C-terminal mini-PEGylation of a marine peptide N6 had potent antibacterial and anti-inflammatory properties against Escherichia coli and Salmonella strains in vitro and in vivo.

BACKGROUND: Enteropathogenic Escherichia coli and Salmonella pullorum are two important groups of zoonotic pathogens. At present, the treatment of intestinal pathogenic bacteria infection mainly relies on antibiotics, which directly inhibit or kill the pathogenic bacteria. However, due to long-term irrational, excessive use or abuse, bacteria have developed different degrees of drug resistance. N6, an arenicin-3 derivative isolated from the lugworm, has potent antibacterial activity and is poorly resistant to enzymatic hydrolysis and distribution in vivo. Polyethylene glycol (PEG) is an extensively studied polymer and commonly used in protein or peptide drugs to improve their therapeutic potential. Here, we modified the N-/C-terminal or Cys residue of N6 with liner PEGn of different lengths (n = 2, 6,12, and 24), and the effects of PEGylation of N6 on the stability, toxicity, bactericidal mechanism, distribution and efficacy were investigated in vitro and in vivo. RESULTS: The antimicrobial activity of the peptide showed that PEGylated N6 at the C-terminus (n = 2, N6-COOH-miniPEG) had potent activity against Gram-negative bacteria; PEGylated N6 at the N-terminus and Cys residues showed low or no activity with increasing lengths of PEG. N6-COOH-miniPEG has higher stability in trypsin than the parent peptide-N6. N6-COOH-miniPEG significantly regulated cytokine expression in lipopolysaccharides (LPS)-induced RAW 264.7 cells, and the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1ß were reduced by 31.21%, 65.62% and 44.12%, respectively, lower than those of N6 (-0.06%, -12.36% and -12.73%); N6-COOH-miniPEG increased the level of IL-10 (37.83%), higher than N6 (-10.21%). The data indicated that N6-COOH-miniPEG has more potent anti-inflammatory and immune-regulatory effect than N6 in LPS-stimulated RAW 264.7 cells. N6-COOH-miniPEG exhibited a much wider biodistribution in mice and prolonged in vivo half-time. FITC-labeled N6-COOH-miniPEG was distributed throughout the body of mice in the range of 0.75 - 2 h after injection, while FITC-labeled N6 only concentrated in the abdominal cavity of mice after injection, and the distribution range was narrow. N6-COOH-miniPEG improved the survival rates of mice challenged with E. coli or S. pullorum, downregulated the levels of TNF-α, IL-6, IL-1ß and IL-10 in the serum of LPS-infected mice, and alleviated multiple-organ injuries (the liver, spleen, kidney, and lung), superior to antibiotics, but slightly inferior to N6. CONCLUSIONS: The antibacterial activity, bactericidal mechanism and cytotoxicity of N6-COOH-miniPEG and N6 were similar. N6-COOH-miniPEG has a higher resistance to trysin than N6. The distribution of N6-COOH-miniPEG in mice was superior to that of N6. In exploring the modulatory effects of antimicrobial peptides on cytokines, N6-COOH-miniPEG had stronger anti-inflammatory and immunomodulatory effects than N6. The results suggested that C-terminal PEGylated N6 may provide an opportunity for the development of effective anti-inflammatory and antibacterial peptides.

A novel PIK3R1 mutation of SHORT syndrome in a Chinese female with diffuse thyroid disease: a case report and review of literature.

BACKGROUND: SHORT syndrome is a rare genetic disease named with the acronyms of short stature, hyper-extensibility of joints, ocular depression, Rieger anomaly and teething delay. It is inherited in an autosomal dominant manner confirmed by the identification of heterozygous mutations in PIK3R1. This study hereby presents a 15-year-old female with intrauterine growth restriction, short stature, teething delay, characteristic facial gestalts who was identified a novel de novo nonsense mutation in PIK3R1. CASE PRESENTATION: The proband was admitted to our department due to irregular menstrual cycle and hirsutism with short stature, who had a history of intrauterine growth restriction and presented with short stature, teething delay, characteristic facial gestalts, hirsutism, and thyroid disease. Whole-exome sequencing and Sanger sequencing revealed c.1960C > T, a novel de novo nonsense mutation, leading to the termination of protein translation (p. Gln654*). CONCLUSIONS: This is the first case report of SHORT syndrome complicated with thyroid disease in China, identifying a novel de novo heterozygous nonsense mutation in PIK3R1 gene (p. Gln654*). The phenotypes are mildly different from other cases previously described in the literature, in which our patient presents with lipoatrophy, facial feature, and first reported thyroid disease. Thyroid disease may be a new clinical symptom of patients with SHORT syndrome.

A novel CEP57 variant associated with mosaic variegated aneuploidy syndrome in a Chinese female presenting with short stature, microcephaly, brachydactyly, and small teeth.

