Harnessing non-Watson-Crick's base pairing to enhance CRISPR effectors cleavage activities and enable gene editing in mammalian cells.

Genomic DNA of the cyanophage S-2L virus is composed of 2-aminoadenine (Z), thymine (T), guanine (G), and cytosine (C), forming the genetic alphabet ZTGC, which violates Watson-Crick base pairing rules. The Z-base has an extra amino group on the two position that allows the formation of a third hydrogen bond with thymine in DNA strands. Here, we explored and expanded applications of this non-Watson-Crick base pairing in protein expression and gene editing. Both ZTGC-DNA (Z-DNA) and ZUGC-RNA (Z-RNA) produced in vitro show detectable compatibility and can be decoded in mammalian cells, including Homo sapiens cells. Z-crRNA can guide CRISPR-effectors SpCas9 and LbCas12a to cleave specific DNA through non-Watson-Crick base pairing and boost cleavage activities compared to A-crRNA. Z-crRNA can also allow for efficient gene and base editing in human cells. Together, our results help pave the way for potential strategies for optimizing DNA or RNA payloads for gene editing therapeutics and give insights to understanding the natural Z-DNA genome.

Monovalent SARS-COV-2 mRNA vaccine using optimal UTRs and LNPs is highly immunogenic and broadly protective against Omicron variants.

The emergence of highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) that are resistant to the current COVID-19 vaccines highlights the need for continued development of broadly protective vaccines for the future. Here, we developed two messenger RNA (mRNA)-lipid nanoparticle (LNP) vaccines, TU88mCSA and ALCmCSA, using the ancestral SARS-CoV-2 spike sequence, optimized 5' and 3' untranslated regions (UTRs), and LNP combinations. Our data showed that these nanocomplexes effectively activate CD4+ and CD8+ T cell responses and humoral immune response and provide complete protection against WA1/2020, Omicron BA.1 and BQ.1 infection in hamsters. Critically, in Omicron BQ.1 challenge hamster models, TU88mCSA and ALCmCSA not only induced robust control of virus load in the lungs but also enhanced protective efficacy in the upper respiratory airways. Antigen-specific immune analysis in mice revealed that the observed cross-protection is associated with superior UTRs [Carboxylesterase 1d (Ces1d)/adaptor protein-3ß (AP3B1)] and LNP formulations that elicit robust lung tissue-resident memory T cells. Strong protective effects of TU88mCSA or ALCmCSA against both WA1/2020 and VOCs suggest that this mRNA-LNP combination can be a broadly protective vaccine platform in which mRNA cargo uses the ancestral antigen sequence regardless of the antigenic drift. This approach could be rapidly adapted for clinical use and timely deployment of vaccines against emerging and reemerging VOCs.

Integration of a CRISPR Cas12a-assisted multicolor biosensor and a micropipette tip enables visible point-of-care testing of foodborne Vibrio vulnificus.

Foodborne pathogens cause numerous food safety problems, and as a virulent bacterium falling under this category, Vibrio vulnificus (V. vulnificus) poses a huge threat to public health. The conventional methods used for the detection of V. vulnificus, including culture-based and molecular detection methods, have a variety of drawbacks, including being time-consuming and labor-intensive, the requirement of large-scale equipment, and the lack of professional operators. This paper establishes a visible detection platform for V. vulnificus based on CRISPR/Cas12a, which is integrated with nucleic acid isothermal amplification and ß-galactosidase-catalyzed visible color reaction. The specific vvhA gene and a conservative segment in the 16S rDNA gene of the Vibrio genus were selected as the detection targets. By using spectrum analysis, this CRISPR detection platform achieved sensitive detection of V. vulnificus (1 CFU per reaction) with high specificity. Through the color transformation system, as low as 1 CFU per reaction of V. vulnificus in both bacterial solution and artificially contaminated seafood could be visibly observed with the naked eye. Furthermore, the consistency between our assay and the qPCR assay in the detection of V. vulnificus spiked seafood was confirmed. In general, this visible detection platform is user-friendly, accurate, portable, and equipment-free, and is expected to provide a powerful supplement in point-of-care testing of V. vulnificus and also holds good promise for future application in foodborne pathogen detection.

