functionalities of endogenous regulatory RNAs inherent to typical Gram-positive bacteria, notably Bacillus subtilis. Furthermore, we explore the engineering and practical applications of these regulatory RNA elements in the arena of synthetic biology, employing B. subtilis as a foundational chassis.

Identification macrophage signatures in prostate cancer by single-cell sequencing and machine learning.

BACKGROUND: The tumor microenvironment (TME) encompasses a variety of cells that influence immune responses and tumor growth, with tumor-associated macrophages (TAM) being a crucial component of the TME. TAM can guide prostate cancer in different directions in response to various external stimuli. METHODS: First, we downloaded prostate cancer single-cell sequencing data and second-generation sequencing data from multiple public databases. From these data, we identified characteristic genes associated with TAM clusters. We then employed machine learning techniques to select the most accurate TAM gene set and developed a TAM-related risk label for prostate cancer. We analyzed the tumor-relatedness of the TAM-related risk label and different risk groups within the population. Finally, we validated the accuracy of the prognostic label using single-cell sequencing data, qPCR, and WB assays, among other methods. RESULTS: In this study, the TAM_2 cell cluster has been identified as promoting the progression of prostate cancer, possibly representing M2 macrophages. The 9 TAM feature genes selected through ten machine learning methods and demonstrated their effectiveness in predicting the progression of prostate cancer patients. Additionally, we have linked these TAM feature genes to clinical pathological characteristics, allowing us to construct a nomogram. This nomogram provides clinical practitioners with a quantitative tool for assessing the prognosis of prostate cancer patients. CONCLUSION: This study has analyzed the potential relationship between TAM and PCa and established a TAM-related prognostic model. It holds promise as a valuable tool for the management and treatment of PCa patients.

Short-term exposure to extreme temperature and outpatient visits for respiratory diseases among children in the northern city of China: a time-series study.

BACKGROUND: Although studies have indicated that extreme temperature is strongly associated with respiratory diseases, there is a dearth of studies focused on children, especially in China. We aimed to explore the association between extreme temperature and children's outpatient visits for respiratory diseases and seasonal modification effects in Harbin, China. METHODS: A distributed lag nonlinear model (DLNM) was used to explore the effect of extreme temperature on daily outpatient visits for respiratory diseases among children, as well as lag effects and seasonal modification effects. RESULTS: Extremely low temperatures were defined as the 1st percentile and 2.5th percentile of temperature. Extremely high temperatures were defined as the 97.5th percentile and 99th percentile of temperature. At extremely high temperatures, both 26 °C (97.5th) and 27 °C (99th) showed adverse effects at lag 0-6 days, with relative risks (RRs) of 1.34 [95% confidence interval (CI): 1.21-1.48] and 1.38 (95% CI: 1.24-1.53), respectively. However, at extremely low temperatures, both - 26 °C (1st) and - 23 °C (2.5th) showed protective effects on children's outpatient visits for respiratory diseases at lag 0-10 days, with RRs of 0.86 (95% CI: 0.76-0.97) and 0.85 (95% CI: 0.75-0.95), respectively. We also found seasonal modification effects, with the association being stronger in the warm season than in the cold season at extremely high temperatures. CONCLUSIONS: Our study indicated that extremely hot temperatures increase the risk of children's outpatient visits for respiratory diseases. Efforts to reduce the exposure of children to extremely high temperatures could potentially alleviate the burden of pediatric respiratory diseases, especially during the warm season.

Minimizing endogenous cryptic plasmids to construct antibiotic-free expression systems for Escherichia coli Nissle 1917.

The probiotic bacterium Escherichia coli Nissle 1917 (EcN) holds significant promise for use in clinical and biological industries. However, the reliance on antibiotics to maintain plasmid-borne genes has overshadowed its benefits. In this study, we addressed this issue by engineering the endogenous cryptic plasmids pMUT1 and pMUT2. The non-essential elements were removed to create more stable derivatives pMUT1NR△ and pMUT2HBC△. Synthetic promoters by integrating binding motifs on sigma factors were further constructed and applied for expression of Bacteroides thetaiotaomicron heparinase III and the biosynthesis of ectoine. Compared to traditional antibiotic-dependent expression systems, our newly constructed antibiotic-free expression systems offer considerable advantages for clinical and synthetic biology applications.

