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Highly specialized myrmecophagy (ant- and termite-eating) has independently evolved multiple times in species of various mammalian orders and represents a textbook example of phenotypic evolutionary convergence. We explored the mechanisms involved in this unique dietary adaptation and convergence through multi-omic analyses, including analyses of host genomes and transcriptomes, as well as gut metagenomes, in combination with validating assays of key enzymes’ activities, in the species of three mammalian orders (anteaters, echidnas and pangolins of the orders Xenarthra, Monotremata and Pholidota, respectively) and their relatives. We demonstrate the complex and diverse interactions between hosts and their symbiotic microbiota that have provided adaptive solutions for nutritional and detoxification challenges associated with high levels of protein and lipid metabolisms, trehalose degradation, and toxic substance detoxification. Interestingly, we also reveal their spatially complementary cooperation involved in degradation of ants’ and termites’ chitin exoskeletons. This study contributes new insights into the dietary evolution of mammals and the mechanisms involved in the coordination of physiological functions by animal hosts and their gut commensals.

BACKGROUND A prolonged exposure to ketamine triggers significant neurodegeneration and long-term neurocognitive deficits in the developing brain. Monosialotetrahexosylganglioside (GM1) can limit the neuronal damage from necrosis and apoptosis in neurodegenerative conditions. We aimed to assess whether GM1 can prevent ketamine-induced developmental neurotoxicity. METHODS Postnatal day 7 (P7) rat pups received 5 doses of intraperitoneal ketamine (20 mg/kg per dose) at 90-minute intervals for 6 hours. Cognitive functions, determined by using Morris water maze (MWM) including escape latency (at P32-36) and platform crossing (at P37), were compared among the ketamine-exposed pups treated with or without exogenous GM1 (30 mg/kg; n = 12/group). The effect of GM1 on apoptosis in hippocampus was determined by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL) staining and activated caspase 3 measurement. The hippocampal expression of brain-derived neurotrophic factor (BDNF), along with the phosphorylation of protein kinase B (AKT) and extracellular signal-related kinases 1 and 2 (ERK1/2), was detected by western blotting (n = 6/group). Anti-BDNF antibody (2 μg per rat) administered before GM1 treatment was applied to determine the neuroprotective mechanisms of GM1. RESULTS The rats receiving ketamine exposure experinced cognitive impairment in MWM test compared to the control rats, indicated by prolonged escape latency at P34 (P = .006), P35 (P = .002), and P36 (P = .005). However, in GM1-pretreated rats, ketamine exposure did not induce prolonged escape latency. The exogenous GM1 increased the platform-crossing times at P37 (3.00 ± 2.22 times vs 5.40 ± 1.53 times, mean ± standard deviation; P = .041) and reduced the hippocampal TUNEL-positive cells and cleaved-caspase 3 expression in ketamine-exposed young rats. Ketamine decreased BDNF expression and phosphorylation of AKT and ERK in the hippocampus, whereas exogenous GM1 blocked these ketamine-caused effects. However, for the ketamine-exposed rat pups receiving exogenous GM1, compared to immunoglobulin Y (IgY) isotype control, the BDNF-neutralizing antibody treatment counteracted the exogenous GM1-induced improvement of the escape latency at P36 (41.32 ± 12.37 seconds vs 25.14 ± 8.97 seconds, mean ± standard deviation; P = .036), platform-crossing times at P37 (2.16 ± 1.12 times vs 3.92 ± 1.97 times, mean ± standard deviation; P < .036), apoptotic activity, as well as AKT and ERK1/2 phosphorylation in the hippocampus of ketamine-challenged young rats. CONCLUSIONS Our data suggest that the exogenous GM1 acts on BDNF signaling pathway to ameliorate the cognitive impairment and hippocampal apoptosis induced by ketamine in young rats. Our study may indicate a potential use of GM1 in preventing the cognitive deficits induced by ketamine in the young per se.

