Optimized CRISPR/Cas9 strategy for homology-directed multiple targeted integration of transgenes in CHO cells

Site-specific integration has emerged as a promising strategy for precise Chinese hamster ovary (CHO) cell line engineering and predictable cell line development (CLD). CRISPR/Cas9 with the homology-directed repair (HDR) pathway enables precise integration of transgenes into target genomic sites. However, inherent recalcitrance to HDR-mediated targeted integration (TI) of transgenes results in low targeting efficiency, thus requiring a selection process to find a targeted integrant in CHO cells. Here, we explored several parameters that influence the targeting efficiency using a promoter-trap-based single- or double-knock-in (KI) monitoring system. A simple change in the donor template design by the addition of single-guide RNA recognition sequences strongly increased KI efficiency (2.9–36.0 fold), depending on integration sites and cell culture mode, compared to conventional circular donor plasmids. Furthermore, sequential and simultaneous KI strategies enabled us to obtain populations with ~1–4% of double-KI cells without additional enrichment procedures. Thus, this simple optimized strategy not only allows efficient CRISPR/Cas9-mediated TI in CHO cells but also paves the way for the applicability of multiplexed KIs in one experimental step without the need for sequential and independent CHO–CLD procedures.     

Interaction of aromatic donor molecules with horseradish peroxidase: identification of the binding site and role of heme iron in the binding and activity

The interaction of aromatic substrates with horseradish peroxidase (HRP) was studied. Chemical modification of HRP was performed using diethylpyrocarbonate (DEPC) and for the first time the amino acid involved in binding with these substrates has been identified. The kinetic parameters for this interaction have been calculated and the role of heme iron in the oxidation of aromatic substrates by HRP has been discussed.     

Poly(1,2,3-benzotriazolyl)borate complexes with copper(I) and tri-organophosphane: an unprecedented κ-coordination of [H2B(btz)2] (btz=1,2,3-benzotriazolyl) in the X-ray crystal structure of [Cu(PBn3)2{(btz)BH2(btz)}]

New copper(I) triorganophosphane derivatives [Cu(PR₃)_n{H₂B(btz)₂}] and [Cu(PR₃)_n{HB(btz)₃}] (n=1 or 2) have been synthesized from the reaction of CuCl with PR₃ (R=phenyl, cyclohexyl, benzyl, o-, m-, or p-tolyl) or PMePh₂ and potassium dihydrobis(1,2,3-benzotriazolyl)borate K[H₂B(btz)₂] or potassium hydrotris(1,2,3-benzotriazolyl)borate K[HB(btz)₃]. The complexes obtained have been characterized by elemental analyses and FT-IR in the solid state and by NMR (¹H and ³¹P{¹H}) spectroscopy and conductivity measurements in solution. Solution data are consistent with partial dissociation of complexes occurring throughout breaking of the CuP bond. Single crystal structural characterizations were undertaken for two of them. The structurally authenticated arrays are, (a) [Cu(PBn₃)₂{(btz)BH₂(btz)}] with a three coordinate P₂Cu(N) coordination sphere and the donor [H₂B(btz)₂] coordinated throughout only one N3 atom. (b) [Cu(P-m-tolyl₃)_n{(btz)₃BH}] with a four coordinate PCuN₃ coordination sphere with the tris(benzotriazolyl)borate acting as tripodal donor throughout all its N2 atoms.     

The determination of the carbonic anhydrases activators in vitro effect of mixed donor crown ethers

Carbonic anhydrases (CAs) play an important function in various physiological and pathological processes. Therefore, many researchers work in this field in order to design and synthesize new drugs. Both inhibitors and activators of CAs, which are associated with the diagnosis and treatment of many diseases, are very important. The emergence of the use of CA activators in the treatment of Alzheimer has led many scholars to work on this issue. In this study, CA activators and inhibitors are determined. The crown ethers compounds ( 1 , 2 , 3 , 6 , 7 , 8 , and 9 ) were found to cause activation on enzyme activities of hCA I and II. The AC₅₀ values on hCA I and II of the compounds are in the range of 4.6565–374.979 μM. The 4 (IC₅₀; 1.301 and 3.215 μM for hCA I and II) and 5 (IC₅₀; 73.96 and 378.5 μM for hCA I and II) compounds were found to cause inhibition on enzyme activities of hCA I and II.     

Identification of target proteins of clinical immunity to Plasmodium falciparum in a region of low malaria transmission

The target molecules of antibodies against falciparum malaria remain largely unknown. Recently we have identified multiple proteins as targets of immunity against Plasmodium falciparum using African serum samples. To investigate whether potential targets of clinical immunity differ with transmission intensity, we assessed immune responses in residents of low malaria transmission region in Thailand. Malaria asymptomatic volunteers (Asy: n = 19) and symptomatic patients (Sym: n = 21) were enrolled into the study. Serum immunoreactivity to 186 wheat germ cell-free system (WGCFS)-synthesized recombinant P. falciparum asexual-blood stage proteins were determined by AlphaScreen, and subsequently compared between the study groups. Forty proteins were determined as immunoreactive with antibody responses to 35 proteins being higher in Asy group than in Sym group. Among the 35 proteins, antibodies to MSP3, MSPDBL1, RH2b, and MSP7 were significantly higher in Asy than Sym (unadjusted p < 0.005) suggesting these antigens may have a protective role in clinical malaria. MSP3 reactivity remained significantly different between Asy and Sym groups even after multiple comparison adjustments (adjusted p = 0.033). Interestingly, while our two preceding studies using African sera were conducted differently (e.g., cross-sectional vs. longitudinal design, observed clinical manifestation vs. functional activity), those studies similarly identified MSP3 and MSPDBL1 as potential targets of protective immunity. This study further provides a strong rationale for the application of WGCFS-based immunoprofiling to malaria vaccine candidate and biomarker discovery even in low or reduced malaria transmission settings.     

