Low molecular weight polyethylenimine-graft-Tween 85 for effective gene delivery: synthesis and in vitro characteristics

The development of safe and efficient gene delivery systems is still a challenge for successful gene therapy. In this work, low molecular weight polyethylenimine (PEI 2K) was modified by Tween 85, which bears three oleate chains. Tween 85 modified PEI 2K (TP) could condense DNA efficiently, and TP/DNA complexes (TPCs) showed high resistance to salt-induced aggregation and enzymatic degradation. In addition, TP did not show the obvious cytotoxicity. The introduction of Tween 85 led to a significant increase in the cellular uptake of complexes with higher transfection efficiency, which was strongly inhibited by the addition of free Tween 85 in MCF-7/ADR cells, but not in MCF-7 cells. These results indicated that TP could be a potentially safe and effective copolymer for gene delivery, and TPCs could be taken up mainly by Tween 85-mediated endocytosis in MCF-7/ADR cells.     

Different genetic patterns in avian Toll-like receptor (TLR)5 genes

Toll-like receptors (TLRs) mediate immune response via recognition of pathogen-associated molecular patterns (PAMPs), thus play important roles in host defense. Polymorphisms of TLR5 may affect their recognition of bacterial flagellin, leading to varied host resistance to pathogenic infections. Here, we cloned TLR5 genes from Common Pheasant, Guinea fowl and 9 Chicken breeds and analyzed their sequences. The open reading frames of TLR5 were sequenced. Amino acid analysis indicated that TLR5 from Chicken breeds shared 99.4–99.9% homology. The amino acid homology of TLR5 ranged from 92.1 to 92.5% between Chickens and Guinea fowl, 95.7–96.1% between Chickens and Turkey, 94.3–94.7% between Chickens and Common Pheasant, and 79.9–80.1% between Chickens and Zebra-finch. Different genetic patterns were determined among Chickens, Common Pheasant, Guinea fowl, Turkey and Zebra-finch. It was found that there were 92 amino acid polymorphic sites, among which 5 sites in chicken TLR5, 63 sites in Guinea fowl TLR5 and 44 sites in Common Pheasant TLR5. Our data indicate that the positive Darwinian selection occurred in avian TLR5 genes since frequency of non-synonymous (d _N ) > frequency of synonymous (d _S ). These results also demonstrate that avian TLR5 genes are polymorphic among avian breeds, suggesting a varied resistance among breeds of avian. This information might be of help to improve the health of avian by breeding and vaccination.     

Periodic orbits near heteroclinic cycles in a cyclic replicator system

A species is semelparous if every individual reproduces only once in its life and dies immediately after the reproduction. While the reproduction opportunity is unique per year and the individual’s period from birth to reproduction is just n years, the individuals that reproduce in the ith year (modulo n) are called the ith year class, i = 1, 2, . . . , n. The dynamics of the n year-class system can be described by a differential equation system of Lotka–Volterra type. For the case n = 4, there is a heteroclinic cycle on the boundary as shown in previous works. In this paper, we focus on the case n = 4 and show the existence, growth and disappearance of periodic orbits near the heteroclinic cycle, which is a part of the conjecture by Diekmann and van Gils (SIAM J Appl Dyn Syst 8:1160–1189, 2009). By analyzing the Poincaré map near the heteroclinic cycle and introducing a metric to measure the size of the periodic orbit, we show that (i) when the average competitive degree among subpopulations (year classes) in the system is weak, there exists an asymptotically stable periodic orbit near the heteroclinic cycle which is repelling; (ii) the periodic orbit grows in size when some competitive degree increases, and converges to the heteroclinic cycle when the average competitive degree tends to be strong; (iii) when the average competitive degree is strong, there is no periodic orbit near the heteroclinic cycle which becomes asymptotically stable. Our results provide explanations why periodic solutions expand and disappear and why all but one subpopulation go extinct.     

Bombyx mori DNA/RNA non-specific nuclease: expression of isoforms in insect culture cells, subcellular localization and functional assays

A DNA/RNA non-specific alkaline nuclease (BmdsRNase) was isolated from the digestive juice of Bombyx mori. While originally reported to be produced by the midgut only, in this project it was found that the mRNA of this enzyme was also expressed in the epidermis, fat body, gut, thoracic muscles, Malpighian tubules, brain, and silk glands of 5th instar larvae, indicating additional functions to its reported role in nucleic acid digestion in the midgut. In order to study the functional properties of BmdsRNase, three pEA-BmdsRNase expression constructs were generated, characterized by presence or absence of a signal peptide and a propeptide, and used for expression in lepidopteran Hi5 tissue culture cells. Western blot indicated that these different forms of BmdsRNase protein were not secreted into the growth medium, while they were detected in the pellets and supernatants of Hi5 cell extracts. Nucleic acids cleavage experiments indicated that full-length BmdsRNase could digest dsRNA and that the processed form (absence of signal peptide and propeptide) of BmdsRNase could degrade both DNA and dsRNA in Hi5 cell culture. Using a reporter assay targeted by transfected homologous dsRNA, it was shown that the digestive property of the processed form could interfere with the RNAi response. Immunostaining of processed BmdsRNase protein showed asymmetric localization in the cellular cytoplasm and co-localization with Flag-tagged Dicer-2 was also observed. In conclusion, our in vitro studies indicated that intracellular protein isoforms of BmdsRNase can be functional and involved in the regulation of nucleic acid metabolism in the cytoplasm. In particular, because of its propensity to degrade dsRNA, the enzyme might be involved in the innate immune response against invading nucleic acids such as RNA viruses.     