Deep Learning Based on ResNet-18 for Classification of Prostate Imaging-Reporting and Data System Category 3 Lesions.

RATIONALE AND OBJECTIVES: To explore the classification and prediction efficacy of the deep learning model for benign prostate lesions, non-clinically significant prostate cancer (non-csPCa) and clinically significant prostate cancer (csPCa) in Prostate Imaging-Reporting and Data System (PI-RADS) 3 lesions. MATERIALS AND METHODS: From January 2015 to December 2021, lesions diagnosed with PI-RADS 3 by multi-parametric MRI or bi-parametric MRI were retrospectively included. They were classified as benign prostate lesions, non-csPCa, and csPCa according to the pathological results. T2-weighted images of the lesions were divided into a training set and a test set according to 8:2. ResNet-18 was used for model training. All statistical analyses were performed using Python open-source libraries. The receiver operating characteristic curve (ROC) was used to evaluate the predictive effectiveness of the model. T-SNE was used for image semantic feature visualization. The class activation mapping was used to visualize the area focused by the model. RESULTS: A total of 428 benign prostate lesion images, 158 non-csPCa images and 273 csPCa images were included. The precision in predicting benign prostate disease, non-csPCa and csPCa were 0.882, 0.681 and 0.851, and the area under the ROC were 0.875, 0.89 and 0.929, respectively. Semantic feature analysis showed strong classification separability between csPCa and benign prostate lesions. The class activation map showed that the deep learning model can focus on the area of the prostate or the location of PI-RADS 3 lesions. CONCLUSION: Deep learning model with T2-weighted images based on ResNet-18 can realize accurate classification of PI-RADS 3 lesions.

LI-RADS CT and MRI Ancillary Feature Association with Hepatocellular Carcinoma and Malignancy: An Individual Participant Data Meta-Analysis.

Background The independent contribution of each Liver Imaging Reporting and Data System (LI-RADS) CT or MRI ancillary feature (AF) has not been established. Purpose To evaluate the association of LI-RADS AFs with hepatocellular carcinoma (HCC) and malignancy while adjusting for LI-RADS major features through an individual participant data (IPD) meta-analysis. Materials and Methods Medline, Embase, Cochrane Central Register of Controlled Trials, and Scopus were searched from January 2014 to January 2022 for studies evaluating the diagnostic accuracy of CT and MRI for HCC using LI-RADS version 2014, 2017, or 2018. Using a one-step approach, IPD across studies were pooled. Adjusted odds ratios (ORs) and 95% CIs were derived from multivariable logistic regression models of each AF combined with major features except threshold growth (excluded because of infrequent reporting). Liver observation clustering was addressed at the study and participant levels through random intercepts. Risk of bias was assessed using a composite reference standard and Quality Assessment of Diagnostic Accuracy Studies 2. Results Twenty studies comprising 3091 observations (2456 adult participants; mean age, 59 years ± 11 [SD]; 1849 [75.3%] men) were included. In total, 89% (eight of nine) of AFs favoring malignancy were associated with malignancy and/or HCC, 80% (four of five) of AFs favoring HCC were associated with HCC, and 57% (four of seven) of AFs favoring benignity were negatively associated with HCC and/or malignancy. Nonenhancing capsule (OR = 3.50 [95% CI: 1.53, 8.01]) had the strongest association with HCC. Diffusion restriction (OR = 14.45 [95% CI: 9.82, 21.27]) and mild-moderate T2 hyperintensity (OR = 10.18 [95% CI: 7.17, 14.44]) had the strongest association with malignancy. The strongest negative associations with HCC were parallels blood pool enhancement (OR = 0.07 [95% CI: 0.01, 0.49]) and marked T2 hyperintensity (OR = 0.18 [95% CI: 0.07, 0.45]). Seventeen studies (85%) had a high risk of bias. Conclusion Most LI-RADS AFs were independently associated with HCC, malignancy, or benignity as intended when adjusting for major features. © RSNA, 2024 Supplemental material is available for this article. See also the editorial by Crivellaro in this issue.

The effect of different pterygium surgery techniques on the ocular surface parameters in different durations: a systematic review and meta-analysis.

