Pyridoxal 5’-phosphate (PLP) is an essential cofactor that participates in ~4% enzymatic activities cataloged by the Enzyme Commission. The intracellular level of PLP is usually lower than that demanded in industrial catalysis. To realize the self-supply of PLP cofactor in whole-cell biotransformation, the de novo ribose 5-phosphate (R5P)-dependent PLP synthesis pathway was constructed. The pdxST genes from Bacillus subtilis 168 were introduced into the tyrosine phenol-lyase (TPL)-overexpressing Escherichia coli BL21(DE3) strain. TPL and PdxST were co-expressed with a double-promoter or a compatible double-plasmid system. The 3,4-dihydroxyphenylacetate-L-alanine (L-DOPA) titer did not increase with the increase in the intracellular PLP concentration in these strains with TPL and PdxST co-expression. Therefore, it is necessary to optimize the intracellular PLP metabolism level so as to achieve a higher L-DOPA titer and avoid the formation of L-DOPA–PLP cyclic adducts. The thi riboswitch binds to PLP and forms a complex such that the ribosome cannot have access to the SD sequence. Therefore, this metabolite-sensing regulation system was applied to reduce the translation of mRNA. Riboswitch was introduced into pET–TPL–pdxST-2 to downregulate the expression of PdxST and biosynthesis of PLP at the translation level by sequestering the ribosome-binding site. As a result, the titer and productivity of L-DOPA using the strain BL21–TPLST–Ribo1 improved to 69.8 g/L and 13.96 g/L/h, respectively, with a catechol conversion of 95.9% and intracellular PLP accumulation of 24.8 µM.

Abstract Aim: Traditional artificial microscopic technologies cannot meet the current demands of automated urine detection. Furthermore, the number of cell types detected in previous studies was relatively limited; therefore, previous studies are considered to be insufficient. Methods: The present study proposes a multi-class detection method of urinary particles based on deep learning. First, we obtained an image database containing 15 types of cellular components, i.e., normal, shrinking, glomerular, and abnormal erythrocytes; leukocytes; calcium oxalate, uric acid, other types of crystals; particle and transparent casts; epithelial cells; low-transitional epithelium; Candida; Bacillus; and abnormal epithelium. The image data was then input into Resnet50 basic network and feature pyramid network (FPN) to obtain a multi-layer feature map. Thereafter, the classification sub-networks and regression sub-networks were used to classify and locate the cellular components. The network detection model was obtained after training was completed. Results: The experimental data showed that for the test set, the mean average precision (mAP) of the network model reached 82.86%, and the time required to process a single image sample was 195 ms. Therefore, we were able to perform multi-class analysis and detect urine cells with good results in terms of detection speed. Conclusion: This study applies the deep learning network model for the multi-category detection of urine cells. The method can be used to analyze and detect urinary particles in actual clinical practice and has great reference significance for the detection of other cells in the clinic.

Water transit time and young water fraction are important metrics for characterizing catchment hydrologic function and understanding solute transport. Hydrological and biogeochemical processes in karst environments are strongly controlled by heterogenous fracture-conduit networks. Quantifying the spatio-temporal variability of water transit time and young water fractions in such heterogeneous hydrogeological systems is fundamental linking discharge and water quality dynamics in the karst critical zone. We used a tracer-aided hydrological model to track the fluxes of water parcels that entered a karst catchment as rainfall, time-stamping each hour of rain input individually. Using this approach, the variability of transit times and water age distributions were estimated in the main landscape units in the karst catchment of Chenqi in Guizhou Province, Southwest China. The estimated mean young water (i.e <~2 months old) fractions were 0.39, 0.31 and 0.10 for output fluxes from the hillslope unit, catchment outlet and slow flow reservoirs (matrix and small fractures), respectively. Marked seasonal variability in sources of runoff generation and associated hydrological connectivity between different conceptual stores were the main drivers of young water fraction dynamics in each landscape unit. The water age and travel time distributions were strongly influenced by the water storage dynamics reflecting catchment wetness conditions. Even though the contribution of young water to runoff was greater, the older water turnover was generally accelerated at moderately high flows during wet season.