Analysis of factors related to ELISA-HBsAg negative and HBV DNA positive blood donors

This report analyzed factors relating to ELISA-HBsAg and Hepatitis B virus (HBV) DNA in blood donors. To provide a reference for an accurate screening model for HBV infection in donated blood, we collected rel-evant information from 124 blood donors testing ELISA-HBsAg negative and HBV DNA positive in 2017, including ELISA-HBsAg Max s/co, gender, age, residence, education level, blood donation record and alanine aminotransferase(ALT) value. Meanwhile, 99 blood donors with the results ELISA-HBsAg negative and HBV DNA negative were randomly selected as control. Univariate logistic analysis was conducted for possible correlation factors, then multivariate logistic regression analysis was performed for statistically significant observation indicators. The results showed that the Ct value of HBV DNA mixed test in the observation group donor was higher than that of HBV DNA single test (P<0.05). If one of the two ELISA-HBsAg s/co values was within the range of 0.259 to 0.304, the chance of HBV DNA positive was increased. Univariate logistic regression analysis showed that ELISA-HBsAg Max s/co, gender, age, blood donation history and ALT value were all risk factors in the observation group. Multivariate logistic regression analysis showed that ELISA-HBsAg Max s/co, (OR=5352.448, P<0.05), age (OR= 4.527, P<0.05) and blood donation history (OR=0.441) were risk factors. The study concluded that ELISA-HBsAg Max s/co, age and number of blood donations are risk factors for ELISA-HBsAg negative and HBV DNA positive blood donors, women or donors under 26 years of age had the lowest risk.     

n-acylation of β-amino alcohol by acyl migration following enzyme-catalyzed esterification

Lipase-catalyzed n-acylations of β-amino alcohols such as ethanolamine and l-serine were investigated. To prepare n-acyl derivatives by taking advantage of the acyl migration, we first carried out a screening of suitable enzymes for the desired reaction. As a result, we found a higher activity for n-acylation with Lipase L. This lipase had higher hydrolytic activity for the o-acyl compound but not the n-acyl compound. The observation shows that n-acylation results from the esterification and successive acyl migration into the amino group. Using Lipase L, we then investigated the n-acylation of ethanolamine or l-serine with fatty acids as acyl donors. The reaction parameters for the n-acylation were clarified.     

Bis[dihydrobis(1-pyrazolyl)borate]cadmium(II): a monomeric, severely distorted tetrahedral complex of cadmium(II)

The crystal structure of the complex [H₂B(pz)₂]₂Cd has been determined: orthorhombic, Pbca, A = 16.052(3), B = 13.935(3), C = 14.974(4) Å, V = 3349.4(13) ų, Z = 8, R(F) = 3.61%. It is the first structurally characterized non-porphyrin CdN₄ complex. It is monomeric in the solid state with a pseudotetrahedral geometry about the cadmium atom. The N---Cd---N angles are distorted by the approximate 93° bite angle of the ligand; the interligand N---Cd---N angles also are distorted, ranging from 106.9 to 131.7°. These distortions are the result of intermolecular packing forces and are facilitated by the spherical set of valence orbitals for Cd²⁺.     

Effects of bicarbonate and formate on the donor side of Photosystem 2

Sodium formate at concentration of 5–20 M suppresses electron flow on the donor side of Photosystem 2 (PS 2) in pea subchloroplast membranes (DT-20) which is revealed by inhibition of photoinduced changes of chlorophyll fluorescence yield related to photoreduction of Q_A and pheophytin (the primary and intermediary electron acceptors) and oxygen evolution and the increase of absorbance changes related to photooxidation of P₆₈₀, the primary electron donor, under continuous illumination. These activities are also inhibited upon partial depletion of bicarbonate in the medium and restored by the addition of 0.1–10 mM NaHCO₃. At concentrations higher than 20 M formate induces the known bicarbonate effect on the acceptor side of PS 2 which dominates at millimolar concentrations of the agent. In Tris-treated (Mn-depleted) DT-20 the restoration of electron flow with 0.2 M MnCl₂ (4 Mn atoms per one PS 2 reaction center) in the medium depleted of bicarbonate is efficient only after the addition of 5 mM NaHCO₃. The restoration in the presence of NaHCO₃ is accompanied by an increased functional binding of Mn²⁺ to PS 2 membranes which is confirmed by experiments on removal of added Mn²⁺ by either sedimentation or the addition of EDTA. Pre-illumination increases the Mn binding in the presence of bicarbonate. The data show that the bicarbonate effect on the donor side of PS 2 is related to a relatively low-affinity bound pool of bicarbonate. It is suggested that bicarbonate takes part in the formation of the Mn-cluster capable of water oxidation as an obligatory ligand or through modification of the binding site(s) of Mn.Abbreviations CCCP carbonyl cyanide-m-chlorophenylhydrazone - DCMU 3-(3,4-dichlorphenyl)-1,1-dimethylurea - DPC diphenylcarbazide - EDTA ethylenediaminetetraacetic acid - SiMo silicomolibdate