Monosialoganglioside 1 (GM1) is the main ganglioside subtype and has neuroprotective properties in the central nervous system. In this study, we aimed to determine whether GM1 alleviates neurotoxicity induced by moderate and high concentrations of propofol combined with remifentanil in the immature central nervous system. Hippocampal neural stem cells were isolated from newborn Sprague-Dawley rats and treated with remifentanil (5, 10, 20 ng/mL) and propofol (1.0, 2.5, 5.0 μg/mL), and/or GM1 (12.5, 25, 50 μg/mL). GM1 reversed combined propofol and remifentanil-induced decreases in the percentage of 5-bromodeoxyuridine(+) cells and also reversed the increase in apoptotic cell percentage during neural stem cell proliferation and differentiation. However, GM1 with combined propofol and remifentanil did not affect β-tubulin(+) or glial fibrillary acidic protein(+) cell percentage during neural stem cell differentiation. In conclusion, we show that GM1 alleviates the damaging effects of propofol combined with remifentanil at moderate and high exposure concentrations in neural stem cells in vitro, and exerts protective effects on the immature central nervous system.

Interferon-alpha2b (IFN-alpha2b) and human serum albumin (HSA) fusion protein (IFN-alpha2b-HSA) is a promising long acting formulation of IFN-alpha2b for the treatment of hepatitis C. However, accelerated mechanical and thermal stress tests revealed that IFN-alpha2b-HSA was prone to disulfide-linked aggregation. The formation of aggregates was associated with an increase in immunogenicity in mice. The addition of non-ionic surfactant Tween 80 increased the stability of IFN-alpha2b-HSA against agitation, but its thermal stability was not improved. Moreover, Tween 80 prompted the aggregation of IFN-alpha2b-HSA during quiescent storage. To increase the stability of IFN-alpha2b-HSA, the unpaired cysteine residue in this fusion protein was substituted with serine by site-directed mutagenesis. The resultant fusion protein was designated as IFN-alpha2b-HSA(C34S). IFN-alpha2b-HSA(C34S) had significant higher stability over IFN-alpha2b-HSA, which was evidenced by the facts that after agitation for 72 h or incubation at 60 degrees C for 2 h, more than 90% of IFN-alpha2b-HSA(C34S) remained monomeric. Consistent with its improved stability, the immunogenicity of IFN-alpha2b-HSA(C34S) increased less significantly after agitation. Pharmacokinetics studies in rats revealed that both fusion proteins had similar pharmacokinetic behavior, both with a half-life of about 50 h.

Onconase, an RNAse extracted from embryos of the Northern leopard frog (Rana pipiens), is in a confirmatory phase IIIb clinical trial for the treatment of unresectable malignant mesothelioma. Because the current purification process for onconase is cumbersome and laborious, the development of more efficient and cost-effective alternative sources is imperative. In this study, we assessed the potential of Pichia pastoris as an expression host for the large-scale production of onconase. Because of its specific N-terminal structure, active onconase with a correct N-terminus could not be secreted by an alpha-mating factor (alpha-MF)-prepro secretion signal, and an alpha-MF-pre secretion signal should be used instead. Onconase accumulated to a high concentration (about 300 and 150 mg L(-1) for glycosylated onconase and aglycosylated mutein, respectively) in high cell density fermentation, and was purified to homogeneity with high yields (56% for glycosylated onconase and 67% for aglycosylated mutein) by a simple purification process consisting of cation exchange chromatography and size exclusion chromatography. In vitro activity assays revealed that glycosylation decreased both the RNAse activity and the cytotoxic activity of onconase. The high expression level and subsequent facile purification process make P. pastoris an efficient and cost-effective host for the large-scale production of onconase.

