Production of functional human nerve growth factor from the submandibular glands of mice using a CRISPR/Cas9 genome editing system.

Nerve growth factor (NGF) is an essential trophic factor for the growth and survival of neurons in the central and peripheral nervous systems. For many years, mouse NGF (mNGF) has been used to treat various neuronal and non-neuronal disorders. However, the biological activity of human NGF (hNGF) is significantly higher than that of mNGF in human cells. Using the CRISPR/Cas9 system, we constructed the transgenic mice expressing hNGF specifically in their submandibular glands. As demonstrated by fluorescence immunohistochemical staining, these mice produced hNGF successfully, with 0.8 mg produced per gram of submandibular glands. hNGF with 99% purity was successfully extracted by two-step ion-exchange chromatography and one-step size-exclusion chromatography from the submandibular glands of these transgenic mice. Further, the purified hNGF was verified by LC-MS/MS. We analyzed the NH2-terminus of hNGF using both Edman degradation and LC-MS/MS-based methods. Both results showed that the obtained hNGF lost the NH2-terminal octapeptide (SSSHPIFH). Moreover, the produced hNGF demonstrated a strong promotion in the proliferation of TF1 cells.

Cationic cell-penetrating peptide is bactericidal against Neisseria gonorrhoeae.

OBJECTIVES: Cell-penetrating peptides (CPPs) have been evaluated for intracellular delivery of molecules and several CPPs have bactericidal activity. Our objectives were to determine the effect of a 12 amino acid CPPs on survival and on the invasive and inflammatory potential of Neisseria gonorrhoeae. METHODS: Survival of MDR and human challenge strains of N. gonorrhoeae grown in cell culture medium with 10% FBS was determined after treatment with the CPP and human antimicrobial peptide LL-37 for 4 h. Confocal microscopy was used to examine penetration of FITC-labelled CPP into bacterial cells. The ability of the CPP to prevent invasion of human ME-180 cervical epithelial cells and to reduce the induction of TNF-α in human THP-1 monocytic cells in response to gonococcal infection was assessed. Cytotoxicity of the CPP towards the THP-1 cells was determined. RESULTS: The CPP was bactericidal, with 95%-100% killing of all gonococcal strains at 100 µM. Confocal microscopy of gonococci incubated with FITC-labelled CPP revealed the penetration of the peptide. CPP treatment of N. gonorrhoeae inhibited gonococcal invasion of ME-180 cells and reduced the expression of TNF-α induced in THP-1 cells by gonococci. The CPP showed no cytotoxicity towards human THP-1 cells. CONCLUSIONS: Based on these promising results, future studies will focus on testing of CPP in the presence of other types of host cells and exploration of structural modifications of the CPP that could decrease its susceptibility to proteolysis and increase its potency.

An efficient constitutive expression system for Anti-CEACAM5 nanobody production in the yeast Pichia pastoris.

Nanobodies offer multiple advantages over conventional antibodies in terms of size, stability, solubility, immunogenicity, and production costs, with improved tumor uptake and blood clearance. Additionally, the recombinant expression of nanobodies is robust in various expression systems, such as Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris. P. pastoris is the most widely used microorganism for nanobody production, but all or almost all expression vectors developed for this system are based on the regulated promoter of the alcohol oxidase 1 gene (AOX1) that requires methanol for full induction. In this study, a constitutive anti-CEACAM5 nanobody expression system was constructed under the control of a glyceraldehyde-3-phosphate dehydrogenase promoter (GAP) promoter. The effects of different carbon sources and pH on nanobody expression were evaluated in shaking flask cultures. After 96 h of constitutive expression in shaking flask, a yield of 51.71 mg/L was obtained. In addition, this constitutive expression system produced nanobodies at equivalent yield and affinity to that produced by methanol-induced expression. The results of this study indicated that the use of a constitutive expression system is a promising alternative for the production of nanobodies applied for cancer diagnosis and therapy.

Treatment of human challenge and MDR strains of Neisseria gonorrhoeae with LpxC inhibitors.

