Mathematical modeling of viral infection and the immune response controlled by the circadian clock.

Time of day affects how well the immune system responds to viral or bacterial infections. While it is well known that the immune system is regulated by the circadian clock, the dynamic origin of time-of-day-dependent immunity remains unclear. In this paper, we studied the circadian control of immune response upon infection of influenza A virus through mathematical modeling. Dynamic simulation analyses revealed that the time-of-day-dependent immunity was rooted in the relative phase between the circadian clock and the pulse of viral infection. The relative phase, which depends on the time the infection occurs, plays a crucial role in the immune response. It can drive the immune system to one of two distinct bistable states, a high inflammatory state with a higher mortality rate or a safe state characterized by low inflammation. The mechanism we found here also explained why the same species infected by different viruses has different time-of-day-dependent immunities. Further, the time-of-day-dependent immunity was found to be abolished when the immune system was regulated by an impaired circadian clock with decreased oscillation amplitude or without oscillations.

Landscape of T cell epitopes displays hot mutations of SARS-CoV-2 variant spikes evading cellular immunity.

The continuous evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been accompanied by the emergence of viral mutations that pose a great challenge to existing vaccine strategies. It is not fully understood with regard to the role of mutations on the SARS-CoV-2 spike protein from emerging viral variants in T cell immunity. In the current study, recombinant eukaryotic plasmids were constructed as DNA vaccines to express the spike protein from multiple SARS-CoV-2 strains. These DNA vaccines were used to immunize BALB/c mice, and cross-T cell responses to the spike protein from these viral strains were quantitated using interferon-γ (IFN-γ) Elispot. Peptides covering the full-length spike protein from different viral strains were used to detect epitope-specific IFN-γ+ CD4+ and CD8+ T cell responses by fluorescence-activated cell sorting. SARS-CoV-2 Delta and Omicron BA.1 strains were found to have broad T cell cross-reactivity, followed by the Beta strain. The landscapes of T cell epitopes on the spike protein demonstrated that at least 30 mutations emerging from Alpha to Omicron BA.5 can mediate the escape of T cell immunity. Omicron and its sublineages have 19 out of these 30 mutations, most of which are new, and a few are inherited from ancient circulating variants of concerns. The cross-T cell immunity between SARS-CoV-2 prototype strain and Omicron strains can be attributed to the T cell epitopes located in the N-terminal domain (181-246 aa [amino acids], 271-318 aa) and C-terminal domain (1171-1273 aa) of the spike protein. These findings provide in vivo evidence for optimizing vaccine manufacturing and immunization strategies for current or future viral variants.

The dynamic-process characterization and prediction of synthetic gene circuits by dynamic delay model.

Differential equation models are widely used to describe genetic regulations, predict multicomponent regulatory circuits, and provide quantitative insights. However, it is still challenging to quantitatively link the dynamic behaviors with measured parameters in synthetic circuits. Here, we propose a dynamic delay model (DDM) which includes two simple parts: the dynamic determining part and the doses-related steady-state-determining part. The dynamic determining part is usually supposed as the delay time but without a clear formula. For the first time, we give the detail formula of the dynamic determining function and provide a method for measuring all parameters of synthetic elements (include 8 activators and 5 repressors) by microfluidic system. Three synthetic circuits were built to show that the DDM can notably improve the prediction accuracy and can be used in various synthetic biology applications.

Applying thallium isotopic compositions as novel and sensitive proxy for Tl(I)/Tl(III) transformation and source apportionment.