Una delle principali caratteristiche che si attribuisce all’odore è quella della soggettività. Misurare, quindi, la concentrazione di un odore all’interno di un contenitore chiuso può apparire un’impresa difficile. Esistono, però, da molti anni, tecniche e strumenti che consentono di determinare la quantità di odore presente all’interno di un campione di aria prelevato da una sorgente gassosa, liquida o addirittura solida. Molte di queste tecniche sono basate sull’uso dei nasi umani come strumento di misurazione. La maggior parte degli studi scientifici attuali affronta, proprio, il problema della soggettività: l’uso del naso umano, infatti, influenza la misurazione dell’odore perché entrano in gioco molteplici fattori, come lo stato di salute, l’età della persona, la provenienza geografica, le abitudini, etc. Ma esistono altri fattori di natura più tecnica che, spesso, vengono sottovalutati. Infatti, sperimentazioni scientifiche dimostrano che l’utilizzo di diversi dispositivi di campionamento e di contenitori costituiti da diverso materiale possono influenzare in maniera differente l’odore contenuto al loro interno. In questo studio vengono, infatti, descritti e confrontati gli effetti che le sacche di campionamento e di conservazione del campione di aria, costituite da tre diversi materiali plastici (Nalophan, Teflon e Tedlar), possono avere sullo stesso campione gassoso. La valutazione di questi effetti si basa sul confronto della concentrazione di odore di tali campioni misurata in diversi intervalli temporali intercorsi tra il momento del campionamento e l’analisi in laboratorio. I risultati mostrano che i tre materiali utilizzati per le sacche influenzano in maniera differente il campione di aria al loro interno. Questi risultati devono essere presi in considerazione per capire come e in quali condizioni utilizzarli, allo scopo di migliorare le tecniche e le tecnologie di misurazione dell’odore.

Complementarity in resource use leading to increased resource partitioning is the most commonly proposed mechanism for explaining the positive relationship between plant diversity and productivity. However, we still have a poor understanding of the relationship between plant diversity and root biomass. We test whether the hypotheses of spatial resource partitioning and symmetric proliferation are responsible for the phenomena that aboveground tree species richness (SRA) increases fine root (≤ 2 mm in diameter) biomass. We found that increasing SRA led to higher belowground biomass and a support for symmetric root proliferation strategies, but this pattern only appeared in the more nutrient-rich upper soil layer. Fine root biomass depended on the SRA × tree density interaction, with lower biomass at lower density and low richness, and this effect disappeared in mixtures with high density. The results indicate that density-dependent biotic interactions affecting tree recruitment are an important driver to influence productivity.

Tropical birds are purported to be longer lived than their temperate counterparts, but it has not been shown whether avian survival rates covary with latitude worldwide. Here, we perform a global-scale meta-analysis of 1,007 estimates from 249 studies of avian survival and demonstrate that a latitudinal survival gradient exists in the northern hemisphere, is dampened or absent for southern hemisphere species, and that differences between passerines and nonpasserines largely drive these trends. We also show that while extrinsic factors related to climate were poor predictors of survival compared to latitude alone, the relationship between survival and latitude is strongly mediated by intrinsic traits ― larger species with smaller clutch size had the highest survival. Taken together, our results suggest that interactions between intrinsic traits and lineage-specific effects surpass latitude and its underlying climatic factors in explaining global patterns of avian survival.

Pollinators utilize different land-uses via the plants they visit, however these connections vary within and among land-uses. Identifying which insects are carrying pollen and from where can elucidate key plant-pollinator interactions and identify the most important sites for maintaining community-level interactions in different land-use types. We developed a novel interaction-site bipartite network approach to identify which land-use types at the field- and landscape-scale best conserve plant-pollinator interactions. We identified distinct pollen-insect interactions that were highly specialised to both natural and modified land-uses. Many interactions involved flies, wasps and beetles; groups requiring greater research effort. Field-scale land-use best predicted interaction richness, uniqueness and strength. Management at this scale may provide the best outcomes for conserving or restoring plant-pollinator interactions in modified landscapes. This novel, intuitive approach could inform land-use planning, whereby priority is afforded to conservation areas that represent significant links between plant and pollinator communities within mosaic landscapes.

A key goal of sustainable agriculture is to produce sufficient food whilst minimising environmental damage. To achieve this we need to understand the role of agricultural landscapes in providing diverse ecosystem services and how these affect crop production and resilience, i.e. maintaining crop yields despite environmental perturbation. We used ten years of English wheat yield data to derive three metrics of resilience (relative function, yield stability and resistance to an extreme event). We explored their relationships with aspects of landscape composition and configuration (10km × 10km scale) known to affect ecosystem service components (e.g. beneficial invertebrates) and delivery (e.g. pest control). We found that resilience was uniformly enhanced in landscapes with higher coverage of semi-natural habitats. However, this was most pronounced for resilience metrics derived over shorter timescales (e.g. resistance) and metrics showed contrasting responses to landscape configuration, suggesting trade-offs if managing landscapes for resilience over shorter vs. longer timescales.