PURPOSE: To evaluate the effects of different pterygium surgery techniques on ocular surface (OS) in different follow-up periods. METHODS: PubMed, Cochrane Library, Web of Science, China National Knowledge Infrastructure, Wan Fang, and China Biology Medicine disc were searched for studies reporting pre- and post-operative OS parameters in pterygium. RESULTS: A total of 33 articles were finally included. Three OS parameters showed relatively consistent changing trends after surgery including ocular surface disease index (OSDI), tear film break-up time (BUT), and score of corneal fluorescein staining (SCFS). They worsened significantly at 1w post-operation and then gradually improved: OSDI and BUT showed obvious improvement in 1 m post-operation (SMD = - 0.58, 95%CI = [- 1.04, - 0.13]; SMD = 0.42, 95%CI = [0.06, 0.78]); SCFS was restored to preoperative levels in 3 m after surgery (SMD = - 0.54, 95%CI = [- 1.16, 0.07]). Another parameter, Schirmer test without anesthesia (SIT), presented transient increase at 1w post-operation (SMD = 0.87, 95%CI = [0.27, 1.47]) and presented a relatively stable improvement after 1 m post-operation (SMD = 0.52, 95%CI = [0.16, 0.89]). All parameters in amniotic membrane graft (AMT) showed better improvement in early stage and they showed non-inferior improvements in the long term compared with conjunctival autograft (CAG). Limbal-conjunctival autograft (LCAG) made excellent improvement to OS in the long term while pterygium excision (PE) showed the worst OS. The type of pterygium (primary and secondary), diabetes mellitus (DM) status, and fixation method had certain effects on the results. CONCLUSIONS: OS of pterygium is deteriorated at 1w post-operation then gradually improved to preoperative levels after 1 m post-operation. Among various surgery techniques, LCAG had the best improvement to OS which especially displayed in the long-term outcomes.

The unique properties of tear film breakup process in patients with nasal unilateral pterygium.

SIGNIFICANCE: This study found that the unique properties of tear film breakup process in eyes with pterygium, combined with ocular surface parameters, further revealed specific dynamic mechanism. It suggested that the thickness of pterygium was especially valuable in deciding the necessity of surgical management. PURPOSE: This study aimed to explore the dynamic mechanism of tear film instability in eyes with pterygium. METHODS: A paired-eye controlled cross-sectional study was conducted. Seventy-eight patients with nasal pterygium were enrolled. Fluorescein tear film breakup was observed. Several key parameters related to tear film quality were defined and analyzed, including total breakup area (mathematically derived from pixel size using MATLAB), breakup velocity, fluorescein breakup time, breakup location and pattern, tear meniscus height, score of fluorescein corneal staining, and meiboscore. RESULTS: With comparable tear meniscus height, score of fluorescein corneal staining, and meiboscore between paired eyes (p > 0.05), eyes with pterygium had shorter breakup time, larger breakup area, and faster breakup velocity (p < 0.05). In eyes with pterygium, a positive correlation between meiboscore and pterygium parameters including length, thickness, and size was observed (p > 0.001). As the thickness increased, difference of breakup time and area between paired eyes increased (p = 0.02 and 0.046). Eyes with pterygium had more fixed inferonasal breakup location and often presented as dimple break (60%), whereas random break was the most common in contralateral normal eyes (62%). A unique breakup pattern named pterygium-induced local dimple break was found. It displayed as an irregular but vertical line-like shape appearing after lipid layer spreading, which was adjacent to the lower margin of pterygium and presented with unique properties including inferonasal breakup location, local breakup area, shorten breakup time, and faster breakup velocity. CONCLUSIONS: Eyes with pterygium showed a unique tear film breakup process and novel breakup pattern named pterygium-induced local dimple break . Dynamic mechanism played a significant role in tear film instability of eyes with pterygium rather than aqueous deficiency and increased evaporation.

Advances in the phytochemistry and pharmacology of plant-derived phthalides.

