A genetic mammalian proportional-integral feedback control circuit for robust and precise gene regulation.

The processes that keep a cell alive are constantly challenged by unpredictable changes in its environment. Cells manage to counteract these changes by employing sophisticated regulatory strategies that maintain a steady internal milieu. Recently, the antithetic integral feedback motif has been demonstrated to be a minimal and universal biological regulatory strategy that can guarantee robust perfect adaptation for noisy gene regulatory networks in Escherichia coli. Here, we present a realization of the antithetic integral feedback motif in a synthetic gene circuit in mammalian cells. We show that the motif robustly maintains the expression of a synthetic transcription factor at tunable levels even when it is perturbed by increased degradation or its interaction network structure is perturbed by a negative feedback loop with an RNA-binding protein. We further demonstrate an improved regulatory strategy by augmenting the antithetic integral motif with additional negative feedback to realize antithetic proportional-integral control. We show that this motif produces robust perfect adaptation while also reducing the variance of the regulated synthetic transcription factor. We demonstrate that the integral and proportional-integral feedback motifs can mitigate the impact of gene expression burden, and we computationally explore their use in cell therapy. We believe that the engineering of precise and robust perfect adaptation will enable substantial advances in industrial biotechnology and cell-based therapeutics.

DeepSARS: simultaneous diagnostic detection and genomic surveillance of SARS-CoV-2.

BACKGROUND: The continued spread of SARS-CoV-2 and emergence of new variants with higher transmission rates and/or partial resistance to vaccines has further highlighted the need for large-scale testing and genomic surveillance. However, current diagnostic testing (e.g., PCR) and genomic surveillance methods (e.g., whole genome sequencing) are performed separately, thus limiting the detection and tracing of SARS-CoV-2 and emerging variants. RESULTS: Here, we developed DeepSARS, a high-throughput platform for simultaneous diagnostic detection and genomic surveillance of SARS-CoV-2 by the integration of molecular barcoding, targeted deep sequencing, and computational phylogenetics. DeepSARS enables highly sensitive viral detection, while also capturing genomic diversity and viral evolution. We show that DeepSARS can be rapidly adapted for identification of emerging variants, such as alpha, beta, gamma, and delta strains, and profile mutational changes at the population level. CONCLUSIONS: DeepSARS sets the foundation for quantitative diagnostics that capture viral evolution and diversity. DeepSARS uses molecular barcodes (BCs) and multiplexed targeted deep sequencing (NGS) to enable simultaneous diagnostic detection and genomic surveillance of SARS-CoV-2. Image was created using Biorender.com .

Enhancing the efficiency of the Pichia pastoris AOX1 promoter via the synthetic positive feedback circuit of transcription factor Mxr1.

BACKGROUND: The methanol-regulated AOX1 promoter (PAOX1) is the most widely used promoter in the production of recombinant proteins in the methylotrophic yeast Pichia pastoris. However, as the tight regulation and methanol dependence of PAOX1 restricts its application, it is necessary to develop a flexible induction system to avoid the problems of methanol without losing the advantages of PAOX1. The availability of synthetic biology tools enables researchers to reprogram the cellular behaviour of P. pastoris to achieve this goal. RESULTS: The characteristics of PAOX1 are highly related to the expression profile of methanol expression regulator 1 (Mxr1). In this study, we applied a biologically inspired strategy to reprogram regulatory networks in P. pastoris. A reprogrammed P. pastoris was constructed by inserting a synthetic positive feedback circuit of Mxr1 driven by a weak AOX2 promoter (PAOX2). This novel approach enhanced PAOX1 efficiency by providing extra Mxr1 and generated switchable Mxr1 expression to allow PAOX1 to be induced under glycerol starvation or carbon-free conditions. Additionally, the inhibitory effect of glycerol on PAOX1 was retained because the synthetic circuit was not activated in response to glycerol. Using green fluorescent protein as a demonstration, this reprogrammed P. pastoris strain displayed stronger fluorescence intensity than non-reprogrammed cells under both methanol induction and glycerol starvation. Moreover, with single-chain variable fragment (scFv) as the model protein, increases in extracellular scFv productivity of 98 and 269% were observed in Mxr1-reprogrammed cells under methanol induction and glycerol starvation, respectively, compared to productivity in non-reprogrammed cells under methanol induction. CONCLUSIONS: We successfully demonstrate that the synthetic positive feedback circuit of Mxr1 enhances recombinant protein production efficiency in P. pastoris and create a methanol-free induction system to eliminate the potential risks of methanol.

Incidence of Subsequent Injuries Associated with a New Diagnosis of Benign Paroxysmal Positional Vertigo and Effects of Treatment: A Nationwide Cohort Study.

