SYMBIOSIS: synthetic manipulable biobricks via orthogonal serine integrase systems.

Serine integrases are emerging as one of the most powerful biological tools for synthetic biology. They have been widely used across genome engineering and genetic circuit design. However, developing serine integrase-based tools for directly/precisely manipulating synthetic biobricks is still missing. Here, we report SYMBIOSIS, a versatile method that can robustly manipulate DNA parts in vivo and in vitro. First, we propose a 'keys match locks' model to demonstrate that three orthogonal serine integrases are able to irreversibly and stably switch on seven synthetic biobricks with high accuracy in vivo. Then, we demonstrate that purified integrases can facilitate the assembly of 'donor' and 'acceptor' plasmids in vitro to construct composite plasmids. Finally, we use SYMBIOSIS to assemble different chromoprotein genes and create novel colored Escherichia coli. We anticipate that our SYMBIOSIS strategy will accelerate synthetic biobrick manipulation, genetic circuit design and multiple plasmid assembly for synthetic biology with broad potential applications.

Stapled NRPS enhances the production of valinomycin in Escherichia coli.

Nonribosomal peptides (NRPs) are a large family of secondary metabolites with notable bioactivities, which distribute widely in natural resources across microbes and plants. To obtain these molecules, heterologous production of NRPs in robust surrogate hosts like Escherichia coli represent a feasible approach. However, reconstitution of the full biosynthetic pathway in a host often leads to low productivity, which is at least in part due to the low efficiency of enzyme interaction in vivo except for the well-known reasons of metabolic burden (e.g., expression of large NRP synthetases-NRPSs with molecular weights of >100 kDa) and cellular toxicity on host cells. To enhance the catalytic efficiency of large NRPSs in vivo, here we propose to staple NRPS enzymes by using short peptide/protein pairs (e.g., SpyTag/SpyCatcher) for enhanced NRP production. We achieve this goal by introducing a stapled NRPS system for the biosynthesis of the antibiotic NRP valinomycin in E. coli. The results indicate that stapled valinomycin synthetase (Vlm1 and Vlm2) enables higher product accumulation than those two free enzymes (e.g., the maximum improvement is nearly fourfold). After further optimization by strain and bioprocess engineering, the final valinomycin titer maximally reaches about 2800 µg/L, which is 73 times higher than the initial titer of 38 µg/L. We expect that stapling NRPS enzymes will be a promising catalytic strategy for high-level biosynthesis of NRP natural products.

Quantitative Susceptibility Mapping Changes Relate to Gait Issues in Parkinson's Disease.

BACKGROUND: Quantitative susceptibility mapping (QSM) demonstrates elevated iron content in Parkinson's disease (PD) patients within the basal ganglia, though it has infrequently been studied in relation to gait difficulties including freezing of gait (FOG). Our purpose was to relate QSM of basal ganglia and extra-basal ganglia structures with qualitative and quantitative gait measures in PD. METHODS: This case-control study included PD and cognitively unimpaired (CU) participants from the Comprehensive Assessment of Neurodegeneration and Dementia study. Whole brain QSM was acquired at 3T. Region of interests (ROIs) were drawn blinded manually in the caudate nucleus, putamen, globus pallidus, pulvinar nucleus of the thalamus, red nucleus, substantia nigra, and dentate nucleus. Susceptibilities of ROIs were compared between PD and CU. Items from the FOG questionnaire and quantitative gait measures from PD participants were compared to susceptibilities. RESULTS: Twenty-nine participants with PD and 27 CU participants were included. There was no difference in susceptibility values in any ROI when comparing CU versus PD (p > 0.05 for all). PD participants with gait impairment (n = 23) had significantly higher susceptibility in the putamen (p = 0.008), red nucleus (p = 0.01), and caudate nucleus (p = 0.03) compared to those without gait impairment (n = 6). PD participants with FOG (n = 12) had significantly higher susceptibility in the globus pallidus (p = 0.03) compared to those without FOG (n = 17). Among quantitative gait measures, only stride time variability was significantly different between those with and without FOG (p = 0.04). CONCLUSION: Susceptibilities in basal ganglia and extra-basal ganglia structures are related to qualitative measures of gait impairment and FOG in PD.

Programmable and printable Bacillus subtilis biofilms as engineered living materials.