Thallium is a rare metal known for its highly toxic nature. Recent research has indicated that the precise determination of Tl isotopic compositions using Multi-Collector Inductively Coupled Plasma Mass Spectrometry (MC-ICP MS) provides new opportunities for understanding Tl geochemical behavior. While isotopic fractionation of Tl derived from anthropogenic activities (e.g., mining, smelting) have been reported, there is limited information regarding Tl influenced by both natural weathering processes and anthropogenic origins. Herein, we investigated, for the first time, the Tl isotopic compositions in soils across a representative Tl-rich depth profile from the Lanmuchang (LMC) quicksilver mine (southwest China) in the low-temperature metallogenesis zone. The results showed significant variations in Tl isotope signatures (ε205Tl) among different soil layers, ranging from -0.23 to 3.79, with heavier isotope-205Tl enrichment observed in the bottom layers of the profile (ε205Tl = 2.18-3.79). This enrichment of 205Tl was not solely correlated with the degree of soil weathering but was also partially associated with oxidation of Tl(I) by Fe (hydr)oxide minerals. Quantitative calculation using ε205Tl vs. 1/Tl data further indicated that the Tl enrichment across the soil depth profile was predominantly derived from anthropogenic origins. All these findings highlight that the robustness and reliability of Tl isotopes as a proxy for identifying both anthropogenic and geogenic sources, as well as tracing chemical alterations and redox-controlled mineralogical processes of Tl in soils. The nascent application of Tl isotopes herein not only offers valuable insights into the behavior of Tl in surface environments, but also establishes a framework for source apportionment in soils under similar circumstances.

Dynamical modelling of viral infection and cooperative immune protection in COVID-19 patients.

Once challenged by the SARS-CoV-2 virus, the human host immune system triggers a dynamic process against infection. We constructed a mathematical model to describe host innate and adaptive immune response to viral challenge. Based on the dynamic properties of viral load and immune response, we classified the resulting dynamics into four modes, reflecting increasing severity of COVID-19 disease. We found the numerical product of immune system's ability to clear the virus and to kill the infected cells, namely immune efficacy, to be predictive of disease severity. We also investigated vaccine-induced protection against SARS-CoV-2 infection. Results suggested that immune efficacy based on memory T cells and neutralizing antibody titers could be used to predict population vaccine protection rates. Finally, we analyzed infection dynamics of SARS-CoV-2 variants within the construct of our mathematical model. Overall, our results provide a systematic framework for understanding the dynamics of host response upon challenge by SARS-CoV-2 infection, and this framework can be used to predict vaccine protection and perform clinical diagnosis.

Determining subunit-subunit interaction from statistics of cryo-EM images: observation of nearest-neighbor coupling in a circadian clock protein complex.

Biological processes are typically actuated by dynamic multi-subunit molecular complexes. However, interactions between subunits, which govern the functions of these complexes, are hard to measure directly. Here, we develop a general approach combining cryo-EM imaging technology and statistical modeling and apply it to study the hexameric clock protein KaiC in Cyanobacteria. By clustering millions of KaiC monomer images, we identify two major conformational states of KaiC monomers. We then classify the conformational states of (>160,000) KaiC hexamers by the thirteen distinct spatial arrangements of these two subunit states in the hexamer ring. We find that distributions of the thirteen hexamer conformational patterns for two KaiC phosphorylation mutants can be fitted quantitatively by an Ising model, which reveals a significant cooperativity between neighboring subunits with phosphorylation shifting the probability of subunit conformation. Our results show that a KaiC hexamer can respond in a switch-like manner to changes in its phosphorylation level.

Molecular mechanism for activation of the 26S proteasome by ZFAND5.

Various hormones, kinases, and stressors (fasting, heat shock) stimulate 26S proteasome activity. To understand how its capacity to degrade ubiquitylated proteins can increase, we studied mouse ZFAND5, which promotes protein degradation during muscle atrophy. Cryo-electron microscopy showed that ZFAND5 induces large conformational changes in the 19S regulatory particle. ZFAND5's AN1 Zn-finger domain interacts with the Rpt5 ATPase and its C terminus with Rpt1 ATPase and Rpn1, a ubiquitin-binding subunit. Upon proteasome binding, ZFAND5 widens the entrance of the substrate translocation channel, yet it associates only transiently with the proteasome. Dissociation of ZFAND5 then stimulates opening of the 20S proteasome gate. Using single-molecule microscopy, we showed that ZFAND5 binds ubiquitylated substrates, prolongs their association with proteasomes, and increases the likelihood that bound substrates undergo degradation, even though ZFAND5 dissociates before substrate deubiquitylation. These changes in proteasome conformation and reaction cycle can explain the accelerated degradation and suggest how other proteasome activators may stimulate proteolysis.