Objectives: Inhibitors of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC), which catalyses the second step in the biosynthesis of lipid A, have been developed as potential antibiotics for Gram-negative infections. Our objectives were to determine the effect of LpxC inhibition on the in vitro survival and inflammatory potential of Neisseria gonorrhoeae. Methods: Survival of four human challenge strains was determined after treatment with two LpxC inhibitors for 2 and 4 h. To confirm results from treatment and assess their anti-inflammatory effect, the expression of TNF-α by human THP-1 monocytic cells infected with bacteria in the presence of the LpxC inhibitors was quantified. Cytotoxicity of inhibitors for THP-1 cells was evaluated by release of lactate dehydrogenase. Survival of five MDR strains was determined after 2 h of treatment with an LpxC inhibitor and the effect of co-treatment on MICs of ceftriaxone and azithromycin was examined. Results: The inhibitors had bactericidal activity against the four human challenge and five MDR strains with one compound exhibiting complete killing at ≥5 mg/L after either 2 or 4 h of treatment. Treatment of gonococci infecting THP-1 monocytic cells reduced the levels of TNF-α probably owing to reduced numbers of bacteria and a lower level of expression of lipooligosaccharide. Neither inhibitor exhibited cytotoxicity for THP-1 cells. The MIC of azithromycin was slightly lowered by sublethal treatment of two MDR strains with an LpxC inhibitor. Conclusions: Our in vitro results demonstrated promising efficacy of LpxC inhibition of N. gonorrhoeae that warrants further investigation particularly owing to the rise in MDR gonorrhoea.

Colon-Targeted Delivery of IgY Against Clostridium difficile Toxin A and B by Encapsulation in Chitosan-Ca Pectinate Microbeads.

This study investigated the use of a newly developed chitosan-Ca pectinate microbead formulation for the colon-targeted delivery of anti-A/B toxin immunoglobulin of egg yolk (IgY) to inhibit toxin binding to colon mucosa cells. The effect of the three components (pectinate, calcium chloride, and chitosan) used for the microbead production was examined with the aim of identifying the optimal levels to improve drug encapsulation efficiency, swelling ratio, and cumulative IgY release rate. The optimized IgY-loaded bead component was pectin 5% (w/v), CaCl2 3% (w/v), and chitosan 0.5% (w/v). Formulated beads were spherical with 1.2-mm diameter, and the drug loading was 45%. An in vitro release study revealed that chitosan-Ca pectinate microbeads inhibited IgY release in the upper gastrointestinal tract and significantly improved the site-specific release of IgY in the colon. An in vivo rat study demonstrated that 72.6% of biologically active IgY was released specifically in the colon. These results demonstrated that anti-A/B toxin IgY-loaded chitosan-Ca pectinate oral microbeads improved IgY release behavior in vivo, which could be used as an effective oral delivery platform for the biological treatment of Clostridium difficile infection (CDI).

Investigation of the Penetration Performance of the Radial Forging Process for Wrought Aluminium Alloy.

With the wide application potential of wrought aluminium alloy in aerospace, automobile and electronic products, high-quality aluminium bars prepared by the radial forging (RF) process have received extensive attention. Penetration performance refers to the depth of radial plastic deformation of forgings, which is the key factor in determining the quality of forging. In this work, the penetration performance of the radial forging process for 6063 wrought aluminium bars is investigated by simulation using FORGE software. The minimum reduction amount of the hammer is calculated based on the forging penetration theory of forging. The influence of process parameters including forging ratio (FR) and billet temperature on the effective stress and hammer load in the RF process are investigated. The RF-deformed billet is then produced with the optimal process parameters obtained from the simulation results. The average grain size of aluminium alloy semi-solid spherical material is used to evaluate the forging penetration. Simulation results showed that the effective strain at the edge and the centre of the RF-deformed billet gradually increases, but the increasing speed of the effective strain at the edge becomes low. The hammer load first decreases quickly and then gradually maintains stability by increasing the FR. It is found that low billet temperature and high FR should be selected as appropriate process parameters under the allowable tonnage range of RF equipment. Under an isothermal temperature of 630 °C and a sustaining time of 10 min, the difference in the average grain dimension between the edge and the centre positions of the starting extruded blank is 186.43 µm, while the difference in the average grain dimension between the edge and the centre positions of the RF-deformed blank is 15.09 µm. The improvement ratio of penetration performance for the RF-deformed blank is obtained as 91.19%.