Expression of recombinant protein HSA-AX15(R13K) in Pichia pastoris GS115 strain produced both the intact protein and its two degradation products with molecular weights of around 43 kDa and 66.2 kDa, respectively. To reduce or avoid the degradation, a modified P. pastoris GS115 stain, in which YPS1 gene was disrupted, was constructed via homologous recombination and used as a host strain for the HSA-AX15(R13K) expression. After 60 h of induction during culture, it was found that the degradation product of around 66.2 kDa was reduced significantly in the supernatant of yps1-disrupted strain compared with that in the supernatant of wild-type strain. By the Western blot analysis of culture supernatants from wild-type and yps1-disrupted strains expressing HSA-AX15(R13K), the significant improvement was also seen in the degradation product of around 43 kDa. Comparison of cell growth between the two strains demonstrated a similar growth tendency, thereby indicating that the disruption of YPS1 gene has no effect on the normal physiology of GS115 strain. Following induction for 60 h, the yield of intact HSA-AX15(R13K) in the yps1 disruptant was three-fold higher than that in the wild-type strain. Therefore, such a P. pastoris mutant deficient in YPS1 activity is suitable for the high-level expression of recombinant protein HSA-AX15(R13K).

In this study, we assessed the potential of PMR1-disrupted Pichia pastoris (Pppmr1) expressing human serum albumin and interferon alpha 2b fusion protein (HSA-IFN-alpha 2b) in large-scale fermentation. The high osmotic pressure of standard basal salts medium (BSM) was detrimental to the growth and viability of Pppmr1. HSA-IFN-alpha 2b was secreted into a supernatant with a concentration of up to 112 mg/L after 20 h of induction and then began to decline. In vitro stability tests indicated that the disappearance of HSA-IFN-alpha 2b was ascribed to proteolytic degradation. Decreasing the salt concentration of BSM medium to one quarter of the original formula improved the growth and viability of Pppmr1. As a result of reduced cell lysis and protease release, HSA-IFN-alpha 2b was stable in the supernatant, which enabled a longer production phase (30 h) and a higher expression level (215 mg/L). Lowering the culture temperature to 20 degrees C increased the cell viability during carbon source transition and alleviated the oxygen and methanol limitation, which extended the production phase to 40 h and increased the expression level to 680 mg/L. The addition of 2% Soytone prolonged the production phase to 60 h and increased the expression level to 1,260 mg/L, which was more than tenfold higher than that of Pppmr1 cultured under the conditions recommended by Invitrogen.

Albuferon is a novel long-acting interferon resulted from the direct genetic fusion of human albumin and interferon-alpha2b (HSA-IFN-alpha2b). Albuferon, co-developed by Human Genome Sciences Inc. and Novartis, is currently in late stage development for the treatment of hepatitis C. It was unexpected that HSA-IFN-alpha2b secreted from Pichia pastoris migrated as doublets on non-reducing SDS-PAGE and was prone to form covalent aggregates in aqueous solution. The heterogeneity and instability of HSA-IFN-alpha2b lowered its recovery rate to about 10% and necessitated lyophilized formulation. Site-directed mutagenesis revealed that the heterogeneity and instability of HSA-IFN-alpha2b was caused by the incomplete disulfide bridge formation between Cys1 and Cys98 of IFN-alpha2b. To alleviate the structural perturbation of IFN-alpha2b by HSA, IFN-alpha2b-HSA fusion protein, in which IFN-alpha2b was located at the N-terminus, was created. IFN-alpha2b-HSA was shown to be homogeneous and stable at 37 degrees C for at least 10 days. The improved homogeneity and stability of IFN-alpha2b-HSA increased the recovery rate by 2.5-fold and made the development of stable solution formulation possible. In vitro antiviral assays showed that both fusion proteins retained the activity of IFN-alpha2b, and the EC(50) of HSA-IFN-alpha2b, and IFN-alpha2b-HSA was calculated to be 120+/-12.5, and 160+/-1 1.3ng/ml, respectively. The increased recovery rate and the possibility of solution formulation of IFN-alpha2b-HSA may compensate for its slightly decreased in vitro activity, and makes it to be a promising therapeutic agent that deserves further evaluation.

