Context-dependent neocentromere activity in synthetic yeast chromosome VIII.

Pioneering advances in genome engineering, and specifically in genome writing, have revolutionized the field of synthetic biology, propelling us toward the creation of synthetic genomes. The Sc2.0 project aims to build the first fully synthetic eukaryotic organism by assembling the genome of Saccharomyces cerevisiae. With the completion of synthetic chromosome VIII (synVIII) described here, this goal is within reach. In addition to writing the yeast genome, we sought to manipulate an essential functional element: the point centromere. By relocating the native centromere sequence to various positions along chromosome VIII, we discovered that the minimal 118-bp CEN8 sequence is insufficient for conferring chromosomal stability at ectopic locations. Expanding the transplanted sequence to include a small segment (∼500 bp) of the CDEIII-proximal pericentromere improved chromosome stability, demonstrating that minimal centromeres display context-dependent functionality.

Diagnosing the benign paroxysmal positional vertigo via 1D and deep-learning composite model.

BACKGROUND: Benign Paroxysmal Positional Vertigo (BPPV) is the leading cause of vertigo, and its characteristic nystagmus induced by positional maneuvers makes it a good model for Artificial Intelligence (AI) diagnosis. However, during the testing procedure, up to 10 min of indivisible long-range temporal correlation data are produced, making the AI-informed real-time diagnosing unlikely in clinical practice. METHODS: A combined 1D and Deep-Learning (DL) composite model was proposed. Two separate cohorts were recruited, with one for model generation and the other for evaluation of model's real-world generalizability. Eight features, including two head traces and three eye traces and their corresponding slow phase velocity (SPV) value, were served as the inputs. Three candidate models were tested, and a sensitivity study was conducted to determine the saliently important features. RESULTS: The study included 2671 patients in the training cohort and 703 in the test cohort. A hybrid DL model achieved a micro-area under the receiver operating curve (AUROC) of 0.982 (95% CI 0.965, 0.994) and macro-AUROC of 0.965 (95% CI 0.898, 0.999) for overall classification. The highest accuracy was observed for right posterior BPPV, with an AUROC of 0.991 (95% CI 0.972, 1.000), followed by left posterior BPPV, with an AUROC of 0.979 (95% CI 0.940, 0.998), the lowest AUROC was 0.928 (95% CI 0.878, 0.966) for lateral BPPV. The SPV was consistently identified as the most predictive feature in the models. If the model process is carried out 100 times for a 10-min data, one single running takes 0.79 ± 0.06 s. CONCLUSION: This study designed DL models which can accurately detect and categorize the subtype of BPPV, enabling a quick and straightforward diagnosis of BPPV in clinical setting. The critical feature identified in the model helps expand our understanding of this disorder.

Social distancing cut down the prevalence of acute otitis media in children.

Objectives: To evaluate the additional, unintended benefits of social distancing in cutting down the prevalence of acute otitis media (AOM) in children, especially during coronavirus disease 2019 (COVID-19) periods. Methods: The daily outpatient attendance of AOM for childhood (from 6 months to 12 years) was compared in the tertiary hospital in Shanghai during pre-COVID-19 and COVID-19 year. Results: A total of 24,543 AOM cases were included from 2015 to 2020. When age was taken into account, children in kindergarten (aged 4-6) constitute 66.2% (16,236/24,543) of all case, followed by primary school students (6,441/24,543, 26.2%) and preschoolers <3 years old (1,866/24,543, 7.6%). There was an estimated 63.6% (54.32-70.36%) reduction in the daily outpatient attendance of AOM associated with the introduction of social distancing in 2020 (COVID-19 year). The epidemic trend of AOM in 2015-2019 was characterized by seasonal fluctuations, with highest incidence in December (18.8 ± 0.5%) and lower in February (4.5 ± 0.2%), June (3.7 ± 0.7%) and August (3.5 ± 0.5%). And distribution characteristics of different ages in COVID-19 period broadly in line with that in non-pandemic period. Conclusion: Seasonal fluctuation in the prevalence of AOM was observed in pre-COVID-19 period (2015-2019), with a peak in winter and a nadir in summer. The >50% drop of outpatient attendance of AOM in 2020 (COVID-19 year) suggest that social distancing, mask effects and good hand hygiene can significantly reduce the incidence of AOM, which provides a preventive and therapeutic point of view for AOM.

A Questionnaire-Based Ensemble Learning Model to Predict the Diagnosis of Vertigo: Model Development and Validation Study.