Dual nucleases-assisted cyclic amplification using polydopamine nanospheres-based biosensors for one-pot detection of microRNAs.

The accurate detection of microRNAs (miRNAs) is essential in the early diagnosis and treatment of cancers. Existing miRNA detection methods represented by nucleic acid amplification (NAA) techniques, such as qRT-PCR, suffer from the small size of miRNAs and lead to limited practicability. CRISPR Cas13a system, another valuable toolbox for nucleic acid detection, relies heavily on the behaviors of accompanying isothermal NAA techniques, which prompts similar deficiencies in miRNA detection. In this study, a dual nucleases-assisted cyclic amplification (DUNCAN) strategy has been established to replace NAA techniques for one-pot detection of miRNAs. The DUNCAN strategy contained an initial reaction based on CRISPR Cas13a for target recognition, and an accompanied cyclic reaction using DNA probes protected by polydopamine nanospheres (PDANSs) for signal amplification and result readout. Exemplified by miR-19b, which has been confirmed to be related to several tumors, the quantitative detection through the DUNCAN strategy was achieved in the dynamic range of 10-106 fM, with a calculated detection limit of 1.27 fM. Besides, the DUNCAN strategy presented well selectivity and anti-interference performance for accurate detection of miR-19b in complex miRNA mixtures, different cell lines and clinical samples compared with qRT-PCR. All these performances demonstrated the promising potential of the DUNCAN strategy in clinical miRNA detection and diagnosis.

Positive remediation on sedimentary P by combination of capping with calcium hydroxide and oxidation with perhydrol.

The capping is called passive remediation because the sedimentary P was released and then immobilized by the capping materials. However, the release depends on the environmental conditions. Therefore, a hypothesis was proposed that the oxidant was used to accelerate sedimentary P release and the capping material was used to capture those released P. It is positive remediation to reduce sedimentary P amount. The results show that soluble reactive phosphorus (SRP) concentration in the overlying water and pore water increased and then decreased gradually under the combination of sodium percarbonate (SPC) and Ca(OH)2, similar as that under the capping with single Ca(OH)2. The sedimentary P amount was reduced considerably and P concentration in the capping layer increased obviously after 60 days, compared with the capping with single Ca(OH)2. All these indicated that oxidation improved the sedimentary P release and the released P was captured and immobilized by the capping material of Ca(OH)2. However, the acceleration of sedimentary P release due to the oxidation is obviously different from the traditional mechanism that the oxic condition in the sediment is favor of the immobilization on sedimentary P. It is attributed to the oxidizability of ·OH from SPC. The reduction of mobile-P and the increase of Ca-P under the combined use are observed. This is similar as traditional mechanism of oxic condition. It is sure that positive remediation reduced sedimentary P amount, resulting in the decrease of P release risk in a long time.

[Effects of Sediment Microenvironment on Sedimentary Phosphorus Release Under Capping].

The sediment microenvironment has an important effect on the release of endogenous phosphorus. In this study, the influence of two different in-situ covering materials on the sediment microenvironment were compared, and the controlling effect of endogenous phosphorus release were studied. The sediment microenvironment was represented by the concentration of NH4+-N, Fe2+ in interstitial water, and microbial activity. The results showed that the concentrations of NH4+-N and Fe2+ were lower under ACPM coverage than those of the Phoslock® group, and the microbial activity was higher, indicating that the redox potential of ACPM coverage was higher than that of Phoslock®. Furthermore, the bottom sediment microenvironment was in an aerobic state. Compared with the Phoslock® group, the DIP concentrations in both the overcover water and porewater in the ACPM group were higher, indicating that Phoslock® was superior to ACPM with respect to the phosphate adsorption capacity, suggesting that the sediment microenvironment was not the only criterion for phosphorus adsorption. In the fixation process of endogenous phosphorus, both of the covering materials were conducive to the fixation of inter phosphorus, and Ca-P increased significantly. On the other hand, oxidizing ACPM led to an increase in NH4Cl-P and a significant decrease in Fe/Al-P. This promotes the release of active phosphorus, which is conducive to the cleaning of the sediment and phosphorus release.