In Pichia pastoris, secretory proteins are folded and assembled in the endoplasmic reticulum (ER). However, upon introduction of foreign proteins, heterologous proteins are often retained in the cytoplasm or in the ER as a result of suboptimal folding conditions, leading to protein aggregation. The Hsp70 and Hsp40 chaperone families in the cytoplasm or in ER importantly regulate the folding and secretion of heterologous proteins. However, it is not clear which single chaperone is most important or which combination optimally cooperates in this process. In the present study we evaluated the role of the chaperones Kar2p, Sec63, YDJ1p, Ssalp, and PDI from Saccharomyces cerevisiae. We found that the introduction of Kar2p, Ssalp, or PDI improves protein secretion 4-7 times. In addition, we found that the combination chaperones of YDJlp/ PDI, YDJlp/Sec63, and Kar2p/PDI synergistically increase secretion levels 8.7, 7.6, and 6.5 times, respectively. Therefore, additional integration of chaperone genes can improve the secretory expression of the heterologous protein. Western blot experiments revealed that the chaperones partly relieved the secretion bottleneck resulting from foreign protein introduction in P. pastoris. Therefore, the findings from the present study demonstrate the presence of a network of chaperones in vivo, which may act synergistically to increase recombinant protein yields.

Contactin associated protein (Caspr), an adhesion molecule, plays roles in formation of paranodal junctions in myelinated axons, neurite outgrowth, synaptic plasticity in nervous system. Here we have shown a novel function of Caspr in pathogenesis of Alzheimer's disease (AD). Caspr distributes around amyloid plaques in APP/PS1 mice. Levels of Caspr increase in the cerebral cortex of 7-month-old APP/PS1 mice comparing to wild-type littermates. Caspr decreased protein levels of APP in both HEK-293 cells stably transfected with Indiana mutant APP (V717F; HEK-APP) and CHO cells which express endogenous APP, while it did not alter mRNA levels of APP. Furthermore, Caspr co-localizes and interacts with APP. Amyloid-β (Aβ) 40 and Aβ42 generation were also reduced in HEK-APP cells by Caspr overexpression.

The characteristics, such as morphologic and phenotypic characteristics and neural transdifferentiation ability, of mesenchymal stromal cells (MSC) derived from different origins have yet to be reported for cases isolated from the same individual.The proliferation capacity, secretion ability of neurotrophins (NT) and neural differentiation ability in rat MSC isolated from bone marrow (BMSC) and adipose tissue (ADSC) were compared from the same animal.The ADSC had a significantly higher proliferation capacity than BMSC according to cell cycle and cumulative population doubling analyses. The proportion of cells expressing neural markers was greater in differentiated ADSC than in differentiated BMSC. Furthermore, the single neurosphere derived from ADSC showed stronger expansion and differentiation abilities than that derived from BMSC. The findings from Western blot lent further support to the immunocytochemical data. The mRNA and protein levels of nerve growth factor (NGF) and brain-derived growth factor (BDNF) expressed in ADSC were significantly higher than those in BMSC at different stages before and following induction.Our study suggests that the proliferation ability of ADSC is superior to that of BMSC. Furthermore, differentiated ADSC expressed higher percentages of neural markers. As one possible alternative source of BMSC, ADSC may have wide potential for treating central nervous system (CNS) diseases.

γ-Aminobutyric acid (GABA) is the principle inhibitory neurotransmitter in adult mammalian brain. GABA receptors B subtype (GABA(B)Rs) are abundantly expressed at presynaptic and postsynaptic neuronal structures in the rat ventrolateral periaqueductal gray (PAG), an area related to pain regulation. Activation of GABA(B)Rs by baclofen, a selective agonist, induces presynaptic inhibition by decreasing presynaptic glutamate release. At the same time, baclofen induces a postsynaptic inhibitory membrane current or potential. We here report that in the ventrolateral PAG, the postsynaptic inhibition is mediated by activation of G protein-coupled inwardly rectifying K(+) (GIRK) channels. Blockade of K(+) channels largely prevents postsynaptic action of baclofen. In contrast, presynaptic inhibition of baclofen is insensitive to K(+) channel blockade. The data indicate that potassium channels play different roles in GABA(B)R-mediated presynaptic and postsynaptic inhibition on PAG neurons.