Characterization of Non-heading Mutation in Heading Chinese Cabbage (Brassica rapa L. ssp. pekinensis).

Heading is a key agronomic trait of Chinese cabbage. A non-heading mutant with flat growth of heading leaves (fg-1) was isolated from an EMS-induced mutant population of the heading Chinese cabbage inbred line A03. In fg-1 mutant plants, the heading leaves are flat similar to rosette leaves. The epidermal cells on the adaxial surface of these leaves are significantly smaller, while those on the abaxial surface are much larger than in A03 plants. The segregation of the heading phenotype in the F2 and BC1 population suggests that the mutant trait is controlled by a pair of recessive alleles. Phytohormone analysis at the early heading stage showed significant decreases in IAA, ABA, JA and SA, with increases in methyl IAA and trans-Zeatin levels, suggesting they may coordinate leaf adaxial-abaxial polarity, development and morphology in fg-1. RNA-sequencing analysis at the early heading stage showed a decrease in expression levels of several auxin transport (BrAUX1, BrLAXs, and BrPINs) and responsive genes. Transcript levels of important ABA responsive genes, including BrABF3, were up-regulated in mid-leaf sections suggesting that both auxin and ABA signaling pathways play important roles in regulating leaf heading. In addition, a significant reduction in BrIAMT1 transcripts in fg-1 might contribute to leaf epinastic growth. The expression profiles of 19 genes with known roles in leaf polarity were significantly different in fg-1 leaves compared to wild type, suggesting that these genes might also regulate leaf heading in Chinese cabbage. In conclusion, leaf heading in Chinese cabbage is controlled through a complex network of hormone signaling and abaxial-adaxial patterning pathways. These findings increase our understanding of the molecular basis of head formation in Chinese cabbage.

Recent advances in the selection and identification of antigen-specific nanobodies.

Nanobodies represent the next-generation antibody-derived biologics with significant advances over conventional antibodies. Several rapid and robust techniques for isolating highly specific nanobodies have been developed. Antigen specific nanobodies are selected from constructed nanobody libraries, which can be classified into 3 main types: immune library, naïve library, and semisynthetic/synthetic library. The immune library is the most widely used strategy for nanobody screening. Target specific nanobodies are highly enriched in immune libraries than in non-immune libraries; however, it is largely limited by the natural antigenicity of antigens. The naïve library is thus developed. Despite the lack of somatic maturation, protein engineering can be employed to significantly increase the affinities of selected binders. However, a substantial amount of blood samples collected from a large number of individual animals is a prerequisite to ensure the diversity of the naïve library. With this issue considered, the semisynthetic/synthetic library may be a promising path toward obtaining a limitless source of nanobodies against a variety of antigens without the need of animals. In this review, we summarize the state-of-the-art screening technologies with different libraries. The approaches presented here can further boost the diverse applications of nanobodies in biomedicine and biotechnology.

[Change of the chromosome conformation of human alpha-globin gene locus in transgenic mice].

