[SARS-CoV-2 with transcription regulatory sequence motif mutation poses a greater threat].

OBJECTIVE: To analyze the mutations in transcription regulatory sequences (TRSs) of coronaviruss (CoV) to provide the basis for exploring the patterns of SARS-CoV-2 transmission and outbreak. METHODS: A combined evolutionary and molecular functional analysis of all sets of publicly available genomic data of viruses was performed. RESULTS: A leader transcription regulatory sequence (TRS-L) usually comprises the first 60-70 nts of the 5' UTR in a CoV genome, and the body transcription regulatory sequences (TRS-Bs) are located immediately upstream of the genes other than ORF1a and 1b. In each CoV genome, the TRS-L and TRS-Bs share a specific consensus sequence, namely the TRS motif. Any changes of nucleotide residues in the TRS motifs are defined as TRS motif mutations. Mutations in the TRS-L or multiple TRS-Bs result in superattenuated variants. The spread of super-attenuated variants may cause an increase in asymptomatic or mild infections, prolonged incubation periods and a decreased detection rate of the viruses, thus posing new challenges to SARS-CoV-2 prevention and control. The super-attenuated variants also increase their possibility of long-term coexistence with humans. The Delta variant is significantly different from all the previous variants and may lead to a large-scale transmission. The Delta variant (B.1.617.2) with TRS motif mutation has already appeared and shown signs of spreading in Singapore, which, and even the Southeast Asia, may become the new epicenter of the next wave of SARS-CoV-2 outbreak. CONCLUSION: TRS motif mutation will occur in all variants of SARS-CoV-2 and may result in super-attenuated variants. Only super-attenuated variants with TRS motif mutations will eventually lose the abilities of cross-species transmission and causing outbreaks.

CDK20 interacts with KEAP1 to activate NRF2 and promotes radiochemoresistance in lung cancer cells.

Radiochemoresistance is considered the main cause of local recurrence and distant metastasis in lung cancer. However, the underlying mechanisms of radiochemoresistance remain to be uncovered. In this study, we determine the functions of cell cycle-related kinase (CDK20) in radiochemoresistance. CDK20 is a newly identified protein kinase, which plays critical roles in cell growth and proliferation in several types of cancer. Using tandem affinity purification technology, we provide evidences that CDK20 binds to the ubiquitin ligase Kelch-like ECH-associated protein 1 (KEAP1), which targets transcriptional factor nuclear factor erythroid-2-related factor 2 (NRF2) for degradation. We show that this interaction is mediated by an evolutionarily conserved ETGE motif on CDK20. Furthermore, we demonstrate that CDK20 competes with NRF2 for KEAP1 binding, enhances the transcriptional activity of NRF2 and lowers the cellular reactive oxygen species level. Moreover, CDK20-depleted cells display impaired cell proliferation, defective G2/M arrest and increased radiochemosensitivity in lung cancer. These phenotypes induced by CDK20 knockdown are partially dependent on NRF2 inactivation. More importantly, CDK20 is overexpressed in human lung cancer tissues, as determined by immunostaining. Collectively, our results suggest that CDK20 positively modulate the KEAP1-NRF2 cytoprotective pathway to regulate tumor progression and radiochemoresistance, implying that CDK20 is a novel, promising therapeutic target for lung cancer.

Signal transductions and nonalcoholic fatty liver: a mini-review.

Nonalcoholic fatty liver disease (NAFLD) is a common liver disease, and the incidence increases year by year. The pathogenesis of NAFLD is correlated with insulin resistant (IR), and oxidative stress which induces varied inflammatory cytokines (TNF-α, IL-1, IL-6, etc). Different signal transductions such as MAPK, NF-κB, AMPK, JAK2/STAT3, PPAR, PI3K/Akt, TLR were activated by the pathogenic factors to regulate correlative reactions. Thus, in-depth study of the signal transductions will probably provide new suitable solutions for the prevention and therapy of NAFLD.

Feline atopic dermatitis: a retrospective study of 45 cases (2001-2012).