[Effect of Oxidation Strengthening on In-situ Phosphorus Immobilization of Calcium Hydroxide].

In this research, calcium hydroxide[Ca(OH)2] and hydrogen peroxide (H2O2) were injected into the bottom mud in the form of plum blossom scatterers to investigate the effect on the control of endogenous phosphorus. The results showed that Ca(OH)2 used singly effectively immobilized in the order of 90% of endogenous phosphorus approximately 20 mm below the sediment-water interface (SWI); however, at the same time, the anaerobic environment was enhanced, resulting in the transformation of stable phosphorus to easily released phosphorus and the accumulation of potential active phosphorus. Nevertheless, the addition of H2O2 greatly reduced the amount of potential active phosphorus in deep sediments after adding Ca(OH)2. The vertical diffusion depth of Ca(OH)2 in the sediments was significantly increased, having an influence across the depth range of 0-40mm below the SWI; the improvement at depths greater than 40 mm was not notable, which was mainly attributed to an 18-fold increase of redox potential due to the addition of the oxidant. The change of phosphorus forms in the sediment also demonstrated the excellent immobilization effect of the oxidant on phosphorus. In the 0-20 mm layer, the content of readily released phosphorus decreased significantly, while compared with a control test, Ca-P increased by approximately 10%. However, at greater depths, the amount of easily released phosphorus decreased and the rate of Ca-P increase gradually slowed.

[Adsorption Behavior of Phosphate by CaO2 Remolded Sediment].

This study simulated the state of CaO2 loss after in situ coverage and examined the bottom 2 cm of sediment after restoration. Observations and elemental analysis of the sediment using scanning electron microscopy (SEM) and X-ray energy spectrometry (EDX) were also performed. The CaO2 remodeling notably changed the structure of the sediment; most of the organic matter and iron-manganese oxide attached to the sediment surface was removed, the porosity of the sediment particles increased, and the Ca2+ content was also increased. CaO2 remodeling stabilized the endogenous phosphorus in the sediment; total phosphorous (TP) was reduced by approximately 20% and potential active phosphorus content was reduced by approximately 30%. Furthermore, the contents of Ca-P and Res-P were significantly increased. The amount of phosphorus released from the remodeled sediment under anaerobic conditions was significantly lower than the original sediment, indicating that the CaO2 remodeling greatly reduced the risk of endogenous phosphorus release. The Langmuir model was more suitable than the Freundlich and Dubinin-Radushkevich models for describing the isothermal adsorption behavior of the CaO2 remodeling, which significantly improved the adsorption capacity of the sediment with respect to phosphate from 1.44 mg·g-1 to 20.91 mg·g-1. The mechanism of adsorption was switched from chemical adsorption to physicochemical adsorption. In addition, the adsorption kinetics of the CaO2 remodeled sediment with respect to phosphate could be best described using the quasi-second-order kinetic model.

Using sediment resuspension to immobilize sedimentary phosphorus.

The response of the transformation of internal phosphorus (P) to resuspended sediment was investigated in the sediment-water system under different disturbance intensity. The sediments and overlying water were collected from Taihu Lake, a typical shallow lake. The concentrations of particulate P (PP) and dissolved inorganic P (DIP) in the water phase and algal available P (AAP) and P fractions in the sediments and suspended particle characteristics were evaluated in laboratory-simulated experiments. The results show that dissolved oxygen (DO) in the overlying water increased continuously and pH decreased slightly under sediment resuspension. The concentration of ammonia nitrogen (NH4+-N) showed a distinct decline, indicating that sediment resuspension promotes the penetration of DO into the sediments. It was also favor of the formation of metal oxides and hydroxides, inducing soluble amorphous metal compounds oxidized to insoluble crystalline metal compounds under disturbance condition. This resulted in the increase of refractory P in sediment compared with the static conditions. Sediment resuspension is beneficial to long-term P retention. This can be confirmed by the increase of maximum P adsorption amount (Qmax) and the decrease of the degree of P saturation (DPS) and equilibrium P concentration (EPC0). This is the main explanation of DIP decrease in the overlying water. It is indicating that sediment resuspension not only improves the redox environment in the sediment-water system but also enhances P retention capacity.