BACKGROUND: Mosaic variegated aneuploidy (MVA) syndrome is a rare, autosomal recessive genetic disease. Here, we report an ultra-rare case of MVA syndrome associated with a CEP57 variant. METHODS: We retrospectively analyzed the clinical data of a 9-year-old female patient and surveyed her family members. Whole-exome sequencing and karyotype analysis were performed; suspected mutations were verified using Sanger sequencing. RESULTS: The patient presented with intrauterine growth restriction, short stature, microcephaly, facial dysmorphism, brachydactyly, and small teeth, and she showed unsatisfactory response to GH replacement therapy. Laboratory tests revealed high insulin-like growth factor-1 levels. Karyotype analysis of the peripheral blood showed mosaic variegated aneuploidies. Whole-exome and Sanger sequencing revealed a novel homozygous nonsense variant, NM_014679.4: c.312 T > G, in CEP57 that leads to translation termination (p.Tyr104*). The parents were heterozygous carriers of the identified variant. CONCLUSION: This study presents an ultra-rare case of CEP57-driven MVA syndrome, identifying a novel homozygous nonsense variant of CEP57 (p.Tyr104*). Our findings enrich the CEP57 mutational spectrum and emphasize the importance of genetic testing in patients with microcephaly and short stature. Furthermore, we conclude that growth hormone treatment is ineffective in such patients.

Identification of SOFT syndrome caused by a pathogenic homozygous splicing variant of POC1A: a case report.

BACKGROUND: Pathogenic variants in POC1A led to SOFT syndrome and variant POC1A-related (vPOC1A) syndrome. SOFT syndrome is a rare primordial dwarfism condition characterized by short stature, onychodysplasia, facial dysmorphism and hypotrichosis.The main clinical differences between SOFT and vPOC1A syndrome include dyslipidemia with insulin resistance and acanthosis nigricans. To our knowledge, this is the first report of a SOFT syndrome patient diagnosed with a homozygous splicing variant, which could help to extend our understanding of the genotypic and phenotypic information of the disease. CASE PRESENTATION: We reported a seven-year-old boy with SOFT syndrome. The patient presented symmetrical short stature and facial features, including prominent forehead, inverted triangular face, epicanthal fold, small teeth and enlarged ears. Laboratory tests displayed mild insulin resistance. Whole-exome sequencing (WES) led to the identification of a homozygous splicing variant (c.981+1G>A) in POC1A gene of the patient, which was inherited from his heterozygous parents confirmed by Sanger sequencing. Further transcriptional experiments of the splicing variant revealed aberrant percentage of exon 9 skipping transcripts. CONCLUSIONS: This is the firstly reported case of a SOFT syndrome patient with a novel homozygous splicing variant and detailed delineation of the aberrant transcript in proband and carrier of the variant in Chinese. Our study enriched mutational spectrum of POC1A which could help in further genetic diagnosis and counselling of SOFT syndrome patients.

Polyethylenimine: An Intranasal Adjuvant for Liposomal Peptide-Based Subunit Vaccine against Group A Streptococcus.

Group A Streptococcus (GAS) and GAS-related infections are a worldwide challenge, with no commercial GAS vaccine available. Polyethylenimine (PEI) attaches to the cells' surface and delivers cargo into endosomal and cytosolic compartments. We hypothesized that this will confer mucosal adjuvant properties for peptide antigens against group A Streptococcus (GAS). In this study, we successfully demonstrated the development of PEI incorporated liposomes for the delivery of a lipopeptide-based vaccine (LCP-1) against GAS. Outbred mice were administrated with the vaccine formulations intranasally, and immunological investigation showed that the PEI liposomes elicited significant mucosal and systemic immunity with the production of IgA and IgG antibodies. Antibodies were shown to effectively opsonize multiple isolates of clinically isolated GAS. This proof-of-concept study showed the capability for PEI liposomes to act as a safe vehicle for the delivery of GAS peptide antigens to elicit immune responses against GAS infection, making PEI a promising addition to liposomal mucosal vaccines.

Recent progress in preparation and agricultural application of microcapsules.

Recent advances in life science technology have prompted the need to develop microcapsule delivery systems that can encapsulate many different functional or active materials such as drugs, peptides, and live cells, etc. The encapsulation technology is now commonly used in medicine, agriculture, food, and other many fields. The application of biodegradable microcapsule systems can not only effectively prevent the degradation of core materials in the body or the biological environment, but also improve the bioavailability, control the release and prolong the halftime or storage of core active materials. Various wall materials, preparation methods, encapsulation processes, and release mechanisms are covered in this review, as well as several main factors including pH values, temperatures, particle sizes, and additives, which can strongly influence the encapsulation efficiency, the strength, and release of microcapsules. The improvement of coating materials, preparation techniques, and challenges are also highlighted, as well as application prospects.

[Study on pharmacokinetics of PVP coated beta-elemene liposome in rats].

OBJECTIVE: To study the pharmacokinetics of PVP costed beta-elemente liposmes in rats. METHODS: Gas chromatography was established to determine the concentration of beta-elemene in plasma of rats after administered through i.g. RESULTS: The pharmacokinetics parameters was: T(1/2) = 95.07 +/- 20.46 min, AUC = 348.72 +/- 32.49 microg x min/ml, Cmax = 4.39 +/- 0.33 microg/ml, Tmax = 60 min. CONCLUSION: The bioavailability of PVP coated beta-elemene liposomes is 140.2 +/- 7.5% compared with conventional liposomes.