BACKGROUND: Atopic dermatitis (AD) is recognized as a common cause of pruritus in cats, but it remains incompletely characterized. HYPOTHESIS/OBJECTIVES: The aim of the study was to evaluate cases of confirmed feline AD. ANIMALS: Fourty-five cats from a dermatology referral practice (2001-2012). METHODS: A retrospective case record review was carried out using strict diagnostic criteria, including exclusion of flea-bite hypersensitivity and adverse food reaction. RESULTS: Disease prevalence was 12.5%, with domestic mixed (n = 24), Abyssinian (n = 6) and Devon rex (n = 3) cat breeds predisposed. Median age of onset was 2 years (62% <3 years; 22% >7 years; range 3 months to 12 years). Common presentations were severe (82%), nonseasonal (82%), waxing/waning (36%) pruritus, with alopecia/crusting/excoriations and/or erosions/ulceration (73%). Miliary dermatitis (20%) and eosinophilic granuloma complex lesions (27%) occurred. The face/head (71%), ventral abdomen (51%), neck (51%), limbs (38%), pinnae (31%), dorsum/rump (31%) and feet (16%) were frequently affected sites; lesions were restricted to the head/neck in only five cats (11%). Concurrent otitis externa (16%), superficial bacterial pyoderma (49%), Malassezia dermatitis (7%), flea-bite hypersensitivity (24%) and adverse food reaction (13%) occurred. Strong reactions on intradermal allergen testing were common (68%; 19 of 30), most frequently to pollens (61%) and/or insects (46%). Good response to ciclosporin (100%; 10 of 10), systemic glucocorticoids (55%; 22 of 40) and allergen-specific immunotherapy (57%; 13 of 23) and good/partial response to antihistamines (67%; 22 of 33) were reported. CONCLUSIONS AND CLINICAL IMPORTANCE: The prevalence of feline AD was higher than previously suggested, and breed predispositions were confirmed. Severe nonseasonal pruritus was most common, with a varied spectrum of lesions affecting a range of body areas.

HLA-B*13:01 and the dapsone hypersensitivity syndrome.

BACKGROUND: Dapsone is used in the treatment of infections and inflammatory diseases. The dapsone hypersensitivity syndrome, which is associated with a reported mortality of 9.9%, develops in about 0.5 to 3.6% of persons treated with the drug. Currently, no tests are available to predict the risk of the dapsone hypersensitivity syndrome. METHODS: We performed a genomewide association study involving 872 participants who had received dapsone as part of multidrug therapy for leprosy (39 participants with the dapsone hypersensitivity syndrome and 833 controls), using log-additive tests of single-nucleotide polymorphisms (SNPs) and imputed HLA molecules. For a replication analysis, we genotyped 24 SNPs in an additional 31 participants with the dapsone hypersensitivity syndrome and 1089 controls and performed next-generation sequencing for HLA-B and HLA-C typing at four-digit resolution in an independent series of 37 participants with the dapsone hypersensitivity syndrome and 201 controls. RESULTS: Genomewide association analysis showed that SNP rs2844573, located between the HLA-B and MICA loci, was significantly associated with the dapsone hypersensitivity syndrome among patients with leprosy (odds ratio, 6.18; P=3.84×10(-13)). HLA-B*13:01 was confirmed to be a risk factor for the dapsone hypersensitivity syndrome (odds ratio, 20.53; P=6.84×10(-25)). The presence of HLA-B*13:01 had a sensitivity of 85.5% and a specificity of 85.7% as a predictor of the dapsone hypersensitivity syndrome, and its absence was associated with a reduction in risk by a factor of 7 (from 1.4% to 0.2%). HLA-B*13:01 is present in about 2 to 20% of Chinese persons, 1.5% of Japanese persons, 1 to 12% of Indians, and 2 to 4% of Southeast Asians but is largely absent in Europeans and Africans. CONCLUSIONS: HLA-B*13:01 was associated with the development of the dapsone hypersensitivity syndrome among patients with leprosy. (Funded by the National Natural Science Foundation of China and others.).

In vitro and in vivo evaluation of sanguinarine liposomes prepared by a remote loading method with three different ammonium salts.