Mapping and fine mapping of quantitative trait loci for the number of vertebrae in a White Duroc × Chinese Erhualian intercross resource population

The number of vertebrae is associated with body size and meat production in pigs. To identify quantitative trait loci (QTL) for the number of vertebrae, phenotypic values were measured in 1029 individuals from a White Duroc × Chinese Erhualian intercross F₂ population. A whole genome scan was performed with 194 microsatellite markers in the F₂ population. Four genome‐wide significant QTL and eight chromosome‐wide significant QTL for the number of vertebrae were identified on pig chromosomes (SSC) 1, 2, 6, 7, 10 and 12. The most significant QTL was detected on SSC7 with a confidence interval of 1 cM, explaining 42.32% of the phenotypic variance in the thoracic vertebral number. The significant QTL on SSC1, 2 and 7 confirmed previous reports. A panel of 276 animals representing seven Western and Chinese breeds was genotyped with 34 microsatellite markers in the SSC7 QTL region. No obvious selective sweep effect was observed in the tested breeds, indicating that intensive selection for enlarged body size in Western commercial breeds did not wipe out the genetic variability in the QTL region. The Q alleles for increased vertebral number originated from both Chinese Erhualian and White Duroc founder animals. A haplotype block of approximately 900 kb was found to be shared by all Q‐bearing chromosomes of F₁ sires except for one distinct Q chromosome. The critical region harbours the newly reported VRTN gene associated with vertebral number. Further investigations are required to confirm whether VRTN or two other positional candidate genes, PROX2 and FOS, cause the QTL effect.     

The production of soluble C-type lectin-like receptor 2 is a regulated process

C-type lectin-like receptor 2 (CLEC-2) is a newly identified type II transmembrane protein belonging to the C-type lectin family molecules, which acts as a cell-surface receptor for snake venom toxin rhodocytin and tumor antigen podoplanin. We previously demonstrated that the full-length mouse CLEC-2 (mCLEC-2) can be cleaved into soluble form. Elevated levels of soluble forms of membrane proteins in circulating blood may reflect increased expression of membrane proteins and disease activities. In the present study, we clarified the domain and sites contributing to the production of soluble mCLEC-2. The shedding process can be positively regulated by protein kinase C (PKC). Moreover, we explored the possibility that human CLEC-2 (hCLEC-2) may also be proteolyticly cleaved and released as a soluble form. We have observed that the production of soluble hCLEC-2 could be induced by phorbol ester (PMA) in cells stably transfected with hCLEC-2 cDNA. Further studies may explore therapeutic and diagnostic applications of soluble hCLEC-2 in platelet-related diseases.     

Primary hyperparathyroidism and familial hypocalciuric hypercalcemia: relationships and clinical implications

ObjectiveTo discuss the unusual occurrence of both familial hypocalciuric hypercalcemia (FHH) and primary hyperparathyroidism in the same patient and to explore potential mechanisms of association and issues related to clinical management.MethodsWe discuss the diagnosis, compare the clinical presentations of FHH and primary hyperparathyroidism, review the literature regarding patients who have presented with both disorders, and discuss management considerations. We also describe 2 patients who have both FHH (confirmed by genetic testing for a mutation in the gene encoding the calcium-sensing receptor [CASR]) and primary hyperparathyroidism.ResultsThe occurrence of both FHH and primary hyperparathyroidism in the same patient has been reported in a few cases, including 2 patients described here, one of whom was documented to have a novel CASR mutation. Inthose with clinical sequelae of hyperparathyroidism, parathyroidectomy has led to reduction, but not normalization, of serum calcium levels.ConclusionsThe coexistence of FHH and primary hyperparathyroidism should be considered in patients with hypercalcemia, hypophosphatemia, frankly elevated parathyroid hormone levels, and low urinary calcium excretion. Genetic testing for inactivating CASR gene mutations can confirm the diagnosis of FHH. Although surgical intervention does not resolve hypercalcemia, it may be beneficial by reducing the degree of hypercalcemia, alleviating the symptoms, and preventing potential complications of hyperparathyroidism. (Endocr Pract. 2012;18:412-417)     

Inhibition of type I interferon production via suppressing IKK-gamma expression: a new strategy for counteracting host antiviral defense by influenza A viruses?

Blockage of the induction of type I interferons (IFNs) is essential for the success of influenza virus proliferation in host cells. Several molecular mechanisms by which influenza viruses inhibit IFN induction have been characterized. Here we report a potentially new strategy influenza viruses employ to inhibit IFN production during viral infection. Through a two-dimensional gel electrophoresis based proteomic approach, we found that the expression of IκB kinase-gamma (IKKγ) was suppressed by influenza A virus infection in human lung epithelial A549 cells. Silencing of cellular IKKγ by small interfering RNA led to enhanced replication of influenza viruses. Concomitantly, overexpression of IKKγ resulted in increased production of IFNα/β, whereas influenza virus infection completely eliminated the IKKγ-overexpression-induced production of IFNα/β. Our results suggest that IKKγ and influenza virus are mutually inhibitory, and influenza viruses may inhibit IFN production through suppressing the expression of IKKγ during viral infection.