In an attempt to increase the secretion capacity of Pichia pastoris (Pp), PpPMR1 gene was disrupted with GS115 as parent strain, and the resultant mutant was designated as Pppmr1. Pppmr1 displayed a Ca2+-dependent growth defect, which was consistent with the PMR1 mutation in other yeasts. HSA-L5-IFNalpha2b, a human serum albumin (HSA) and inferferon-alpha2b (IFNalpha2b) fusion protein with a flexible linker of 5 amino acid residues, was employed as a reporter to study the effects of PpPMR1 disruption on the secretion of heterologous protein. Because of its decreased viability after induction, Pppmr1 secreted more HSA-L5-IFNalpha2b only during the early phase (the first 15 hours) of induction. Although HSA-L5-IFNalpha2b secreted from GS115 and Pppmr1 had similar antiviral activity, the latter was heterogeneous (migrated as doublets on non-reducing SDS-PAGE) and unstable (prone to aggregation at neutral to mild alkaline pH). Site-directed mutagenesis revealed that the heterogeneity of HSA-L5-IFNalpha2b secreted from Pppmr1 was originated from the incomplete disulphide bridge pairing between Cys1 and Cys98 of IFNalpha2b. To be secreted homogeneously from Pppmr1 and to be stable in aqueous solution, the linker of the fusion protein should be extended to 10 amino acid residues.

Previous studies in our laboratory have shown that when the N-terminus of interferon-alpha2b (IFN-alpha2b) was directly fused of to the C-terminus of human serum albumin (HSA), the resultant fusion protein (HSA-IFN-alpha2b) was heterogeneous (migrated as doublets on non-reducing SDS-PAGE) and unstable (prone to form covalent aggregates). The heterogeneity and instability of HSA-IFN-alpha2b was ascribed to the structural disturbance between HSA and IFN-alpha2b. To alleviate such structural disturbance, linkers with different lengths (1, 2, 5, 10 amino acid residues) or different conformation (flexible linker (FL, GGGGS), rigid linker (RL, PAPAP) or helix-forming linker (HL, AEAAAKEAAAKA)) were inserted between HSA and IFN-alpha2b. It was demonstrated that linker with 5 amino acid residues was sufficient to separated HSA and IFN-alpha2b effectively, as fusion protein with this linker migrated as single band on non-reducing SDS-PAGE. The fusion proteins with FL, RL and HL linkers were purified to homogeneity with yields of 20%, while the recovery rate of HSA-IFN-alpha2b was only 10%. Accelerated thermal stress tests showed that in contrast to HSA-IFN-alpha2b, fusion proteins with FL, RL and HL linkers were free of aggregates after stored at 37 degrees C for 10 days. Stability tests also revealed that fusion proteins with FL, RL and HL linkers had different susceptibility to hydrolysis, with HSA-RL-IFN-alpha2b being the least susceptible to hydrolysis at pH 6 and 7. Activity assay revealed that the insertion of FL, RL and HL linkers increased the anti-viral activity of fusion protein by 39%, 68% and 115%, respectively.

Many studies have provided evidence that heat shock protein 65 (Hsp65) can elicit potent specific cellular adaptive immune responses (e.g. CD8(+) cytotoxic T-cell effectors or classic CTLs) based on their ability to chaperone antigenic peptides. Hsp65 is thus an effective carrier for heterologous peptide epitopes for therapeutic vaccines against cancer or chronic infectious diseases. The core antigen of hepatitis B virus (HBcAg) is extremely immunogenic, and functions as both a T-cell-dependent and a T-cell-independent antigen. Therefore, HBcAg may be a promising candidate target for therapeutic vaccine control of chronic HBV infection. Here, a chimeric protein, Hsp65Bc, was created by fusing the HBcAg sequence to the carboxyl terminus of the Hsp65 sequence in E. coli. Analysis of its antigenicity and immunogenicity revealed that HBc epitopes are surface accessible. Hsp65Bc induced moderate anti-HBc immune responses as well as a strong specific T-cell response in BALB/c mice. These results indicate that Hsp65Bc may have potential as a vaccine for treatment of HBV chronic infection.