Bacterial biofilms can be programmed to produce living materials with self-healing and evolvable functionalities. However, the wider use of artificial biofilms has been hindered by limitations on processability and functional protein secretion capacity. We describe a highly flexible and tunable living functional materials platform based on the TasA amyloid machinery of the bacterium Bacillus subtilis. We demonstrate that genetically programmable TasA fusion proteins harboring diverse functional proteins or domains can be secreted and can assemble into diverse extracellular nano-architectures with tunable physicochemical properties. Our engineered biofilms have the viscoelastic behaviors of hydrogels and can be precisely fabricated into microstructures having a diversity of three-dimensional (3D) shapes using 3D printing and microencapsulation techniques. Notably, these long-lasting and environmentally responsive fabricated living materials remain alive, self-regenerative, and functional. This new tunable platform offers previously unattainable properties for a variety of living functional materials having potential applications in biomaterials, biotechnology, and biomedicine.

Falls in Synucleinopathies.

Parkinson's disease (PD) and other synucleinopathies, namely dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), are common degenerative neurological disorders that share synuclein pathology. Although certain cardinal features of parkinsonism, including bradykinesia and rigidity, respond well to levodopa, axial features, such as gait and balance impairment, are less reliably responsive to dopaminergic therapy and surgical interventions. Consequently, falls are common in PD and other synucleinopathies and are a major contributor toward injury and loss of independence. This underscores the need for appropriate fall risk assessment and implementation of preventative measures in all patients with parkinsonism. The aim of this review is therefore to explore modifiable and non-modifiable risk factors for falls in synucleinopathies. We next review and evaluate the evidence for pharmacological, nonpharmacological, and surgical approaches for fall prevention, and emphasize individualized and multifaceted approaches.

Les risques de chute dans le cas des synucléinopathies. La maladie de Parkinson (MP), de même que d'autres synucléinopathies comme la démence à corps de Lewy (DCL) et l'atrophie multi-systématisée (AMS), sont des troubles neurologiques dégénératifs courants qui ont en commun l'accumulation anormale de protéine synucléine. Bien que certains des principaux symptômes caractéristiques de la MP, par exemple la bradykinésie et la rigidité, répondent bien à la lévodopa, d'autres signes axiaux, par exemple une altération de l'équilibre et de la démarche, vont répondre de façon moins efficace à un traitement dopaminergique et à des interventions chirurgicales. Il s'ensuit que les chutes de patients atteints de la MP et d'autres synucléinopathies contribuent grandement à leur perte d'autonomie, et ce, en raison de blessures. Cette situation met en évidence la nécessité de procéder à une évaluation appropriée des risques de chute chez ces patients et de mettre en œuvre des mesures préventives destinées à tous les patients souffrant de parkinsonisme. L'objectif de cette étude consiste donc, dans le cas des synucléinopathies, à examiner les facteurs de risque modifiables et non-modifiables liés aux chutes. Nous passerons ainsi en revue et évaluerons les approches pharmacologiques, non-pharmacologiques et chirurgicales dans la prévention des chutes pour ensuite mettre en relief des approches individuelles et multidimensionnelles.

Repetitive transcranial magnetic stimulation protects mice against 6-OHDA-induced Parkinson's disease symptoms by regulating brain amyloid ß1-42 level.

Repetitive transcranial magnetic stimulation (rTMS) is a technique protecting neurons against diverse neurodegenerative disorders by delivering magnetic stimuli into the brain through the intact scalp. In the current study, the protection effect of rTMS on Parkinson's disease (PD) and the associated mechanism driving the treatment were explored. The PD symptoms were induced using 6-OHDA in mice, and the effect of rTMS of two frequencies (1 Hz and 10 Hz) on the cognitive behaviors and neuron viability was detected. Afterwards, the level of Aß1-42 and activity of MKK7-ERK-Fos-APP axis under the administration of rTMS were recorded as well. The intracranial injection of 6-OHDA impaired the cognitive behaviors of the mice in the test of Morris water maze as well as reducing the viability and number of neurons in PD mice. After the treatment of rTMS of both frequencies, the cognitive function of mice was improved and the neuron viability and number were restored in mice brain tissues. The administration of rTMS also increased the cerebrospinal fluid (CSF) level of Aß1-42 in PD mice, which was accompanied by the suppressed levels of p-MKK7, p-ERK1/2, p-c-Fos, and APP. Moreover, the effect of rTMS on mice nerve system was all exerted in a frequency-dependent manner. In conclusion, the findings outlined in the current study affirmed the protection effect of rTMS against PD. The anti-PD function of rTMS was associated with the suppression of MKK7-ERK-Fos-APP axis, which subsequently resulted in the increased CSF Aß1-42 level and decreased brain Aß1-42 level.