Phthalides are a class of unique compounds such as ligustilide, butylphthalide and butyldenephthalide, which have shown to possess multiple bioactivities in new drug research and development. Phthalides are naturally distributed in different plants that have been utilized as herbal treatments for various ailments with a long history in Asia, Europe and North America. Their extensive biological activity has led to a dramatic increase in the study of phthalide compounds in recent years. This review summarizes the latest research progress of plant-derived phthalides, and a total of 133 phthalide compounds are described based on the characteristics of chemical structures. Pharmacological properties of plant-derived phthalides are associated with hemorheological improvement, vascular function modulation and central nervous system protection. Potential treatments for a variety of diseases mainly including cardio-cerebrovascular disorders and neurological complications such as Alzheimer's disease are also concluded. In addition, key metabolic pathways have been clearly elucidated. Further investigations on the molecular mechanisms involved in biological activity are recommended for offering new insights into profound comprehension of phthalide applications.

Individual Participant Data Meta-Analysis of LR-5 in LI-RADS Version 2018 versus Revised LI-RADS for Hepatocellular Carcinoma Diagnosis.

Background A simplification of the Liver Imaging Reporting and Data System (LI-RADS) version 2018 (v2018), revised LI-RADS (rLI-RADS), has been proposed for imaging-based diagnosis of hepatocellular carcinoma (HCC). Single-site data suggest that rLI-RADS category 5 (rLR-5) improves sensitivity while maintaining positive predictive value (PPV) of the LI-RADS v2018 category 5 (LR-5), which indicates definite HCC. Purpose To compare the diagnostic performance of LI-RADS v2018 and rLI-RADS in a multicenter data set of patients at risk for HCC by performing an individual patient data meta-analysis. Materials and Methods Multiple databases were searched for studies published from January 2014 to January 2022 that evaluated the diagnostic performance of any version of LI-RADS at CT or MRI for diagnosing HCC. An individual patient data meta-analysis method was applied to observations from the identified studies. Quality Assessment of Diagnostic Accuracy Studies version 2 was applied to determine study risk of bias. Observations were categorized according to major features and either LI-RADS v2018 or rLI-RADS assignments. Diagnostic accuracies of category 5 for each system were calculated using generalized linear mixed models and compared using the likelihood ratio test for sensitivity and the Wald test for PPV. Results Twenty-four studies, including 3840 patients and 4727 observations, were analyzed. The median observation size was 19 mm (IQR, 11-30 mm). rLR-5 showed higher sensitivity compared with LR-5 (70.6% [95% CI: 60.7, 78.9] vs 61.3% [95% CI: 45.9, 74.7]; P < .001), with similar PPV (90.7% vs 92.3%; P = .55). In studies with low risk of bias (n = 4; 1031 observations), rLR-5 also achieved a higher sensitivity than LR-5 (72.3% [95% CI: 63.9, 80.1] vs 66.9% [95% CI: 58.2, 74.5]; P = .02), with similar PPV (83.1% vs 88.7%; P = .47). Conclusion rLR-5 achieved a higher sensitivity for identifying HCC than LR-5 while maintaining a comparable PPV at 90% or more, matching the results presented in the original rLI-RADS study. © RSNA, 2023 Supplemental material is available for this article. See also the editorial by Sirlin and Chernyak in this issue.

Engineering sulfonate group donor regeneration systems to boost biosynthesis of sulfated compounds.

Sulfonation as one of the most important modification reactions in nature is essential for many biological macromolecules to function. Development of green sulfonate group donor regeneration systems to efficiently sulfonate compounds of interest is always attractive. Here, we design and engineer two different sulfonate group donor regeneration systems to boost the biosynthesis of sulfated compounds. First, we assemble three modules to construct a 3'-phosphoadenosine-5'-phosphosulfate (PAPS) regeneration system and demonstrate its applicability for living cells. After discovering adenosine 5'-phosphosulfate (APS) as another active sulfonate group donor, we engineer a more simplified APS regeneration system that couples specific sulfotransferase. Next, we develop a rapid indicating system for characterizing the activity of APS-mediated sulfotransferase to rapidly screen sulfotransferase variants with increased activity towards APS. Eventually, the active sulfonate group equivalent values of the APS regeneration systems towards trehalose and p-coumaric acid reach 3.26 and 4.03, respectively. The present PAPS and APS regeneration systems are environmentally friendly and applicable for scaling up the biomanufacturing of sulfated products.

Management Strategy for Prostate Imaging Reporting and Data System Category 3 Lesions.