Free energy dissipation enhances spatial accuracy and robustness of self-positioned Turing pattern in small biochemical systems.

Accurate and robust spatial orders are ubiquitous in living systems. In 1952, Turing proposed a general mechanism for pattern formation exemplified by a reaction-diffusion model with two chemical species in a large system. However, in small biological systems such as a cell, the existence of multiple Turing patterns and strong noise can lower the spatial order. Recently, a modified reaction-diffusion model with an additional chemical species is shown to stabilize the Turing pattern. Here, we study non-equilibrium thermodynamics of this three-species reaction-diffusion model to understand the relationship between energy cost and the performance of self-positioning. By using computational and analytical approaches, we show that beyond the onset of pattern formation the positioning error decreases as energy dissipation increases. In a finite system, we find that a specific Turing pattern exists only within a finite range of total molecule number. Energy dissipation broadens this range, which enhances the robustness of Turing pattern against molecule number fluctuations in living cells. The generality of these results is verified in a realistic model of the Muk system underlying DNA segregation in Escherichia coli, and testable predictions are made for the dependence of the accuracy and robustness of the spatial pattern on the ATP/ADP ratio.

Molecular mechanisms for activation of the 26S proteasome.

Various hormones, kinases, and stressors (fasting, heat shock) stimulate 26S proteasome activity. To understand how its capacity to degrade ubiquitylated protein can increase, we studied ZFAND5, which promotes protein degradation during muscle atrophy. Cryo-electron microscopy showed that ZFAND5 induces large conformational changes in the 19S regulatory particle. ZFAND5's AN1 Zn finger interacts with the Rpt5 ATPase and its C-terminus with Rpt1 ATPase and Rpn1, a ubiquitin-binding subunit. Surprisingly, these C-terminal interactions are sufficient to activate proteolysis. With ZFAND5 bound, entry into the proteasome's protein translocation channel is wider, and ZFAND5 dissociation causes opening of the 20S gate for substrate entry. Using single-molecular microscopy, we showed that ZFAND5 binds ubiquitylated substrates, prolongs their association with proteasomes, and increases the likelihood that bound substrates undergo degradation, even though ZFAND5 dissociates before substrate deubiquitylation. These changes in proteasome conformation and reaction cycle can explain the accelerated degradation and suggest how other proteasome activators may stimulate proteolysis.

Plant-on-chip: Core morphogenesis processes in the tiny plant Wolffia australiana.

A plant can be thought of as a colony comprising numerous growth buds, each developing to its own rhythm. Such lack of synchrony impedes efforts to describe core principles of plant morphogenesis, dissect the underlying mechanisms, and identify regulators. Here, we use the minimalist known angiosperm to overcome this challenge and provide a model system for plant morphogenesis. We present a detailed morphological description of the monocot Wolffia australiana, as well as high-quality genome information. Further, we developed the plant-on-chip culture system and demonstrate the application of advanced technologies such as single-nucleus RNA-sequencing, protein structure prediction, and gene editing. We provide proof-of-concept examples that illustrate how W. australiana can decipher the core regulatory mechanisms of plant morphogenesis.

Coupled oscillator cooperativity as a control mechanism in chronobiology.