[Phosphate Control Effect and Water Body Remediation of Magnesium Modified Reed Biochar].

Adding biochar from harvested reed to sediments is a new method to control the release of sedimentary phosphorus. Three types of Mg-modified biochars were prepared by pyrolysis of reed modified by magnesium chloride at 300, 450, and 600℃.The phosphate adsorption characteristics of the three types of biochars were analyzed by isothermal adsorption experiments. Biochar MBC-450 with good phosphate adsorption effects was selected as the material for the following experiments. Taking the sediment and overlying water of a campus river as the experimental object, we studied the adsorption of phosphate in overlying water and the control of sedimentary phosphorus by Mg-modified biochar under different dosage modes (mixing and covering). The concentration of DIP in the overlying water could be effectively reduced by biochar mixing and covering, and the accumulative phosphorus adsorption capacity increased by 17.3% (mixing) and 11.7% (covering) compared with that of the control. The control effect of sedimentary phosphorus by biochar mixing was more obvious; the DIP in sedimentary water decreased by 14.7%, 18.9%, and 35.36% from 0-2 cm to 4-6 cm compared with the control. The DIP in sedimentary water decreased by 33.3%, -28.2%, and 12.9% when covered with biochar. Compared with the control, the proportion of NH4 Cl-P in the sediment of 0-2 cm and 2-4 cm increased by 15% and 15% (mixing) and 12% and 2% (covering), respectively, while BD-P in TP decreased by 7% and 9% (mixing) and 6% and 3% (covering), respectively, and the Al-P in TP decreased by 7% and 6% (mixing) and 7% and -1% (covering), respectively. The other forms of phosphorus did not change significantly. Biochar mixing and covering can both improve the microbial activity in surface sediment, and biochar mixing can improve the microbial activity in deeper sediments more significantly.

[Adsorption Properties of Sludge-hydrochar for Methylene Blue].

A low-cost and efficient adsorbent, based on sewage sludge, was prepared by hydrothermal carbonization. The sludge-hydrochar was prepared at different temperatures (160, 190, 220, and 250℃) and different reaction times (1, 4, 8, and 16 h). It was applied to the adsorption of methylene blue (MB) in water. This study analyzed the structure and physicochemical properties of hydrochar by BET, FT-IR, and zero charge points. In addition, we investigated the optimum hydrothermal conditions for the preparation of adsorbents and adsorption properties for MB by experiments. Studies have shown that 190℃ and 4 h were the best hydrothermal conditions for the preparation of adsorbent (SS190-4). SS190-4 has the largest specific surface area (11.916 m2·g-1) and the best removal rate (96.44%) for methylene blue (MB). The adsorption of MB is more favorable when the solution is alkaline. The adsorption conforms to the Langmuir isotherm equation, and the maximum model adsorption capacity for MB is 400 mg·g-1. When the concentration of hydrochar is 0.5 g·L-1, it is more economical and reasonable. When there are coexisting ions in the solution, the adsorption capacity of hydrochar to MB is inhibited. The adsorption process of MB by hydrochar conforms to the quasi-secondary kinetic model, which is a spontaneous exothermic reaction.

[Phosphate Adsorption from Water on CaO2-loaded Magnetic Diatomite].