OBJECTIVE: To establish chromosome conformation capture (3C) strategy and to use this method for exploring the effect of chromosome conformation on human alpha-globin gene expression in the human alpha-globin transgenic mouse. METHODS: Homozygous human alpha-globin transgenic male mouse was crossed with KM female mouse. The 14.5-day post-coitum (dpc) embryos were used for the isolation of fetal liver and fetal brain cells. Homogeneous single-cell suspension was treated with formaldehyde to crosslink the chromatin conformation in the nuclear. The cross-linked chromatin compound was digested with Nco I and then ligated with T4 DNA ligase. The ligated compound was reversely cross-linked and then the ligated genomic DNA was purified for PCR analysis. The primers were designed along the two sides of cut and ligated sites. Semi-quantitative PCR was used to analyze the chromosome conformation of the whole human alpha-globin gene locus in fetal liver and fetal brain cells. RESULTS: When HS40 fragment was used as the fixed fragment, in fetal brain cells, the ligation frequencies of HS40 fragment with other fragments were decreased as the linear distances to HS40 fragment were increasing; while in fetal liver cells, two active genes (alpha1 and alpha2) fragments showed higher ligation frequencies with HS40 fragment than other fragments. However, the fragment containing an inactive gene (xi) displayed the comparable low ligation frequency as that in fetal brain. When alpha2 fragment was used as the fixed fragment, similarly, in fetal brain cells the ligation frequencies of alpha2 fragment with other ones were decreased as the linear distances increasing; when in fetal liver cells, it showed higher ligation frequencies with two upstream regulatory elements (HS 40 and 33). However, it showed a little bit lower ligation frequency with another two upstream regulatory elements (HS10 and 8) than those in fetal brain. CONCLUSION: In fetal liver cells, the distant regulatory elements are in close proximity to the downstream of the expressed globin genes through looping out, the interval region; however, in fetal brain, they were not in vicinity to the expressed globin genes.

Oral delivery of human growth hormone: Preparation, characterization, and pharmacokinetics.

Daily subcutaneous injection of human growth hormone has been used for the treatment of growth hormone deficiency and growth failure but has led to poor patient compliance and renal toxicity. Thus, it is crucial to develop favorable growth hormone delivery systems to improve patient compliance. In the present study, to increase the oral bioavailability of growth hormone and improve patient compliance, enteric-coated capsules filled with monomethoxyl poly(ethylene glycol)-b-poly(L-lactide-co-glycolide) nanoparticles were prepared to facilitate oral growth hormone delivery. The nanoparticles were less than 100 nm in size, exhibited narrow polydispersity indices < 0.3, and showed a zeta potential of -4.87 mV. The highest efficiency of growth hormone encapsulation achieved in this study was nearly 70%. An in vitro release experiment showed that adequate amounts of growth hormone were retained under simulated gastric conditions and significant amounts of growth hormone were released under simulated intestinal conditions. The bioavailability of encapsulated growth hormone relative to subcutaneously injected growth hormone in Sprague-Dawley rats was 11.06%. Thus, the use of poly(ethylene glycol)-b-poly(L-lactide-co-glycolide) nanoparticles yielded promising results, and these agents should be investigated further regarding their potential as an oral growth hormone delivery system in the future.

Development of microbeads of chicken yolk antibodies against Clostridium difficile toxin A for colonic-specific delivery.

The incidence of Clostridium difficile infection has increased in Western world in the past 10 years, similar infection rates are also reported in developing countries such as China. Current antibiotics treatments have recurrence rates between 15% and 30%. IgY antibodies against toxin A of C. difficile could protect animal models from the challenge of lethal dose of C. difficile spores. However, IgY is sensitive to the low pH environment of the stomach and proteinases in the intestine. The objective of this study was to prepare colonic-specific delivery system of toxin A antigen-specific IgY to block the recognition of toxin A to the colon mucosa cells. Egg-laying hens were immunized with purified C. difficile toxin A C-terminal domain for 3 times, then egg IgY against the recombinant ToxA-C protein was purified from immunized egg yolk and frozen dried. IgY-loaded microbeads were prepared using mini fluid bed system; the loading efficiency was 21%. The pH and temperature stabilities of the microbeads were assayed. The IgY-loaded microbeads coated with 35% Eudragit S100 had colonic-specific IgY release specificity both in vitro and in vivo, the colonic-specific release of biological active IgY was 87.5% in the rat. Our study provides a new option for the biological treatment C. difficile infection.