Physicochemical and Biocompatibility Properties of Type I Collagen from the Skin of Nile Tilapia (Oreochromis niloticus) for Biomedical Applications.

The aim of this study is to investigate the physicochemical properties, biosafety, and biocompatibility of the collagen extract from the skin of Nile tilapia, and evaluate its use as a potential material for biomedical applications. Two extraction methods were used to obtain acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) from tilapia skin. Amino acid composition, FTIR, and SDS-PAGE results showed that ASC and PSC were type I collagen. The molecular form of ASC and PSC is (α1)2α2. The FTIR spectra of ASC and PSC were similar, and the characteristic peaks corresponding to amide A, amide B, amide I, amide II, and amide III were 3323 cm-1, 2931 cm-1, 1677 cm-1, 1546 cm-1, and 1242 cm-1, respectively. Denaturation temperatures (Td) were 36.1 °C and 34.4 °C, respectively. SEM images showed the loose and porous structure of collagen, indicting its physical foundation for use in applications of biomedical materials. Negative results were obtained in an endotoxin test. Proliferation rates of osteoblastic (MC3T3E1) cells and fibroblast (L929) cells from mouse and human umbilical vein endothelial cells (HUVEC) were increased in the collagen-treated group compared with the controls. Furthermore, the acute systemic toxicity test showed no acute systemic toxicity of the ASC and PSC collagen sponges. These findings indicated that the collagen from Nile tilapia skin is highly biocompatible in nature and could be used as a suitable biomedical material.

Imaging appearance of myelopathy secondary to nitrous oxide abuse: a case report and review of the literature.

Purpose The abuse of nitrous oxide (N2O) can induce Vitamin B12 deficiency that subsequently leads to central nervous demyelination, myelopathy and peripheral neuropathy. Although myelopathy has been reported in the past, the specific locations and prognosis of the disease are still unclear. MATERIALS AND METHODS: We report the case of a 22-year-old male who presented with quadriplegia that began after a 3-month history of inhalation of N2O. We summarized the clinical data of this entity and performed a comprehensive literature review of various presentations and MRI features of myelopathy secondary to N2O abuse. RESULTS: In combination with previous reports of 14 cases, we found that the onset of the disease was usually subacute, and the majority of patients (92.85%) were young men. There was no definite relationship between myelopathy and the amount or duration of N2O inhalation. The most common clinical manifestation was sensory ataxia, and the cervical spinal cord was the most frequently impaired area of the whole spinal cord. The spinal cord lesions had a high signal intensity on T2-weighted MRI and usually involved more than three spinal segments and impaired the posterior column more significantly. Most patients recovered well after vitamin B12 supplementation. CONCLUSIONS: Myelopathy secondary to N2O abuse is generally seen in young men. The clinical diagnosis mainly depends on a history of N2O inhalation and the characteristic imaging changes in the posterior cervical spinal cord. Early diagnosis and intervention are important for a satisfactory prognosis.

Parkinson Disease: The Relationship Between Non-motor Symptoms and Motor Phenotype.

BACKGROUND: Parkinson disease (PD) presents with motor and non-motor symptoms (NMS). The NMS often precede the onset of motor symptoms, but may progress throughout the disease course. Tremor dominant, postural instability gait difficulty (PIGD), and indeterminate phenotypes can be distinguished using Unified PD Rating scales (UPDRS-III). We hypothesized that the PIGD phenotype would be more likely to develop NMS, and that the non-dopamine-responsive axial signs would correlate with NMS severity. METHODS: We conducted a retrospective cross-sectional chart review to assess the relationship between NMS and PD motor phenotypes. PD patients were administered the NMS Questionnaire, the UPDRS-III, and the Mini-Mental State Examination score. The relationship between NMS burden and PD subtypes was examined using linear regression models. The prevalence of each NMS among difference PD motor subtypes was analyzed using chi-square test. RESULTS: PD patients with more advanced disease based on their UPDRS-III had higher NMS Questionnaire scores. The axial component of UPDRS-III correlated with higher NMS. There was no correlation between NMS and tremor scores. There was a significant correlation between PIGD score and higher NMS burden. PIGD group had higher prevalence in most NMS domains when compared with tremor dominant and indeterminate groups independent of disease duration and severity. CONCLUSIONS: NMS profile and severity vary according to motor phenotype. We conclude that in the PD population, patients with a PIGD phenotype who have more axial involvement, associated with advanced disease and poor motor response, have a higher risk for a higher NMS burden.