Control of dynamical processes is vital for maintaining correct cell regulation and cell-fate decisions. Numerous regulatory networks show oscillatory behavior; however, our knowledge of how one oscillator behaves when stimulated by two or more external oscillatory signals is still missing. We explore this problem by constructing a synthetic oscillatory system in yeast and stimulate it with two external oscillatory signals. Letting model verification and prediction operate in a tight interplay with experimental observations, we find that stimulation with two external signals expands the plateau of entrainment and reduces the fluctuations of oscillations. Furthermore, by adjusting the phase differences of external signals, one can control the amplitude of oscillations, which is understood through the signal delay of the unperturbed oscillatory network. With this we reveal a direct amplitude dependency of downstream gene transcription. Taken together, these results suggest a new path to control oscillatory systems by coupled oscillator cooperativity.

A 3D Scalable Chamber-Array Chip for Digital LAMP.

As an absolute quantification method at the single-molecule level, digital PCR (dPCR) offers the highest accuracy. In this work, we developed a 3D scalable chamber-array chip that multiplied the number of partitions by stacking chamber-array layers and realized digital loop-mediated isothermal amplification to quantify DNA molecules. It greatly increases the number of partitions to improve the performance of dPCR without increasing the chip size, the operation workflow complicity, and operation time. For the three-chamber-array-layer chip which contains 200,000 reactors of a 0.125 nL volume, it has been proved that the reagent filling and partition were finished within 3 min, and the whole detection could be finished within 1 h. The method demonstrated that it could be scalable to a six-chamber-array layer, which contains 400,000 reactors without increasing the size of the chip and the complication of filling/partition workflow but only takes an additional hour for scanning. Due to its potential for high throughput, low cost, and simple operation, our device may significantly expand the clinical application range of dPCR.

A novel application of thallium isotopes in tracing metal(loid)s migration and related sources in contaminated paddy soils.

Thallium (Tl) is a highly toxic heavy metal, which is harmful to plants and animals even in trace amounts. Migration behaviors of Tl in paddy soils system remain largely unknown. Herein, Tl isotopic compositions have been employed for the first time to explore Tl transfer and pathway in paddy soil system. The results showed considerably large Tl isotopic variations (ε205Tl = -0.99 ± 0.45 ~ 24.57 ± 0.27), which may result from interconversion between Tl(I) and Tl(III) under alternative redox conditions in the paddy system. Overall higher ε205Tl values of paddy soils in the deeper layers were probably attributed to abundant presence of Fe/Mn (hydr)oxides and occasionally extreme redox conditions during alternative dry-wet process which oxidized Tl(I) to Tl(III). A ternary mixing model using Tl isotopic compositions further disclosed that industrial waste contributed predominantly to Tl contamination in the studied soil, with an average contribution rate of 73.23%. All these findings indicate that Tl isotopes can be used as an efficient tracer for fingerprinting Tl pathway in complicated scenarios even under varied redox conditions, providing significant prospect in diverse environmental applications.

Precise programming of multigene expression stoichiometry in mammalian cells by a modular and programmable transcriptional system.

Context-dependency of mammalian transcriptional elements has hindered the quantitative investigation of multigene expression stoichiometry and its biological functions. Here, we describe a host- and local DNA context-independent transcription system to gradually fine-tune single and multiple gene expression with predictable stoichiometries. The mammalian transcription system is composed of a library of modular and programmable promoters from bacteriophage and its cognate RNA polymerase (RNAP) fused to a capping enzyme. The relative expression of single genes is quantitatively determined by the relative binding affinity of the RNAP to the promoters, while multigene expression stoichiometry is predicted by a simple biochemical model with resource competition. We use these programmable and modular promoters to predictably tune the expression of three components of an influenza A virus-like particle (VLP). Optimized stoichiometry leads to a 2-fold yield of intact VLP complexes. The host-independent orthogonal transcription system provides a platform for dose-dependent control of multiple protein expression which may be applied for advanced vaccine engineering, cell-fate programming and other therapeutic applications.

In vitro activity of tetracycline analogs against multidrug-resistant and extensive drug resistance clinical isolates of Mycobacterium tuberculosis.