Background/Objectives: Benign paroxysmal positional vertigo (BPPV) is the most common cause of recurrent vertigo and the most common peripheral vestibular disorder. It is characterized by intense vertigo triggered by head and position changes. This study investigates the risk of subsequent injury in BPPV patients and the effects of treatment. Methods: A population-based retrospective cohort study was conducted using data from the Longitudinal Health Insurance Database 2005 in Taiwan. Patients with and without BPPV were identified between 2000 and 2017. The study outcomes were diagnoses of all-cause injuries. The Kaplan-Meier method determined the cumulative incidence rates of injury in both cohorts, and a log-rank test analyzed the differences. Cox proportional hazard models calculated each cohort's 18-year hazard ratios (HRs). Results: We enrolled 50,675 patients with newly diagnosed BPPV and 202,700 matched individuals without BPPV. During follow-up, 47,636 patients were diagnosed with injuries (13,215 from the BPPV cohort and 34,421 from the non-BPPV cohort). The adjusted HR for injury in BPPV patients was 2.63 (95% CI, 2.49-2.88). Subgroup analysis showed an increased incidence of unintentional and intentional injuries in BPPV patients (aHR 2.86; 95% CI, 2.70-3.13 and 1.10; 95% CI, 1.04-1.21, respectively). A positive dose-response relationship was observed with increasing BPPV diagnoses. Treatment with canalith repositioning therapy (CRT) or medications reduced the risk of injury slightly but not significantly (aHR, 0.78; 95% CI, 0.37-1.29, 0.88; 95% CI, 0.40-1.40, respectively). Conclusions: BPPV is independently associated with an increased risk of injuries. CRT or medications have limited effects on mitigating this risk. Physicians should advise BPPV patients to take precautions to prevent injuries even after treatment.

Biomolecular feedback controllers: from theory to applications.

Billions of years of evolution have led to the creation of sophisticated genetic regulatory mechanisms that control various biological processes in a timely and precise fashion, despite their uncertain and noisy environments. Understanding such naturally existing mechanisms and even designing novel ones will have direct implications in various fields such as biotechnology, medicine, and synthetic biology. In particular, many studies have revealed that feedback-based control mechanisms inside the living cells endow the overall system with multiple attractive features, including homeostasis, noise reduction, and high dynamic performance. The remarkable interdisciplinary nature of these studies has brought together disparate disciplines such as systems/synthetic biology and control theory in an effort to design and build more powerful and reliable biomolecular control systems. Here, we review various biomolecular feedback controllers, highlight their characteristics, and point out their promising impact.

A cybergenetic framework for engineering intein-mediated integral feedback control systems.

The ability of biological systems to tightly regulate targeted variables, despite external and internal disturbances, is known as Robust Perfect Adaptation (RPA). Achieved frequently through biomolecular integral feedback controllers at the cellular level, RPA has important implications for biotechnology and its various applications. In this study, we identify inteins as a versatile class of genetic components suitable for implementing these controllers and present a systematic approach for their design. We develop a theoretical foundation for screening intein-based RPA-achieving controllers and a simplified approach for modeling them. We then genetically engineer and test intein-based controllers using commonly used transcription factors in mammalian cells and demonstrate their exceptional adaptation properties over a wide dynamic range. The small size, flexibility, and applicability of inteins across life forms allow us to create a diversity of genetic RPA-achieving integral feedback control systems that can be used in various applications, including metabolic engineering and cell-based therapy.

Diya - A universal light illumination platform for multiwell plate cultures.

Recent progress in protein engineering has established optogenetics as one of the leading external non-invasive stimulation strategies, with many optogenetic tools being designed for in vivo operation. Characterization and optimization of these tools require a high-throughput and versatile light delivery system targeting micro-titer culture volumes. Here, we present a universal light illumination platform - Diya, compatible with a wide range of cell culture plates and dishes. Diya hosts specially designed features ensuring active thermal management, homogeneous illumination, and minimal light bleedthrough. It offers light induction programming via a user-friendly custom-designed GUI. Through extensive characterization experiments with multiple optogenetic tools in diverse model organisms (bacteria, yeast, and human cell lines), we show that Diya maintains viable conditions for cell cultures undergoing light induction. Finally, we demonstrate an optogenetic strategy for in vivo biomolecular controller operation. With a custom-designed antithetic integral feedback circuit, we exhibit robust perfect adaptation and light-controlled set-point variation using Diya.

Different synaptic mechanisms of intermittent and continuous theta-burst stimulations in a severe foot-shock induced and treatment-resistant depression in a rat model.

Treatment-resistant depression (TRD) is a condition wherein patients with depression fail to respond to antidepressant trials. A new form of repetitive transcranial magnetic stimulation (rTMS), called theta-burst stimulation (TBS), which includes intermittent theta-burst stimulation (iTBS) and continuous theta-burst stimulation (cTBS), is non-inferior to rTMS in TRD treatment. However, the mechanism of iTBS and cTBS underlying the treatment of TRD in the prefrontal cortex (PFC) remains unclear. Hence, we applied foot-shock stress as a traumatic event to develop a TRD rat model and investigated the different mechanisms of iTBS and cTBS. The iTBS and cTBS treatment were effective in depressive-like behavior and active coping behavior. The iTBS treatments improved impaired long-term potentiation and long-term depression (LTD), whereas the cTBS treatment only improved aberrant LTD. Moreover, the decrease in mature brain-derived neurotrophic factor (BDNF)-related protein levels were reversed by iTBS treatment. The decrease in proBDNF-related protein expression was improved by iTBS and cTBS treatment. Both iTBS and cTBS improved the decreased α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and downregulation of mammalian target of the rapamycin (mTOR) signaling pathway. The iTBS produces both excitatory and inhibitory synaptic effects, and the cTBS only produces inhibitory synaptic effects in the PFC.