BACKGROUND: Questionnaires have been used in the past 2 decades to predict the diagnosis of vertigo and assist clinical decision-making. A questionnaire-based machine learning model is expected to improve the efficiency of diagnosis of vestibular disorders. OBJECTIVE: This study aims to develop and validate a questionnaire-based machine learning model that predicts the diagnosis of vertigo. METHODS: In this multicenter prospective study, patients presenting with vertigo entered a consecutive cohort at their first visit to the ENT and vertigo clinics of 7 tertiary referral centers from August 2019 to March 2021, with a follow-up period of 2 months. All participants completed a diagnostic questionnaire after eligibility screening. Patients who received only 1 final diagnosis by their treating specialists for their primary complaint were included in model development and validation. The data of patients enrolled before February 1, 2021 were used for modeling and cross-validation, while patients enrolled afterward entered external validation. RESULTS: A total of 1693 patients were enrolled, with a response rate of 96.2% (1693/1760). The median age was 51 (IQR 38-61) years, with 991 (58.5%) females; 1041 (61.5%) patients received the final diagnosis during the study period. Among them, 928 (54.8%) patients were included in model development and validation, and 113 (6.7%) patients who enrolled later were used as a test set for external validation. They were classified into 5 diagnostic categories. We compared 9 candidate machine learning methods, and the recalibrated model of light gradient boosting machine achieved the best performance, with an area under the curve of 0.937 (95% CI 0.917-0.962) in cross-validation and 0.954 (95% CI 0.944-0.967) in external validation. CONCLUSIONS: The questionnaire-based light gradient boosting machine was able to predict common vestibular disorders and assist decision-making in ENT and vertigo clinics. Further studies with a larger sample size and the participation of neurologists will help assess the generalization and robustness of this machine learning method.

The effect of fluoxetine combined with repetitive transcranial magnetic stimulation on the psychological emotions and cognitive and neurological functions of acute post-stroke depression patients.

OBJECTIVE: This research was designed to probe into the effects of fluoxetine combined with repetitive transcranial magnetic stimulation (rTMS) on the psychological emotions and the cognitive and neurological functions of acute post-stroke depression patients. METHODS: This experiment recruited 115 acute post-stroke depression patients who were treated in our hospital from February 2018 to April 2020 as the study cohort. 55 of the patients were treated with fluoxetine, and 60 were treated with fluoxetine combined with rTMS. Both groups were treated for 2 months. The self-rating anxiety scale (SAS), the self-rating depression scale (SDS), the National Institutes of Health stroke scale (NIHSS), the mini mental state scale (MMSE), the Barthel index, and the quality of life scale (SF-36) scores were observed. RESULTS: Compared with the control group (CG), the SAS, SDS, and NIHSS scores in the research group (RG) decreased, while the MMSE and Barthel index scores increased (P < 0.05). After the treatment, the SF-36 scores in the RG were higher than they were in the CG (P < 0.05). CONCLUSION: Fluoxetine combined with rTMS can effectively improve the psychological emotions and the cognitive and neurological functions of acute post-stroke depression patients, so it is worthy of clinical promotion.

Sequential enzyme-activated macrotheranostic probe for selective tumor mitochondria targeting.

Subcellular organelle targeted imaging and therapy are of enormous interest in cancer theranostics. However, the lack of tumor-selective organelle targeting has compromised their efficacy and safety. In this work, we found that the near-infrared (NIR) fluorophore hemicyanine (CyNH2) can selectively target mitochondria with strong cytotoxicity through decreasing the mitochondrial membrane potential and increasing the intracellular reactive oxygen species (ROS) levels. A macrotheranostic probe (denoted as PLCy) based on conjugating CyNH2 with an acetylated lysine group was developed with masked fluorescence and cytotoxicity, which could both be unmasked through sequential activation by cancer cells overexpressing histone deacetylases (HDACs) and cathepsin L (CTSL) enzymes for selective cancer cell mitochondria-targeted imaging and therapy. In vitro and in vivo studies confirmed that the specific fluorescence turn-on and toxicity were restored in cancer cells and efficiently inhibited tumor growth. This macrotheranostic probe with sequential enzyme activation and mitochondrial targeting is expected to have promising applications in imaging-guided cancer therapy with high specificity and efficiency. STATEMENT OF SIGNIFICANCE: To improve the targeting efficiency and enhance the anti-cancer activities of macrotheranostic probe. We designed macrotheranostic probe PLCy that can be activated via sequential enzymes for selective tumor mitochondria targeting. More importantly, the activated CyNH2 can decrease the mitochondrial membrane potential and elevate the reactive oxygen species level in cancer cells without light irradiation, which can further induce apoptosis of tumor cells for chemotherapy. Therefore, the use of sequential enzyme activation and mitochondria targeting strategies in the context of enzymatic activation may provide a general strategy for organelle-targeted imaging and therapy with high specificity and efficiency.