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) has become a big threaten to global health. The current strategy for treatment of MDR-TB and extensive drug resistant tuberculosis (XDR-TB) is with low efficacy and high side effect. While new drug is fundamental for cure MDR-TB, repurposing the Food and Drug Administration (FDA)-approved drugs represents an alternative soluation with less cost. METHODS: The activity of 8 tetracycline-class antibiotics against mycobacterium tuberculosis (M.tb) were determined by Minimum Inhibitory Concentration (MIC) in vitro. A transposon M.smeg libraries was generated by using the Harm phage and then used to isolate the conditional growth mutants in doxycycline containing plate. Eleven mutants were isolated and genomic DNAs were extracted using the cetyltrimethyl ammonium bromide (CTAB) method and analyzed by whole genome sequencing. RESULTS: We found that three of eight drugs efficiently inhibited mycobacteria growth under the peak plasma concentration in the human body. Further tests showed these three tetracycline analogs (demeclocycline, doxycycline and methacycline) had antimicrobial activity against seven clinical isolates, including MDR and XDR strains. Among them, Doxycycline had the lowest MICs in all mycobacteria strains tested in this study. By using a transposon library, we identify the insertion of transposon in two genes, porin and MshA, associatewith the resistant to doxycycline. CONCLUSIONS: Our findings show that tetracycline analogs such as doxycycline, has bactericidal activity against not only drug sensitive M.tb, but also clinical MDR and XDR strains, provided proof of concept to repurpose doxycycline to fight MDR-TB and XDR-TB. Further investigations are warranted to clarify the underlying mechanism and optimize the strategy in combination with other anti-TB drugs.

Structure of the Spring Viraemia of Carp Virus Ribonucleoprotein Complex Reveals Its Assembly Mechanism and Application in Antiviral Drug Screening.

Spring viremia of carp virus (SVCV) is a highly pathogenic Vesiculovirus infecting the common carp, yet neither a vaccine nor effective therapies are available to treat spring viremia of carp (SVC). Like all negative-sense viruses, SVCV contains an RNA genome that is encapsidated by the nucleoprotein (N) in the form of a ribonucleoprotein (RNP) complex, which serves as the template for viral replication and transcription. Here, the three-dimensional (3D) structure of SVCV RNP was resolved through cryo-electron microscopy (cryo-EM) at a resolution of 3.7 Å. RNP assembly was stabilized by N and C loops; RNA was wrapped in the groove between the N and C lobes with 9 nt nucleotide per protomer. Combined with mutational analysis, our results elucidated the mechanism of RNP formation. The RNA binding groove of SVCV N was used as a target for drug virtual screening, and it was found suramin had a good antiviral effect. This study provided insights into RNP assembly, and anti-SVCV drug screening was performed on the basis of this structure, providing a theoretical basis and efficient drug screening method for the prevention and treatment of SVC. IMPORTANCE Aquaculture accounts for about 70% of global aquatic products, and viral diseases severely harm the development of aquaculture industry. Spring viremia of carp virus (SVCV) is the pathogen causing highly contagious spring viremia of carp (SVC) disease in cyprinids, especially common carp (Cyprinus carpio), yet neither a vaccine nor effective therapies are available to treat this disease. In this study, we have elucidated the mechanism of SVCV ribonucleoprotein complex (RNP) formation by resolving the 3D structure of SVCV RNP and screened antiviral drugs based on the structure. It is found that suramin could competitively bind to the RNA binding groove and has good antiviral effects both in vivo and in vitro. Our study provides a template for rational drug discovery efforts to treat and prevent SVCV infections.

Mobile and Self-Sustained Data Storage in an Extremophile Genomic DNA.