Multivesicular liposome formulation for the sustained delivery of breviscapine.

Breviscapine, a well-known bioactive flavonoid ingredient extracted from the traditional Chinese medicine, has been extensively used in clinic to treat ischemic cerebrovascular and cardiovascular diseases in China. In order to prolong the duration of the drug in the circulation, reduce the frequency of injection administration and subsequently afford patient compliance, multivesicular liposome (MVL, namely DepoFoam) was utilized as a sustained-delivery system for breviscapine. In vitro release and in vivo pharmacokinetics of MVLs containing breviscapine (bre-MVLs) following intramuscular injection to rats were investigated compared with those of traditional liposomes containing breviscapine (bre-TLs). The drug durations both in vitro and in vivo were significantly prolonged for the bre-MVL, and that the drug release in vitro and the absorption in vivo showed a good linear correlation (R=0.9834), which provided an evidence for the suitability to select human plasma as the medium of drug release from MVLs in vitro. Drug release from bre-MVLs (triolein/tricaprylin, 10/0) in vitro extended a long period of 5-6 days, while the bre-TLs released 80% within only 4h. The mean residence time (MRT) obtained from the pharmacokinetics study of bre-MVL was about 16.6- and 5.04-fold longer than those of breviscapine solution (BS) and bre-TL, respectively. A duration in vivo for a period of 4-5 days was fulfilled for bre-MVL. In conclusion, MVL can be successfully used as a sustained delivery system of breviscapine.

In vitro and in vivo studies of different liposomes containing topotecan.

Liposome as a carrier of topotecan (TPT), a promising anticancer drug, has been reported in attempt to improve the stability and antitumor activity of TPT. However, the biodistribution pattern of TPT liposome in vivo and PEG-modified liposome containing TPT have not been studied systemically. In this paper, the in vitro stability and in vivo biodistribution behavior of several liposomes containing TPT with different lipid compositions and PEG-modification were studied. Compared with the 'fluid' liposome (S-Lip) composed of soybean phosphatidylcholine (SPC), the 'solid' liposome (H-Lip) composed of hydrogenated soybean phosphatidylcholine HSPC decreased the leaking efficiency of TPT from liposome and enhanced the stability of liposome in fetal bovine serum (FBS) or human blood plasma (HBP). The results of biodistribution studies in S180 tumor-bearing mice showed that liposomal encapsulation increased the concentrations of total TPT and the ratio of lactone form in plasma. Compared with free TPT, S-Lip and H-Lip resulted in 5- and 19-fold increase in the area under the curve (AUC(0-->infinity)), respectively. PEG-modified H-Lip (H-PEG) showed 3.7-fold increase in AUC(0-->infinity) compared with H-Lip, but there was no significant increase in t(1/2) and AUC(0-->infinity) for PEG-modified S-Lip (S-PEG) compared with S-Lip. Moreover, the liposomal encapsulation changed the biodistribution behavior, and H-Lip and H-PEG dramatically increased the accumulation of TPT in tumor, and the relative tumor uptake ratios were 3.4 and 4.3 compared with free drug, respectively. There was also a marked increase in the distribution of TPT in lung when the drug was encapsulated into H-Lip and H-PEG. Moreover, H-PEG decreased the accumulation of TPT in bone marrow compared with unmodified H-Lip. All these results indicated that the membrane fluidity of liposome has an important effect on in vitro stability and in vivo biodistribution pattern of liposomes containing TPT, and PEG-modified 'solid' liposome may be an efficient carrier of TPT.

PEG-rhGH therapy for small for gestational age rats and effects on their metabolic parameters.

AIMS: To determine whether administration of long-acting polyethylene glycol moiety covalently linked to recombinant human growth hormone (PEG-rhGH) can be efficacious in the treatment of small for gestational age (SGA) rats and to characterize its effects on metabolic parameters. METHODS: 20 pregnant rats were randomly divided into undernourished and standard nourished groups. SGA or appropriate for gestational age (AGA) offspring from each were then randomly assigned to receive either PEG-rhGH or normal saline (NS). Once-weekly subcutaneous injections of PEG-rhGH (2 µg/g/week) were administered starting at 21-42 days. Glycometabolism, blood pressure (BP), hormone and biochemical levels were analyzed at 21, 42 and 70 days. RESULTS: SGA rats at 21 days were lighter and shorter than AGAs (34.77 ± 2.11 vs. 48.83 ± 1.78 g, 10.42 ± 0.22 vs. 12.99 ± 0.17 cm, p < 0.05). At 42 days, SGA animals experienced a greater body weight gain. BP was higher in NS-treated SGA than in the AGA group at 70 days (138.16 ± 3.02 vs. 112.26 ± 5.42 mm Hg, p < 0.05). Meanwhile, NS-treated SGA exhibited higher glucose levels at 60 and 90 min than the AGA groups. No differences in hormone levels between the SGA and AGA groups were found at the end of PEG-rhGH treatment. CONCLUSION: These data suggest that PEG-rhGH treatment does not increase the risk of developing metabolic syndrome in adolescent aged rats.