PURPOSE OF REVIEW: Prostate Imaging Reporting and Data System (PI-RADS) category 3 lesions present a clinical dilemma due to their uncertain nature, which complicates the development of a definitive management strategy. These lesions have an incidence rate of approximately 22-32%, with clinically significant prostate cancer (csPCa) accounting for about 10-30%. Therefore, a thorough evaluation is warranted. RECENT FINDINGS: This review highlights the need for radiology peer review, including the confirmation of dynamic contrast-enhanced (DCE) compliance, as the initial step. Additional MRI models such as VERDICT or Tofts need to be verified. Current evidence shows that imaging and clinical indicators can be used for risk stratification of PI-RADS 3 lesions. For low-risk lesions, a safety net monitoring approach involving annual repeat MRI can be employed. In contrast, lesions deemed potentially risky based on prostate-specific antigen density (PSAD), 68 Ga-PSMA PET/CT, MPS, Proclarix, or AI/machine learning models should undergo biopsy. It is recommended to establish a multidisciplinary team that takes into account factors such as age, PSAD, prostate, and lesion size, as well as previous biopsy pathological findings. Combining expert opinions, clinical-imaging indicators, and emerging methods will contribute to the development of management strategies for PI-RADS 3 lesions.

Production of different molecular weight glycosaminoglycans with microbial cell factories.

Glycosaminoglycans (GAGs) are naturally occurring acidic polysaccharides with wide applications in pharmaceuticals, cosmetics, and health foods. The diverse biological activities and physiological functions of GAGs are closely associated with their molecular weights and sulfation patterns. Except for the non-sulfated hyaluronan which can be synthesized naturally by group A Streptococcus, all the other GAGs such as heparin and chondroitin sulfate are mainly acquired from animal tissues. Microbial cell factories provide a more effective platform for the production of structurally homogeneous GAGs. Enhancing the production efficiency of polysaccharides, accurately regulating the GAGs molecular weight, and effectively controlling the sulfation degree of GAGs represent the major challenges of developing GAGs microbial cell factories. Several enzymatic, metabolic engineering, and synthetic biology strategies have been developed to tackle these obstacles and push forward the industrialization of biotechnologically produced GAGs. This review summarizes the recent advances in the construction of GAGs synthesis cell factories, regulation of GAG molecular weight, and modification of GAGs chains. Furthermore, the challenges and prospects for future research in this field are also discussed.

Advances and challenges in biotechnological production of chondroitin sulfate and its oligosaccharides.

Chondroitin sulfate (CS) is a member of glycosaminoglycans (GAGs) and has critical physiological functions. CS is widely applied in medical and clinical fields. Currently, the supply of CS relies on traditional animal tissue extraction methods. From the perspective of medical applications, the biggest drawback of animal-derived CS is its uncontrollable molecular weight and sulfonated patterns, which are key factors affecting CS activities. The advances of cell-free enzyme catalyzed systems and de novo biosynthesis strategies have paved the way to rationally regulate CS sulfonated pattern and molecular weight. In this review, we first present a general overview of biosynthesized CS and its oligosaccharides. Then, the advances in chondroitin biosynthesis, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) synthesis and regeneration, and CS biosynthesis catalyzed by sulfotransferases are discussed. Moreover, the progress of mining and expression of chondroitin depolymerizing enzymes for preparation of CS oligosaccharides is also summarized. Finally, we analyze and discuss the challenges faced in synthesizing CS and its oligosaccharides using microbial and enzymatic methods. In summary, the biotechnological production of CS and its oligosaccharides is a promising method in addressing the drawbacks associated with animal-derived CS and enabling the production of CS oligosaccharides with defined structures.

Effects of a complex probiotic preparation, Fengqiang Shengtai and coccidiosis vaccine on the performance and intestinal microbiota of broilers challenged with Eimeria spp.