Rainbow screening: Chromoproteins enable visualized molecular cloning.

Molecular cloning facilitates the assembly of heterologous DNA fragments with vectors, resulting in the generation of plasmids that can steadily replicate in host cells. To efficiently and accurately screen out the expected plasmid candidates, various methods, such as blue-white screening, have been developed for visualization. However, these methods typically require additional genetic manipulations and costs. To simplify the process of visualized molecular cloning, here we report Rainbow Screening, a method that combines Gibson Assembly with chromoproteins to distinguish Escherichia coli (E. coli) colonies by naked eyes, eliminating the need for additional genetic manipulations or costs. To illustrate the design, we select both E. coli 16s rRNA and sfGFP expression module as two inserted fragments. Using Rainbow Screening, false positive colonies can be easily distinguished on LB-agar plates. Moreover, both the assembly efficiency and the construct accuracy can exceed 80%. We anticipate that Rainbow Screening will enrich the molecular cloning methodology and expand the application of chromoproteins in biotechnology and synthetic biology.

Expanding the toolbox of probiotic Escherichia coli Nissle 1917 for synthetic biology.

Escherichia coli Nissle 1917 (EcN) is a probiotic microbe that has the potential to be developed as a promising chassis for synthetic biology applications. However, the molecular tools and techniques for utilizing EcN remain to be further explored. To address this opportunity, the EcN-based toolbox was systematically expanded, enabling EcN as a powerful platform for more applications. First, two EcN cryptic plasmids and other compatible plasmids were genetically engineered to enrich the manipulable plasmid toolbox for multiple gene coexpression. Next, two EcN-based technologies were developed, including the conjugation strategy for DNA transfer, and quantification of protein expression capability. Finally, the EcN-based applications were further expanded by developing EcN native integrase-mediated genetic engineering and establishing an in vitro cell-free protein synthesis (CFPS) system. Overall, this study expanded the toolbox for manipulating and making full use of EcN as a commonly used probiotic chassis, providing several simplified, dependable, and predictable strategies for researchers working in synthetic biology fields.

An In Vitro Hybrid Biocatalytic System Enabled by a Combination of Surface-Displayed, Purified, and Cell-Free Expressed Enzymes.

Enzymatic cascades have become a green and sustainable approach for the synthesis of valuable chemicals and pharmaceuticals. Using sequential enzymes to construct a multienzyme complex is an effective way to enhance the overall performance of biosynthetic routes. Here we report the design of an efficient in vitro hybrid biocatalytic system by assembling three enzymes that can convert styrene to (S)-1-phenyl-1,2-ethanediol. Specifically, we prepared the three enzymes in different ways, which were cell surface-displayed, purified, and cell-free expressed. To assemble them, we fused two orthogonal peptide-protein pairs (i.e., SpyTag/SpyCatcher and SnoopTag/SnoopCatcher) to the three enzymes, allowing their spatial organization by covalent assembly. By doing this, we constructed a multienzyme complex, which could enhance the production of (S)-1-phenyl-1,2-ethanediol by 3 times compared to the free-floating enzyme system without assembly. After optimization of the reaction system, the final product yield reached 234.6 µM with a substrate conversion rate of 46.9% (based on 0.5 mM styrene). Taken together, our strategy integrates the merits of advanced biochemical engineering techniques, including cellular surface display, spatial enzyme organization, and cell-free expression, which offers a new solution for chemical biosynthesis by enzymatic cascade biotransformation. We, therefore, anticipate that our approach will hold great potential for designing and constructing highly efficient systems to synthesize chemicals of agricultural, industrial, and pharmaceutical significance.

Cell-free biosynthesis and engineering of ribosomally synthesized lanthipeptides.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural products with diverse chemical structures and potent biological activities. A vast majority of RiPP gene clusters remain unexplored in microbial genomes, which is partially due to the lack of rapid and efficient heterologous expression systems for RiPP characterization and biosynthesis. Here, we report a unified biocatalysis (UniBioCat) system based on cell-free gene expression for rapid biosynthesis and engineering of RiPPs. We demonstrate UniBioCat by reconstituting a full biosynthetic pathway for de novo biosynthesis of salivaricin B, a lanthipeptide RiPP. Next, we delete several protease/peptidase genes from the source strain to enhance the performance of UniBioCat, which then can synthesize and screen salivaricin B variants with enhanced antimicrobial activity. Finally, we show that UniBioCat is generalizable by synthesizing and evaluating the bioactivity of ten uncharacterized lanthipeptides. We expect UniBioCat to accelerate the discovery, characterization, and synthesis of RiPPs.