Equilibrium structures of neutral end substituted thia[n]helicenes (n=1-10) and their radical cations in DCM solvent are reported. For both neutral and radical cations of these helicenes, calculated structures are non-planar for n=3-10. Helical structures are obtained for higher helicenes and thia[8]helicene system has a helical structure with one complete turn. Geometries are predicted applying B3LYP-D/6-311++G(d,p) method in conjunction with SMD solvent model. Single point energy calculations are also performed at MP2 level to improve the energy parameters. Excited state calculations are performed using Time-Dependent Density Functional Theory (TDDFT) to predict UV-Visible spectra of neutral and radical cations of thia[n]helicenes in DCM solvent. Thia[n]helicenes radical cation have strong absorption in the near IR region. Calculations also suggest that dimerization of end substituted neutral and radical cation of thia[7]helicene is not a favourable process in DCM solvent. Overall, this study examines the molecular and electronic properties of thia[n]helicenes in search of near infrared electronic devices.

Time-dependent density functional theory approach implemented at hybrid-B3LYP, GGA-PBE and DFTB levels of theory was used to model photoinjection in organic-dye/TiO2 quantum-dot to explore the prospects of improvement of DSSC. The photosensitizer used in this study consisted of six carbazole based organic dyes having acceptor as cyanoacrilic acid group and oligothiophene π-bridge spacer. The modifications were made in the dyes by increasing length of the spacer by adding thiophene and oxadiazole rings at different positions of the donor-acceptor bridge. The structural variations appeared to alter the electronic and optical properties of dyes studied via energy levels and excitation spectra. The UV-Vis spectra calculated for all the dyes in solvents exhibited a red shift in spectral peaks with increase in polarity of the solvents. The findings of the study pointed towards photoinjection of indirect nature studied in dye-(TiO2)96 complex for six different dyes. The substitution of oxadiazole ring in center and addition of a thiophene ring at the edge of the spacer produced two dyes which exhibited lowest injection energies of 0.11eV and 0.17 eV along with the regeneration energies of 1.18 eV and 1.12 eV respectively. The dyes reported herein may have promising applications in photoanode for enhancing the performance of DSSC.

Zr-doped icosahedral Cu nanoparticle (NP) with 54 Cu atoms and one Zr atom is studied by Density functional theory (DFT) calculation for CO2 reduction to CHO which is intermediate converting into methanol. A Cu atom being at the vertex (′v′) and edge (′e′) site of Cu NP’s surface is respectively substituted by a Zr atom, and this doping of Zr atom makes Cu NP becoming one of the potential catalysts for CO2 reduction to methanol. Especially, Cu54Zre NP shows it’s superior activity and selectivity for CO2 reduction to CHO, where Zr atom stays at ′e′ site. This can be well confirmed not only from it’s adsorption behavior for several important intermediates, but also from the lowered reaction barrier of CO2 converting into CHO. Moreover, Cu54Zrv NP also shows it’s relatively good performance in CO2 converting into CHO, where Zr atom is doped at ′v′ site, if compared with pure Cu NP and even bulk Cu with Zr doping. That is to say, the introduction of Zr atom can effectively improve the catalytic properties of Cu NPs for CO2 reduction to methanol, and the fact of Zr atom doping site and it’s relation with catalytic property, to some extent provides the guidance in designing catalysts that capture and convert CO2 to fuels or chemicals.

The equilibrium geometries, stabilities, and electronic properties of MAln(M=F, Cl; n=3-15) are calculated by employing CALYPSO global search technique combined with B3LYP scheme. Optimized geometries for FAln and ClAln clusters displayed that the rule of structural evolution is attaching type, and in the end to the cage-like pattern with Al atom located inside Aln clusters (n=12-15). The analysis of stabilities shown that the FAl7 and ClAl7 clusters are the magic numbers with good chemical stabilities. The analysis of internal charge transfer shown that F or Cl atom is an electronic acceptor and strong sp hybridization exists in the F or Cl atom. Finally, the chemical hardness and polarizabilities are discussed.