To study the secretory expression of human hepatocyte growth factor (hdHGF) gene in Pichia pastoris.The full-length gene of human cDNA encoding the deleted variant of hdHGF was cloned by RT-PCR and overlapping-fragment PCR technique using mRNA of human placenta as a template. The cloned hdHGF cDNA was inserted into the Escherichia coli-yeast shuttle vector of pPIC9. The constructed plasmid, pPIC9-hdHGF, was transformed into the GS115 cells of the methylotrophic yeast, P pastoris, using a chemical method. The Mut(+ ) transformants were screened to obtain high-expression strains by the test and analysis of expressed products of shake-flask culture. A secretory form of rhdHGF was made with the aid of the leader peptide sequence of Saccharomyces cerevisiae alpha-factor.The expressed products, which showed a band of molecular mass of about 80 ku, were observed on 15% SDS-PAGE and identified by Western blotting and N-terminal amino acid sequencing. In the high cell density culture of 5 L fermentor by fed-batch culture protocol, the cell biomass was reached at approximately 135 g (DCW)/L. The productivity of secreted total supernant protein concentration attained a high-level expression of more than 8.0 g/L and the ratio of rhdHGF band area was about 12.3% of the total band area scanned by SDS-PAGE analysis, which estimated that the product of rhdHGF was 500-900 mg/L.The P pastoris system represents an attractive tool of generating large quantities of hdHGF for both research and industrial purposes.

AIM: To construct two kinds of anti-gastrin immunogen based on P64K protein from Neisseria meningitids and to compare their immunogenic effect. METHODS: G17P64K gene was cloned and ligated into pET28a plasmid, then transformed into BL21(DE3). After inoculation of LB medium and IPTG induction, the recombinant protein was solubly expressed at a high level.The purification of G17P64K fusion protein was similar to that of P64K. An initial step of purification consisting of 30 % saturated ammonium sulfate precipitation was done. Additional fine optimizations included phenyl-sepharose, G200 Sephadex gel filtration and Q-sepharose anion exchanger chromatography. Highly purified protein was obtained and sequenced at the N-terminal amino acid residues. Polypeptide was synthesized by Fmoc solid phase chemical method and cross-linked to carrier protein P64K and DT mutant by MBS method and then the rabbit anti-gastrin 17 antibody was prepared by immunizing rabbit with cross-linked and fused protein. The titer and the activity in vitro of antibody were assessed. RESULTS: G17P64K gene and the recombinant bacteria were obtained. After four steps purification, protein sample that has the purity above 90 % was achieved. At the 84th day after the first immunization, the titer of antibody against cross-linked protein reached 51 200. Evaluation of the antibody in vitro manifested that it had a high inhibitory activity on the growth of tumor cell SW480. CONCLUSION: The P64K-polypeptide cross-linked immunogen immunized rabbit and achieved a higher titer antibody against gastrin 17 than the G17P64K fusion protein immunogen, which could inhibit the growth of the tumor cell SW480.

AIM: To observe the reversal effects of wide-type p53 gene on multi-drug resistance to 5-FU (LOVO/5-FU). METHODS: After treatment with Ad-p53, LOVO/5-FU sensitivity to 5-Fu was investigated using tetrazolium dye assay. Multidrug resistance gene-1 (MDR1) gene expression was assayed by semi-quantitative reverse transcription-polymerase chain reaction and the expression of p53 protein was examined by Western blotting. RESULTS: The reversal activity after treatment with wide-type p53 gene was increased up to 4.982 fold at 48 h. The expression of MDR1 gene decreased significantly after treatment with wide-type p53 gene, and the expression of p53 protein lasted for about 5 d, with a peak at 48 h, and began to decrease at 72 h. CONCLUSION: Wide-type p53 gene has a remarkable reversal activity for the high expression of MDR1 gene in colorectal cancers. The reversal effects seem to be in a time dependent manner. It might have good prospects in clinical application.