Dual-Drug Backboned Polyprodrug with a Predefined Drug Combination for Synergistic Chemotherapy.

The codelivery of drugs at specific optimal ratios to cancer cells is vital for combination chemotherapy. However, most of the current strategies are unable to coordinate the loading and release of drug combinations to acquire precise and controllable synergistic ratios. In this work, we designed an innovative dual-drug backboned and reduction-sensitive polyprodrug PEG-P(MTO-ss-CUR) containing the anticancer drugs mitoxantrone (MTO) and curcumin (CUR) at an optimal synergistic ratio to reverse drug resistance. Due to synchronous drug activation and polymer backbone degradation, drug release at the predefined ratio with a synergistic anticancer effect was demonstrated by in vitro and in vivo experiments. Therefore, the dual-drug delivery system developed in this work provides a novel and efficient strategy for combination chemotherapy.

Application of counter-selectable marker PIGA in engineering designer deletion cell lines and characterization of CRISPR deletion efficiency.

The CRISPR/Cas9 system is a technology for genome engineering, which has been applied to indel mutations in genes as well as targeted gene deletion and replacement. Here, we describe paired gRNA deletions along the PIGA locus on the human X chromosome ranging from 17 kb to 2 Mb. We found no compelling linear correlation between deletion size and the deletion efficiency, and there is no substantial impact of topologically associating domains on deletion frequency. Using this precise deletion technique, we have engineered a series of designer deletion cell lines, including one with deletions of two X-chromosomal counterselectable (negative selection) markers, PIGA and HPRT1, and additional cell lines bearing each individual deletion. PIGA encodes a component of the glycosylphosphatidylinositol (GPI) anchor biosynthetic apparatus. The PIGA gene counterselectable marker has unique features, including existing single cell level assays for both function and loss of function of PIGA and the existence of a potent counterselectable agent, proaerolysin, which we use routinely for selection against cells expressing PIGA. These designer cell lines may serve as a general platform with multiple selection markers and may be particularly useful for large scale genome engineering projects such as Genome Project-Write (GP-write).

Linear Well-Defined Polyamines via Anionic Ring-Opening Polymerization of Activated Aziridines: From Mild Desulfonylation to Cell Transfection.

Linear polyethylenimine (L-PEI), a standard for nonviral gene delivery, is usually prepared by hydrolysis from poly(2-oxazoline)s. Lately, anionic polymerization of sulfonamide-activated aziridines had been reported as an alternative pathway toward well-defined L-PEI and linear polyamines. However, desulfonylation of the poly(sulfonyl aziridine)s typically relied on harsh conditions (acid, microwave) or used a toxic solvent (e.g., hexamethylphosphoramide). In addition, the drastic change of polarity requires solvents, which keep poly(sulfonyl aziridine)s as well as L-PEI in solution, and only a limited number of strategies were reported. Herein, we prepared 1-(4-cyanobenzenesulfonyl) 2-methyl-aziridine (1) as a monomer for the anionic ring-opening polymerization. It was polymerized to well-defined and linear poly(sulfonyl aziridine)s. The 4-cyanobenzenesulfonyl-activating groups were removed under mild conditions to linear polypropylenimine (L-PPI). Using dodecanethiol and diazabicyclo-undecene (DBU) allowed ≥98% desulfonylation and a reliable purification toward polyamines with high purity and avoiding main-chain scission. This method represents a fast approach in comparison to previous methods used for postpolymerization desulfonylation and produces linear well-defined polyamines. The high control over molecular weight and dispersities achieved by living anionic polymerization are the key advantages of our strategy, especially if used for biomedical applications, in which molecular weight might correlate with toxicity. The synthesized polypropylenimine was further tested as a cell-transfection agent and proved, with 16.1% transfection efficiency of the cationic nanoparticles, to be an alternative to L-PEI obtained from the 2-oxazoline route. This general strategy will allow the preparation of complex macromolecular architectures containing polyamine segments, which were not accessible before.