The densely populated coastal areas of the Ganges-Brahmaputra-Meghna (GBM) delta within Bangladesh are in danger of losing up to one fourth of their habitable land by 2100 due to relative sea level rise (RSLR). Tidal River Management (TRM) presents an opportunity to combat RSLR by raising the land level through controlled sedimentation in re-opened polder sections. To date, TRM has been applied to tide-dominated coastal regions, but the potential applicability of TRM for river-dominated flow and mixed flow regimes is yet to be assessed. We apply a calibrated 2D numerical hydromorphodynamic model to quantify sediment deposition in a re-opened polder section (‘beel’) under conditions of river-dominated, tide-dominated and mixed flow regimes for different seasons and flow regulations. Simulation results show seasonality in sediment deposition with monsoon season having the highest. The potential for TRM is largest along the reaches of the tide-dominated region where sediment deposition is highest in all three seasons (Pre-monsoon, Monsoon and Dry season), and almost 28 times higher than river-dominated region during monsoon. Regulating flow into a polder increases trapping efficiency, but slightly lower total deposition than without regulation. Our results show that re-establishing polder flooding without regulating the flow into the polder is a promising strategy for the mixed and tide-dominated flow regions in the delta as the sediment deposition can elevate the land more than the yearly rate of RSLR. Application of controlled flooding like TRM therefore provides opportunity to match the rate of RSLR throughout the GBM delta. TRM can potentially be applied to the sinking deltas around the world to counter RSLR.

Rationale, aims and objectives: An outbreak of HAdV-7 infection in a tertiary hospital that led to a death case and nosocomial infection is described in the report. 14 confirmed cases (8 laboratory-confirmed cases and 6 silent-infection cases) were identified. Methods: The epidemiology investigation was carried out and 75 nasopharyngeal swab specimens were collected. Extraction and detection of viral nucleic acids and virus isolation were done to confirm the pathogenic factor. Results: The overall attack rate of this outbreak was 18.7%. 14 samples of 75 specimens were positive for HAdV-DNA, the virus strains belonged to HAdV7 and had high homology with the predominant HAdV7 strain ( HAdV7 0901HZ/ShX/CHN/2009 GU230898), which was isolated in 2009 from Hanzhong, Shaanxi. Conclusion: There were no a standard fever clinic in the hospital and no strict infection control procedures when caring for the patients, which were the main causes of the outbreak. Setting up a standard fever clinic, improving diagnostic capabilities for rapid detection of HAdV infection, isolating the infection sources as soon as possible and adopting infection control precautions strictly were necessary to prevent nosocomial infections.

In this paper, the Shannon entropy and Fisher information are studied for the screened Kratzer potential model (SKP). We calculated the position and momentum entropies for the screened Kratzer potential for its ground states as well as the first excited state. Our result shows that the sum of the position and momentum entropies satisfies the lower bound Berkner, Bialynicki–Birula and Mycieslki (BBM) inequality. Also, our results showed that decreasing Shannon entropy in the position space was complemented with an increasing Shannon entropy in the momentum space. Similarly, we evaluated for Fisher information and show that the Stam, Cramer-Rao inequalities are satisfied. The squeezing phenomena were also observed for certain values of the screening parameter α.

Hemicellulose-rich substrates produced in the lignocellulose biorefinery context can yield macromolecular hemicellulose structures with assorted application in the chemical industry. In the current work, alkaline-active xylanase aided to extract a varied group of xylan fractions from alkaline-sulfite pretreated sugarcane bagasse. Extraction yields (12-44%) depended on the reaction time and the xylanase load used in the extraction procedure. Prepared fractions contained low levels of lignin contamination (4-9%) and 4-O-methyl glucuronic acid arabinoxylan structures. Molar masses of fractions ranged from 2.3 kDa to 34 kDa. Prepared xylans were incorporated onto eucalyptus pulp fibers up to 4.7 g xylan/100 g pulp. The efficiency of xylan incorporation was dependent on the xylan structures, where low molar mass and low substitution degree favored high incorporation levels. Enzymatic xylan extraction proved a distinguishing procedure to add value in the lignocellulosic biorefinery chain of sugarcane bagasse once extracted xylans are suitable for incorporation onto cellulosic fibers.

Changes in root morphological traits of seed plants has been typically associated with transitions from the ancestral arbuscular mycorrhizal (AM) to the alternative ectomycorrhizal (ECM) or non-mycorrhizal (NM) associations. However, changes in root morphology also coincide with changes in leaf physiology and growth habit during the diversification of Angiosperms. To explore the evolution of root systems and their role in the diversification in seed plants, we assembled a 600+ species database to reconstruct historical changes in root, leaf and growth form in seed plants. Our findings show important shifts in diameter, specific root length and tissue density as Angiosperms diversified. For most plants, changes in morphology occurred before the acquisition of novel mycorrhizal affiliations, but along with changes in leaf hydraulics and growth form. These findings suggest that adaptation in root systems was crucial during plant diversification and defined important ecological divergences among phylogenetic clades.