DNA has been pursued as a novel biomaterial for digital data storage. While large-scale data storage and random access have been achieved in DNA oligonucleotide pools, repeated data accessing requires constant data replenishment, and these implementations are confined in professional facilities. Here, a mobile data storage system in the genome of the extremophile Halomonas bluephagenesis, which enables dual-mode storage, dynamic data maintenance, rapid readout, and robust recovery. The system relies on two key components: A versatile genetic toolbox for the integration of 10-100 kb scale synthetic DNA into H. bluephagenesis genome and an efficient error correction coding scheme targeting noisy nanopore sequencing reads. The storage and repeated retrieval of 5 KB data under non-laboratory conditions are demonstrated. The work highlights the potential of DNA data storage in domestic and field scenarios, and expands its application domain from archival data to frequently accessed data.

Stable thallium (Tl) isotopic signature as a reliable source tracer in river sediments impacted by mining activities.

Thallium (Tl) is an extremely toxic metal, whose geochemical behavior remains poorly understood. This study aims to clarify the migration pathway and source apportionment of Tl in sediments from a watershed downstream of an open and large-scale pyrite mine area in south China, using high-precised Tl isotopic compositions. Results showed that Tl isotopic fractionations were mainly influenced by the anthropogenic Tl sources in all the sediments as a whole from the studied watershed, while in situ mineral adsorption and biological activity were limited. Moreover, plot of ε205Tl vs. 1/Tl further illustrated that three possible end-members, viz. background sediments, pyrite tailings, and sewage treatment wastes were ascribed to predominant sources of Tl enrichment in the sediments. A ternary mixing model unveiled that waste from pyrite mining activities (i.e., both pyrite tailings and sewage treatment wastes) affected the downstream sediments up to 10 km. All these findings suggest that Tl isotopic signature is a reliable tool to trace Tl sources in the sediments impacted by mining activities. It is highly critical for further target-oriented and precise remediation of Tl contamination.

The Topological Characteristics of Biological Ratio-Sensing Networks.

Ratio sensing is a fundamental biological function observed in signal transduction and decision making. In the synthetic biology context, ratio sensing presents one of the elementary functions for cellular multi-signal computation. To investigate the mechanism of the ratio-sensing behavior, we explored the topological characteristics of biological ratio-sensing networks. With exhaustive enumeration of three-node enzymatic and transcriptional regulatory networks, we found that robust ratio sensing was highly dependent on network structure rather than network complexity. Specifically, a set of seven minimal core topological structures and four motifs were deduced to be capable of robust ratio sensing. Further investigations on the evolutionary space of robust ratio-sensing networks revealed highly clustered domains surrounding the core motifs which suggested their evolutionary plausibility. Our study revealed the network topological design principles of ratio-sensing behavior and provided a design scheme for constructing regulatory circuits with ratio-sensing behavior in synthetic biology.

Improving cooperativity of transcription activators by oligomerization domains in mammalian cells.

Cooperative activation is critical for the applications of synthetic biology in mammalian cells. In this study, we have developed cooperative transcription factor by fusing oligomerization domain in mammalian cells. Firstly, we demonstrated that two oligomerized domains (CI434 and CI) successfully improved transcription factor cooperativity in bacterial cells but failed to increase cooperativity in mammalian cells, possibly because the additional mammalian activation domain disrupted their oligomerization capability. Therefore, we chose a different type of oligomerized domain (CarHC), whose ability to oligomerize is not dependent on its C-terminal domains, to fuse with a transcription factor (RpaR) and activation domain (VTR3), forming a potential cooperative transcription activator RpaR-CarH-VTR3 for mammalian regulatory systems. Compared with RpaR-VTR3, the cooperativity of RpaR-CarH-VTR3 was significantly improved with higher Hill coefficient and a narrower input range in the inducible switch system in mammalian cells. Moreover, a mathematical model based on statistical mechanics model was developed and the simulation results supported the hypothesis that the tetramer of the CarH domain in mammalian cells was the reason for the cooperative capacity of RpaR-CarH-VTR3.