Gray matter loss relates to dual task gait in Lewy body disorders and aging.

BACKGROUND: Within the spectrum of Lewy body disorders (LBD), both Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are characterized by gait and balance disturbances, which become more prominent under dual-task (DT) conditions. The brain substrates underlying DT gait variations, however, remain poorly understood in LBD. OBJECTIVE: To investigate the relationship between gray matter volume loss and DT gait variations in LBD. METHODS: Seventy-nine participants including cognitively unimpaired PD, PD with mild cognitive impairment, PD with dementia (PDD), or DLB and 20 cognitively unimpaired controls were examined across a multi-site study. PDD and DLB were grouped together for analyses. Differences in gait speed between single and DT conditions were quantified by dual task cost (DTC). Cortical, subcortical, ventricle, and cerebellum brain volumes were obtained using FreeSurfer. Linear regression models were used to examine the relationship between gray matter volumes and DTC. RESULTS: Smaller amygdala and total cortical volumes, and larger ventricle volumes were associated with a higher DTC across LBD and cognitively unimpaired controls. No statistically significant interaction between group and brain volumes were found. Adding cognitive and motor covariates or white matter hyperintensity volumes separately to the models did not affect brain volume and DTC associations. CONCLUSION: Gray matter volume loss is associated with worse DT gait performance compared to single task gait, across cognitively unimpaired controls through and the LBD spectrum. Impairment in DT gait performance may be driven by age-related cortical neurodegeneration.

Gut microbiota changes associated with low-carbohydrate diet intervention for obesity.

Low-carbohydrate diets (LCDs) are frequently recommended for alleviating obesity, and the gut microbiota plays key roles in energy metabolism and weight loss. However, there is limited in-human research on how LCD changes gut microbiota. In this before-after study, 43 participants were assigned to the LCD intervention for 4 weeks. The main objective was to investigate the specific changes that occur in the participants' microbiome in response to the LCD. Changes in gut microbiota were analyzed using 16s rRNA sequencing. Body composition was measured using InBody 770. Remarkably, 35 participants (79.07%) lost more than 5% of their body weight; levels of BMI, body fat, and total cholesterol were significantly decreased, indicating the effectiveness of the LCD intervention. The richness of microbiota significantly increased after the intervention. By taking the intersection of ANOVA and linear discriminant analysis effect size (LEfSe) analysis results, we identified three phyla, three classes, four orders, five families, and six genera that were differentially enriched between baseline and week-4 time points. Among the three phyla, relative abundances of Firmicutes and Actinobacteriota decreased significantly, while Bacteroidetes increased significantly. At the genus level, Ruminococcus, Agathobacter, Streptococcus, and Bifidobacterium showed a significant reduction in relative abundances, whereas Parabacteroides and Bacteroides increased steadily. Our results demonstrate that LCD can effectively alleviate obesity and modify certain taxa of gut microbiota, providing potential insights for personalized dietary interventions against obesity.

Harnessing the potential of long non-coding RNAs in breast cancer: from etiology to treatment resistance and clinical applications.

Breast cancer (BC) is the most common malignancy among women and a leading cause of cancer-related deaths of females worldwide. It is a complex and molecularly heterogeneous disease, with various subtypes that require different treatment strategies. Despite advances in high-resolution single-cell and multinomial technologies, distant metastasis and therapeutic resistance remain major challenges for BC treatment. Long non-coding RNAs (lncRNAs) are non-coding RNAs with more than 200 nucleotides in length. They act as competing endogenous RNAs (ceRNAs) to regulate post-transcriptional gene stability and modulate protein-protein, protein-DNA, and protein-RNA interactions to regulate various biological processes. Emerging evidence suggests that lncRNAs play essential roles in human cancers, including BC. In this review, we focus on the roles and mechanisms of lncRNAs in BC progression, metastasis, and treatment resistance, and discuss their potential value as therapeutic targets. Specifically, we summarize how lncRNAs are involved in the initiation and progression of BC, as well as their roles in metastasis and the development of therapeutic resistance. We also recapitulate the potential of lncRNAs as diagnostic biomarkers and discuss their potential use in personalized medicine. Finally, we provide lncRNA-based strategies to promote the prognosis of breast cancer patients in clinical settings, including the development of novel lncRNA-targeted therapies.