The combinations of taxonomic, functional and phylogenetic method have been frequently advocated to assess how changes in biodiversity affect community structure and ecosystem function. Using a large grassland community database involving 917 species and 118 sites across the eastern and central Tibetan plateau, we found an overall positive biodiversity-productivity relationship in species', functional and phylogenetic space. The relationship, however, was nonlinear, in which biodiversity explained better the variation in community biomass when species diversity was more than a threshold, showing a weak effect of biodiversity on ecosystem function in low species diversity communities. We also found a filled triangle for the limit of the relationship between species' and functional diversity, implying that functional diversity differs significantly among communities when their species diversity is low but finally converges to be a constant with increasing communities' species diversity. Our research suggests that multiple niche processes may structure the Tibetan grassland communities, and their forces tend to balance in high-biodiversity communities.

Alanine is a transfer standard dosimeter using in gamma-ray and electron beam calibration. One of the important factor affecting its dosimetric response is amount of humidity which can deviate the dosimetry expert from the exact value of absorbed doses. Ab initio molecular dynamics calculations were performed to determine the environmental effects on the EPR parameters of L-α-Alanine radicals in acidic and alkaline solutions. Similar to the closed-shell amino acid molecule alanine, the zwitterionic form of alanine radical is the stable form in the gas phase while the non-zwitterionic neutral alanine radical is not a stable structure. Geometric and EPR parameters of radicals in both gas and solution phases are found to be dependent on hydrogen bonding of water molecules with the polar groups and by dynamic solvation. Calculations on the optimized free radicals in the gas phase revealed that for neutral radical, hydrogen bonding to water molecules drives a decrease in the magnitudes of g-tensor components gxx and gyy without affecting neither gzz component nor the HFCCs. For the transfer from the gas to solution phase of the alanine radical anion is accompanied with an increase in the spin density on the carboxylic group’s oxygen atoms. However, for the neutral radical, this transfer from gas to solution phase is accompanied with the decrease in the spin density on oxygen atoms. Calculated isotropic HFCCs and g-tensor of all radicals were in good agreement with their experimental counterparts in both acidic and alkaline solutions, which enhances the confidence in our calculated results.

Background: Amrubicin (AMR), a third-generation anthracycline and potent topoisomerase II inhibitor, has been known to be effective treatment for previously treated Extensive-Disease Small-Cell Lung Cancer (ED-SCLC). However, the efficacy and toxicityof amrubicin remainsconflicting . Thus,this meta-analysis was designed to evaluate amrubicin effectiveness in refractory ED-SCLC patients. Methods: Pubmed, Embase, Cochrane library databases were searched for the relevant articles. After rigorous evaluation on quality, the data extraction process was carried from eligible randomized controlled trials (RCTs). Meta-analysis Revman 5.3 software was used for statistical analysis. Results: A total of 7 RCTs were included in our analysis. The regimen of AMR was associate with better progression-free survival (PFS)(OR=0.81,95%CI=0.71-0.92, P=0.001), and the objective response rate (ORR) (OR=1.83,95%CI=1.37-2.45, P＜0.0001＝. While, there was no statistical significance was found in terms of the overall survival (OS) (OR=0.90,95%CI=0.79-1.03, P=0.12). The frequencies of the most common toxicities were more commonly occurred in the AMR group compared with the control group were neutropenia (OR=1.67, 95%CI=1.10-2.54, P= 0.02) and anemia (OR=0.47,95%CI=0.34-0.66, P＜0.0001＝ , respectively. Conclusion: AMR was associated with better efficacy and acceptable side effects for the treatment-refractory ED-SCLC. The findings revealed that amrubicin may be used as second-line chemotherapy for patients with sensitive-relapse ED-SCLC.