In this study,magnetic diatomite was used as a carrier to load calcium peroxide(CaO2) nanoparticles,fabricating a high efficiency phosphate adsorption and recovery composite material(MDCP).The micromorphology, inner structure,crystalline constituents and element composition of MDCP were characterized by SEM,EDX-mapping,XRD, XDS, and VSM,respectively.The adsorption isotherm data of MDCP exhibited good agreement with the Langmuir isotherm model. According to the Langmuir model,when T=20℃,the maximum monolayer phosphate adsorption capacities can reach 191.84mg·g-1 for MDCP. The isotherm and kinetics studies showed that MDCP has a regulating effect on the pH of the solution,which can maintain the pH of the solution at the level where adsorption of phosphate occurs on MDCP as a chemisorption process. pH plays a important role on the adsorption of phosphate by MDCP,the pH of effective adsorption ranges from 4 to 10,and the pH of the adsorbed solution can still be maintained in the range of 7 to 9.The MDCP exhibited a high selective adsorption for phosphate in the presence of anions,including Cl-, SO42-, CO32-, HCO32-, F-, and NO3-.The recovered MDCP could be desorbed by HCl solution,and after desorption, the phosphate removal rate of MDCP after re-loading CaO2 could still reach 70% of the initial adsorption.

[Effect of Calcium Peroxide Composite Tablets on Water Remediation and Phosphorus Control in Sediment].

Two types of calcium peroxide composite tablets (CPCTs) were prepared, and the inhibition effect on the release of endogenous phosphorus and the influence on the overlying water by mixed-dosing were investigated. The CPCTs were made of calcium peroxide (CaO2), calcined water purification sludge, and hydroxypropyl methylcellulose (HPMC), which were directly pressed into composite tablets, among which Tablet-B (T-B) contained ferrous sulfate (FeSO4) and Tablet-A (T-A) did not. Both the tablets agreed well with the Langmuir and Freundlich isothermal models; the theoretical maximum adsorption capacities of T-A and T-B on phosphorus were 110.908 mg·g-1 and 106.390 mg·g-1, respectively. Compared with the control group, the pH of overlying water was increased, the concentration of Chl-a was decreased by 42.75% and 60.82%, and the DO was increased by 53.73% and 63.30% in group A and B, respectively. The DIP of the overlying water decreased significantly by 54.93% and 25.11% in group A and B, respectively. For the interstitial water in sediment, the DIP in layer Ⅰ (0-2 cm) decreased significantly by 74.81% and 65.66% in group A and B, and the DIP in layer Ⅱ (2-4 cm) decreased significantly by 46.23% in group B, but not obviously in group A. The DIP in layer Ⅲ (4-6 cm) remained unchanged in group A and B. For phosphorus fractions in sediment, the proportion of NH4Cl-P in TP was significantly increased (layer Ⅰ: 16.87% and 13.11%; layer Ⅱ: 12.99% and 11.02%, in group A and B, respectively), and the proportion of Al-P in TP was significantly decreased (layer Ⅰ: 7.58% and 13.91%; layerⅡ: 9.86% and 7.28%, in group A and B, respectively). The other phosphorus fractions did not change significantly. Both T-A and T-B dosing can improve the microbial activity of the surface sediment, though T-A can improve the microbial activity more significantly.

The effect of microenvironment in the sediment on phosphorus immobilization under capping with ACPM and Phoslock®.

Currently, in situ capping is a typical popular geoengineering method for eutrophication control. It is crucial to better understand the effect of microenvironment change due to capping, such as amended calcium peroxide material (ACPM) and Phoslock®, on phosphorus (P) adsorption and immobilization under the addition of external P. The microenvironment in sediment was presented by the concentration of O2, NH4+, and Fe2+ and microbial activity. The P removal and immobilization were also analyzed. The results show that the stronger oxidation in the microenvironment under the capping with ACPM was due to the higher reduction of NH4+ and Fe2+ and the higher increase of microbial activity, compared to Phoslock®. Although, under the capping of ACPM, less amount of external P was removed and there was a faster release of sedimentary P, compared to Phoslock®, ACPM improved the transformation of P from mobile P fractions to inert P fractions. In addition, sedimentary P under the capping of ACPM presents less release than that under the capping of Phoslock® during the anaerobic incubation. However, the settlement of suspended solids decreased the function of capping. All these results indicated that the mechanism of P removal and immobilization was different under the capping of ACPM and Phoslock®.