Effects of Laser Therapy on Rheumatoid Arthritis: A Systematic Review and Meta-analysis.

OBJECTIVE: The aim of the study was to examine the effects of laser therapy on rheumatoid arthritis. DESIGN: A search of controlled trials was conducted in different medical electronic databases. The primary outcome was immediate pain relief after treatment. Secondary outcomes were level of functional disability, morning stiffness duration, and 3-mo follow-up for pain. RESULTS: Ten trials met the inclusion criteria. Rheumatoid arthritis treatment with laser therapy significantly improved immediate pain relief (standardized mean difference = -0.839, 95% confidence interval = -1.336 to -0.343) and overall functional score (standardized mean difference = -0.309, 95% confidence interval = -0.587 to -0.031). There was no significant improvement in morning stiffness duration (standardized mean difference = -0.519, 95% confidence interval = -1.176 to 0.138), and 3-mo follow-up for pain (standardized mean difference = -1.125, 95% confidence interval = -2.311 to 0.061). There was no publication bias (Egger regression, P = 0.277). However, heterogeneity was noted despite the removal of an outlier (Q = 18.646, I2 = 57.096). Results of subgroup analyses suggested that high-intensity laser therapy and nerve irradiation are better suited for immediate pain relief. Meta-regression analyses showed no significant linear relationship between the treatment effect with laser wavelength or number of treatment sessions. CONCLUSIONS: Laser therapies provide significant immediate pain relief and improve function for rheumatoid arthritis patients, but without significant improvement in morning stiffness duration and 3-mo follow-up for pain.Systematic review registration number: CRD42020192906.

Diabetes Is the Most Critical Risk Factor of Adverse Complications After Peritoneal Dialysis Catheter Placement.

Introduction: Peritoneal dialysis (PD) is a kind of renal replacement therapy for end-stage renal disease (ESRD). While PD has many advantages, various complications may arise. Methods: This retrospective study analyzed the complications of ESRD patients who received PD catheter implantation in a single medical center within 15 years. Results: This study collected 707 patients. In the first 14 days after PD implantation, 54 patients experienced bleeding complications, while 47 patients experienced wound infection. Among all complications, catheter-related infections were the most common complication 14 days after PD implantation (incidence: 38.8%). A total of 323 patients experienced PD catheter removal, of which 162 patients were due to infection, while 96 were intentional due to kidney transplantation. Excluding those whose catheters were removed due to transplantation, the median survival of the PD catheter was 4.1 years; among them, patients without diabetes mellitus (DM) were 7.4 years and patients with DM were 2.5 years (p < 0.001). Further, 50% probability of surviving was beyond 3.5 years in DM patients with HbA1CC < 7 and 1.6 years in DM patients with HbA1C <7 (p ≥ 0.001). Conclusions: Catheter-related infections were the most common complications following PD catheter implantation. DM, especially with HbA1C ≥7, significantly impacted on the catheter-related infection and the survival probability of the PD catheter.

Sputtering deposition of P-type SnO films with SnO2 target in hydrogen-containing atmosphere.

In this work, we had investigated sputtering deposition of p-type SnO using the widely used and robust SnO2 target in a hydrogen-containing reducing atmosphere. The effects of the hydrogen-containing sputtering gas on structures, compositions, optical, and electrical properties of deposited SnOx films were studied. Results show that polycrystalline and SnO-dominant films could be readily obtained by carefully controlling the hydrogen gas ratio in the sputtering gas and the extent of reduction reaction. P-type conductivity was unambiguously observed for SnO-dominant films with traceable Sn components, exhibiting a p-type Hall mobility of up to ∼3 cm(2) V(-1) s(-1). P-type SnO thin-film transistors using such SnO-dominant films were also demonstrated.

Implement the RFID position based system of automatic tablets packaging machine for patient safety.

Patient safety has been regarded as the most important quality policy of hospital management. The medicine dispensing definitely plays an influential role in the Joint Commission International Accreditation Standards. The problem we are going to discuss in this paper is that the function of detecting mistakes does not exist in the Automatic Tablets packaging machine (ATPM) in the hospital pharmacy department when the pharmacists implement the replenishment of cassettes. In this situation, there are higher possibilities of placing the wrong cassettes back to the wrong positions, so that the human errors will lead to a crucial impact on total inpatients undoubtedly. Therefore, this study aims to design the RFID (Radio frequency identification) position based system (PBS) for the ATPM with passive high frequency (HF) model. At first, we placed the HF tags on each cassette and installed the HF readers on the cabinets for each position. Then, the system works on the reading loop to verify ID numbers and positions on each cassette. Next, the system would detect whether the orbit opens or not and controls the readers' working power consumption for drug storage temperature. Finally, we use the RFID PBS of the ATPM to achieve the goal of avoiding the medication errors at any time for patient safety.