Background: Lack of time has consistently been reported as a major barrier to effective research evidence-uptake into clinical practice. There has been no research to our knowledge that explores time as a barrier within the Transtheoretical model of Stages of Change (SoC), to better understand the processes of physiotherapists’ uptake of clinical practice guidelines (CPG). This paper explores the concept of lack of time as a barrier for CPG uptake for physiotherapists at different SoC. Methods: A 6-step process is presented to determine the best-fit SoC for 31 physiotherapy interviewees. This process used an amalgamation of interview findings and socio-demographic data, which was layered onto the SoC and previously identified time-barriers to CPG uptake (few staff; high workload; access to CPGs; evidence-based practice as priority in clinical practice; “time is money” attitude; and knowledge on the use of CPGs). Results: The analysis process highlighted the complexities of assigning individuals to a SoC. A model of time management for better CPG uptake is proposed which is a novel approach to assist evidence implementalists and clinicians alike to determine how to progress through the SoC and barriers to improve CPG uptake. Conclusions: To the authors’ knowledge, this is the first attempt at exploring the construct of (lack of) time for CPG-uptake in relation to the physiotherapists’ readiness to behaviour change. This study shows that ‘lack of time’ is a euphemism for quite different barriers, which map to different stages of readiness to embrace current best evidence into physiotherapy practice. By understanding what is meant by ‘lack of time’, it may indicate specific support required by physiotherapists at different stages of changing these behaviours.

ABSTRACT We consider a residue convolution operation INTRODUCTION Consider the definition of a residue $$ Res(f,s) = (z-s)f(z) $$ for a function f with a pole at s. Here the role of (z − s) is to neutralise the infinity at f(z) with a zero. Consider this to actually be a function g(x)=x, and extend the definition to $$ Res(g,f,s) = g(z)f(z) $$ for any function which has a zero at g(s). This could be seen as a form of convolution of the two functions. If multiple residues are to be summed over, it would be interesting to consider functions with coinciding zeros and infinities. For example $$ Res(\sin(\pi \cdot),\Gamma(\cdot),s) = \sin(\pi z)\Gamma(z) $$ The sum over residues is $$ ^\infty {k!} = \pi e $$ for Res(sin²(π ⋅ ), Γ(⋅),s)² we have $$ ^\infty {k!^2} = \pi^2 I_0(2) $$ We can define this into a transform which defines a function like an inverse Mellin transform For g = sin(πx)cos(πx), f = Γ(s), we get πe−x. g f ℳ−1 ---------------- ------- ------------------------- sin(πx) Γ(s) πex sin²(πx) Γ(s)² $\pi^2 I_0(2 )$ sin(πx)cos(πx) Γ(s) πe−x

Technological advancements in the past few decades have made it possible to manufacture nanomaterials at large scale and ENPs are increasingly found in consumer products such as cosmetics, sports products and LED displays. A large amount of these ENPs are in wastewater and potentially impact the performance of wastewater treatment plants (WWTPs). One important function of the WWTP is nitrification, which is carried out by the actions of two groups of bacteria, ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB). Since most ENPs are found to have or are designed to have antimicrobial activities, it is a legitimate concern that ENPs entering WWTPs may have negative impacts on nitrification. In this paper, the effects of ENPs on nitrification is discussed, focusing mainly on autotrophic nitrification by AOBs and NOBs. This review also covers ENPs effects on ANAMMOX. Generally, nitrifiers in pure and mixed culture can be inhibited by a variety of ENPs, but stress response mechanisms may attenuate toxicity. Long-term studies demonstrated that a wide range of NPs can cause severe deterioration of AOBs and/or NOBs when the influent concentration exceeded an inhibition threshold. Proposed mechanisms include the generation of reactive oxygen species, dissolved metals, physical disruption of cell membranes, bacterial engulfment, and intracellular accumulation of ENPs. Future research needs are also discussed.

Homologous recombination over large genomic regions is difficult to achieve due to low efficiencies. Here, we report the successful engineering of a humanized mTert allele, hmTert, in the mouse genome by replacing an 18.1-kb genomic region around the mTert gene with a recombinant fragment of over 45.5-kb, using homologous recombination facilitated by the Crispr/Cas9 technology, in mouse embryonic stem cells (mESCs). In our experiments, with specific sites of DNA double strand breaks (DSBs) by Crispr/Cas9 system, the homologous recombination efficiency was up to 11% and 16% in two mESC lines TC1 and v6.5, respectively. Overall, we obtained a total of 27 mESC clones with heterozygous hmTert/mTert alleles and 3 clones with homozygous hmTert alleles. DSBs induced by Crispr/Cas9 cleavages also caused high rates of genomic DNA deletions and mutations at small guide RNA (sgRNA) target sites. Our results indicated the Crispr/Cas9 system significantly increased the efficiency of homologous recombination-mediated gene editing over a large genomic region in mammal cells, but also inherently caused mutations at unedited target sites. Overall, this strategy provides an efficient and feasible way for manipulating large chromosomal regions.