[Environmental Significance of Phosphorus Fractions of Phytoplankton-and Macrophyte-Dominated Zones in Taihu Lake].

It is of great importance to study the environmental significance of phosphorus fractions in overlying water and sediments of typical phytoplankton-and macrophyte-dominated zones. It will help to clarify the process of phosphorus migration and transformation in the sediment-water interface, and has practical significance for understanding the eutrophication process and its treatment in different regions of Taihu Lake. The investigation was conducted within typical phytoplankton-and macrophyte-dominated zones of Taihu Lake over four seasons to analyze the spatial and temporal differences between phosphorus fractions in water and sediments, and reveal their environmental significance. The results showed that:① Total phosphorus (TP), total soluble phosphorus (DTP), dissolved inorganic phosphorus (DIP), and particulate phosphorus (PP) in the overlying water of phytoplankton-dominated zones were much higher than those in macrophyte-dominated zones. Most of them showed seasonal characteristics, which were higher in summer and autumn than in winter and spring. PP is the main component of TP, accounting for 71.8% to 89.6%. A similar distribution character was found in the content of chlorophyll (Chl-a) compared with phosphorus concentration in overlying water. ② The concentration of TP in the surface sediments of phytoplankton-dominated zones was 372.38-529.64 mg·kg-1, and that in macrophyte-dominated zones was 304.29-454.27 mg·kg-1. In surface sediments, concentrations of TP in phytoplankton-dominated zones were significantly higher than in phytoplankton-dominated zones. The highest TP concentrations appeared in winter, and the lowest in summer. These were owing to the input of exogenous pollution, and the migration and transformation of internal phosphorus between sediments and overlying water under different environmental conditions. ③ The order of the mass fraction of phosphorus in sediments was:NH4Cl-P < Fe-P < Org-P < Res-P < Al-P < Ca-P. Mobile-P=NH4Cl-P+Fe-P+Org-P, accounting for 9.10%-16.93% of TP in phytoplankton-dominated zones, and slightly higher in macrophyte-dominated zones, where it was 8.11%-13.50%. Res-P accounted for 10.06%-14.97% of TP in phytoplankton-dominated zones, and 11.02%-20.28% in macrophyte-dominated zones. The risk of internal phosphorus release in phytoplankton-dominated zones is high, which is not conducive to the fixation and burial of phosphorus. The eutrophication degree of different regions in Taihu Lake is obviously different, and different characteristics of phosphorus release and burial are showed. The phytoplankton-dominated zones deserve special attention because of their high internal phosphorus load and release potential.

Metabolic engineering of Lactococcus lactis for high level accumulation of glutathione and S-adenosyl-L-methionine.

Glutathione (GSH) and S-adenosyl methionine (SAM) have been applied as liver-protective factors to prevent and treat many different liver damages and diseases. Due to their low stability and short half-life, oral administration of GSH or SAM might be replaced by continuous supplying through living lactic bacteria in yogurt. In this study, Lactococcus lactis was engineered via synthetic biology strategies to produce these two important molecules. The bi-functional GSH synthase gene (gshF) and SAM synthase gene (metK) were transformed into food-grade L. lactis together with an adhesion factor gene (cwaA). The highest accumulation of SAM (9.0 mg/L) and GSH (17.3 mg/L) was achieved after 17 h cultivation of the recombinant L. lactis. Meanwhile, the autoaggregation and hydrophobicity were also improved significantly, which suggested that this engineered L. lactis might have an increased colonization-prone ability in human GI. Our studies demonstrated one potential route to self-produce and deliver the liver-healthy factors within living probiotic bacteria.