No existence of translocus balancer to coordinate the expression and regulation of human hemoglobin genes in transgenic mice study.

All mammals use hemoglobin (Hb) to transport oxygen. Each Hb molecule is a tetramer of two pairs of unlike globin polypeptide chains. Equal amount of subunit globin chains derived from the corresponding alpha- and beta-like genes can always result during development though the two separate gene clusters are located on two different chromosomes and spatially transcribed within different nuclear domains. Disturbance of this balance will result in degradation or precipitation of the excessive globin chains, which is the character of various thalassemic syndromes. In previous studies, we had established two kinds of bacterial artificial chromosome (BAC) mediated transgenic mouse models, which contain respectively the entire human alpha- and beta-globin cluster. Here, we investigated the regulatory relationship between the two clusters by interbreeding these two kinds of transgenic mice. The levels of human alpha- and beta-mRNA in the various hybrid lines reflect the levels in the original transgenic lines that contain either the alpha- or beta-globin cluster alone. The results suggested that there is no apparent cross talk or regulatory interaction between the two human globin clusters in transgenic mice.

Excessive expression of the scavenger receptor class A type I can significantly affect the serum lipids.

Scavenger receptor (SR) is characterized by its ability to bind negatively charged macromolecules, particularly the modified lipoproteins that are pertinent to the development of vascular disease. To determine the role of excessive scavenger receptor A in the serum lipoprotein metabolism, transgenic mice lines with mouse scavenger receptor A gene type I (SR-AI) under the control of human SR-AI enhancer and metallothionein gene promotor were established. After zinc induction, the expression of SR-AI in transgenic mice was a little higher than the controls, but the serum lipids levels were significantly different from the controls, especially the cholesterol. These results demonstrated that overexpression of SR-AI significantly affected the serum lipids levels.

Signalling molecules involved in mouse bladder smooth muscle cellular differentiation.

Mouse bladder mesenchyme differentiates into smooth muscle under the influence of urothelium at gestational day 13.5 (E13.5). Sonic hedgehog (Shh) is considered to be the upstream gene arising from the urothelium, which induces smooth muscle in the peripheral bladder mesenchyme. We hypothesize differential gene expression across the full thickness of bladder mesenchyme as a function of proximity to the inducing bladder urothelium and the peripheral location of the smooth muscle. Embryonic bladders from FVB mice were collected at E12.5, 13.5, 15 and 16 and cryosectioned followed by microdissection with a PixCell II laser capture microscope. RNA extraction was performed at the laser captured sites and mRNA expression profiles were measured using SYBR Green quantitative RT-PCR. Smooth muscle a-actin (SMAA) and smooth muscle myosin heavy chain (SM-MHC) were expressed in the E13.5, E15 and E16 bladders in the peripheral layer of mesenchyme, but not in the prospective submucosa. Patched 1 (Ptc1), Gli1 and bone morphogenetic protein (Bmp) 4 expression was consistently elevated in the mesenchymal layer immediately adjacent to the urothelium compared to the peripheral location at E12.5. After E12.5, Ptc1 expression decreased to an undetectable level throughout the bladder mesenchyme. The level of TGF-beta1 was highest in the mesenchymal layer adjacent to the serosa at E13.5. The level of expression of serum response factor (SRF) was also highest at E15 in the peripheral mesenchyme. Genes downstream of Shh are differentially expressed in the prospective submucosa vs. the peripheral bladder mesenchyme as a function gestation age and smooth muscle differentiation.

Cluster specific regulation pattern of upstream regulatory elements in human alpha- and beta-globin gene clusters.