BACKGROUND: Coccidiosis, a prominent intestinal protozoan disease, carries significant economic implications for the poultry industry. The aim of this study was to evaluate the effects of Fengqiang Shengtai (BLES), a probiotics product, and coccidiosis vaccine in modulating the intestinal microbiome and providing insight into mitigating the occurrence and management of avian coccidiosis. METHODS: Broilers included in the study were divided into four pre-treatment groups: the Pre-Con group (commercial diet), Pre-BLES group (BLES supplement), Pre-Vac group (coccidiosis vaccination) and Pre-Vac-BLES group (combined vaccination and BLES). Body weight gain, feed consumption and feed conversion ratio were monitored from age 25 to 55 days. Cecum contents were collected at 8 and 15 days of age for comparative analysis of intestinal microbiomes. In the Pre-BLES and Pre-Vac-BLES groups, probiotics were administered at a dose of 0.01 g per chicken between ages 3 to 6 days and 10-13 days. At 3 days of age, chickens in the Pre-Vac and Pre-Vac-BLES groups were vaccinated with 1700 sporulated oocysts of the live coccidiosis vaccine per chicken. At the age of 25 days, Eimeria spp. challenge experiments were performed based on the aforementioned immunization strategy, and the oocysts per gram (OPG) in the feces, intestinal lesion score and intestinal pathological characteristics were evaluated. Specifically, 30 chickens were randomly selected from each group and orally administered 34,000 sporulated oocysts of Eimeria spp. per chicken, re-defined as Eimeria group, BLES-Eimeria group, Vac-Eimeria group and Vac-BLES-Eimeria group, respectively. Additionally, 30 chickens were randomly selected from the Pre-Con group and included as negative control without Eimeria spp. CHALLENGE: Intestinal microbiota was sequenced and analyzed when the broilers were 32 days old. RESULTS: A significant improvement was observed in body weight gain of the broilers in the Pre-BLES and Pre-Vac-BLES group at 45 days of age. Analysis of the intestinal microbiota revealed a positive correlation between the experimental groups receiving BLES and coccidiosis vaccines at 8 and 15 days of age with the Enterococcus genus and Lachnospiraceae NK4A136 group, respectively. In addition to the reduced lesion score and OPG values, the combination of coccidiosis vaccine and BLES also reduced the intestinal epithelial abscission induced by coccidiosis vaccines. The results of intestinal microbial function prediction demonstrated that N-glycan biosynthesis and ferroptosis were the prominent signal pathways in the Vac-BLES-Eimeria group. CONCLUSIONS: Taken together, the results of the present study suggest that supplementation of BLES with coccidiosis vaccine represents a promising strategy for improving growth performance, alleviating clinical manifestations and inducing favorable alterations to the intestinal microbiota in broiler chickens affected by coccidiosis.

Crosstalk between 5-Aminolevulinic Acid and Abscisic Acid Adjusted Leaf Iron Accumulation and Chlorophyll Synthesis to Enhance the Cold Tolerance in Solanum lycopersicum Seedlings.

Previous studies found that 5-aminolevulinic acid (ALA) and abscisic acid (ABA) can mitigate damage from adversity by enhancing photosynthesis. However, it is not clear whether they have positive effects on iron utilization and chlorophyll synthesis of tomato seedlings under low-temperature stress. To investigate the possible functional relationship between ABA and ALA and elucidate the possible mechanisms of action of ALA to alleviate low-temperature stress in tomato seedlings, this experiment analyzed the effects of ALA and ABA on chlorophyll synthesis in tomato seedling leaves sprayed with exogenous of ALA (25 mg·L-1) or ABA (100 µM) under low-temperature stress (8-18 °C/8-12 °C, day/night). The results show that exogenous ALA increased the Fv/Fm of tomato leaves by 5.31% and increased the accumulation of iron and chlorophyll by 101.15% and 15.18%, respectively, compared to the low-temperature treatment alone, and tomato resistance of low-temperature stress was enhanced. Meanwhile, exogenous application of ALA increased the ABA content by 39.43%, and subsequent application of exogenous ABA revealed that tomato seedlings showed similar effects to exogenous ALA under low-temperature stress, with increased accumulation of iron and chlorophyll in tomato seedlings, which eventually increased the maximum photochemical efficiency of PS II. Under low-temperature stress, application of exogenous ABA significantly reduced ALA content, but the expression of key enzyme genes (PPGD, HEMB1, HEME1, and HEMF1), precursors of chlorophyll synthesis by ALA, was significantly elevated, presumably because the increased activity of these enzymes after external application of ABA accelerated ALA consumption. In conclusion, ABA may crosstalk with ALA to improve the photochemical efficiency and low temperature resistance of tomatoes by regulating chlorophyll synthesis and iron accumulation.