Located in different chromatin contexts and with different developmental switching mode, human alpha- and beta-globin gene clusters are co-regulated temporally and quantitatively to keep balanced expression. Here, by exchanging their key upstream regulatory elements (UREs) in cluster level, and investigating the expression level of exogenous globin genes in the bacterial artificial chromosome (BAC) mediated transgenic mice, we explored the similarities and differences in the regulatory effects between alpha-upstream regulatory element (alpha-URE) and beta-locus control region (beta-LCR). The results showed that, after exchange, the developmental switching modes of human alpha- and beta-like globin genes had changed, with lost expression of epsilon- and alpha1-genes. Their expression levels also decreased. Our study suggests that the regulation of alpha-URE and beta-LCR on the expression level and developmental switching mode of downstream globin genes is cluster specific.

HS-48 alone has no enhancement role on the expression of human alpha-globin gene cluster.

To investigate the in vivo function of the newly defined DNase I hypersensitive site HS-48 on the whole human alpha-globin gene cluster, the region containing all the other known 5 hypersensitive sites HS-4 to HS-40 was deleted from a 117 kb bacterial artificial chromosome clone bearing the whole human alpha-globin gene cluster. Transgenic mice were generated from this construct. The RNase protection assays showed that with HS-48 left and all the other 5 hypersensitive sites deleted, the expression of human alpha-like globin genes was completely silenced in embryonic, fetal and adult stages in all tissues. This finding indicates that HS-48 alone has no enhancer activity on the expression of human alpha-like globin genes, and that the region of HS-4 to HS-40 already contains all the upstream cis-elements needed for regulating human alpha-like globin genes.

Gene order in human alpha-globin locus is required for their temporal specific expressions.

The human alpha-globin cluster represents a unique model of transcriptional regulation and provides challenges to the current understanding of interactions between distal and proximal regulatory elements. Although the gene proximal regions are believed to possess almost all the necessary elements for temporal and spatial specificity of gene transcription, it is still not clear whether the relative distance of embryonic zeta- and fetal/adult alpha-genes to their distal regulatory element alpha-URE plays any role in transcriptional switching. To investigate the role of gene order in regulating temporal expression, we inverted the entire structure gene region of human alpha-globin locus in a BAC clone bringing alpha-genes closest to alpha-URE and zeta-gene the farthest away. Expression analysis of the reverted locus in transgenic mice showed that alpha-globin genes, now relocated closer to alpha-URE, maintained their expression levels through all developmental stages. However, the zeta-globin gene suffered a total loss at both embryonic and fetal/adult stages. It indicates that proximal location of zeta-globin gene to alpha-URE is necessary for its normal embryonic expression and necessary to prevent embryonic expression of the alpha-globin gene. We proved that, in the human alpha-globin gene cluster, the normal order of structural genes relative to alpha-URE plays a crucial role in the regulation of developmental switching.

The binding of the ubiquitous transcription factor Sp1 at the locus control region represses the expression of beta-like globin genes.

To investigate the function of transcription factor Sp1 in beta-like globin gene activation, we analyzed the recruitment of Sp1, fetal Krüppel-like factor 2 (FKLF2), and related factors at the human beta-globin locus in a human fetal liver and mouse erythroleukemia hybrid cell (A181gamma cell) that contains a single copy of human chromosome 11. Sp1 binds at the GT boxes of the cis-elements throughout the beta-locus, but it is phosphorylated and lost over DNase I hypersensitive site (HS)2, HS3, HS4, and the human beta-globin gene promoter after A181gamma cell differentiation. The binding of FKLF2 at HS2 and HS3 was unchanged. Histone deacetylase 1, which could be recruited by Sp1, is also lost over HS2 and HS3 after differentiation, resulting in the acetylation of histones 3 and 4 across the human beta-globin locus. We previously detected in vivo GT footprints over the beta-globin locus after A181gamma differentiation. Here, we report that after differentiation, the p300/CREB-binding protein-associated factor is recruited by FKLF2 to the locus control region to acetylate histones 3 and 4 at the human beta-globin gene locus. Our results suggest that Sp1 is an inhibitor of beta-like globin gene transcription during erythroid terminal differentiation. Its phosphorylation and release allow the erythroid-specific FKLF2 or erythroid Krüppel-like factor to interact with other erythroid-specific transcription factors to initiate the transcription of beta-like globin genes.