Sanguinarine liposomes were prepared by a remote loading method using three different ammonium salts. A series of studies, including in vitro release, in vitro and in vivo anti-tumor effects and pharmacokinetics in rats, were conducted. The three liposomes showed pH-sensitive release characteristics in vitro, but there were obvious variations in their release profiles. Among the three liposomes, the liposomes made using ammonium citrate and phosphate possessed better anti-tumor activity in vitro and in vivo, compared with the liposome using ammonium sulfate. Pharmacokinetics test results in rats indicated that sanguinarine liposomes have notably elevated AUC (P<0.05) and markedly lower CL (P<0.05) compared with the solution, but there were no obvious differences between the three liposomes. The present study may be useful for better understanding and better choice of a suitable ammonium salt for the remote loading method.

Prolongation of corneal allograft survival using cyclosporine in a polylactide-co-glycolide polymer.

PURPOSE: To test for prolongation of corneal transplant survival with cyclosporine in a polymer placed in the anterior chamber of corneal allograft recipients. METHODS: Wistar inbred rats with vascularized corneas were recipients of corneal allografts from Sprague-Dawley donor rats. Grafted rats were randomized into six groups: untreated control animals, cyclosporine-polymer anterior chamber recipients, cyclosporine-polymer subconjunctival recipients, cyclosporine-olive oil drop recipients, polymer-only anterior chamber recipients, and autografted Wistar rats. Grafts were examined by slit lamp every 3 days and the clinical condition scored. The cyclosporine concentration in the aqueous humor was assayed at 1, 2, and 4 weeks. At 2 and 4 weeks after transplantation, the eyes were collected for histopathologic evaluation of the grafts. RESULTS: The median survival time of untreated corneal allografts was 8.2 +/- 1.48 days for grafts treated with topical cyclosporine, 8.5 +/- 1.50 days for polymer-only anterior chamber implants, 10.6 +/- 1.90 days for 1% cyclosporine drops, 11.4 +/- 2.50 days for grafts given subconjunctival cyclosporine-polymer, 17 +/- 3.05 days for grafts given cyclosporine-polymer implants in the anterior chamber, and more than 3 months in autografted rats. There was a statistically significant difference ( p < 0.05) between the survival time of the allografts in the animals treated with the cyclosporine-polymer in the anterior chamber compared with the other groups of graft recipients. Significantly higher concentrations of cyclosporine were found in the eyes given an anterior chamber implant of cyclosporine-polymer than in the other treatment groups or the untreated rats. The cyclosporine-polymer implants placed in the anterior chamber induced a transient inflammatory response in transplanted eyes. CONCLUSIONS: Cyclosporine-polymer placed in the anterior chamber significantly prolongs corneal allograft survival in a high-risk corneal graft rejection. This intraocular delivery system may be a valuable adjunct for the suppression of immune graft rejection in high-risk recipients of corneal transplants.

[Overexpression of artificial synthetic gene of Aspergillus niger NRRL3135 phytase in Pichia pastoris].

The phytase gene of Aspergillus niger NRRL3135 was modified with a deletion of intron and signal coding sequence. Then, according to the codon preference of Pichia pastoris, modified phyA gene was artificially synthesized and cloned into expression vector of pPICZ alpha A. The recombinant plasmid was transformed into chromosome of Pichia pastoris X-33 strain by electroporation. The results of SDS-PAGE and enzymatic kinetic analysis proved that the recombinant phytase was secreted into culture medium with nearly same character of natural phytase. After screening for high level productive yeast strains, a strain named SPAN-III produced recombinant phytase with 165,000 u/mL under the condition of shake cultivation. It will satisfy the demand for industrialized production in some degree.

[Study on improvement of cell affinity of polymer materials--modified poly(D,L-lactide) by anhydrous ammonia gaseous plasma].

OBJECTIVE: To modify the surface of poly(D,L-lactide) film by anhydrous ammonia gaseous plasma treatment. METHODS: The changes of contact angles were measured and surface energy were calculated. Mouse 3T3 fibroblast cells were cultured on plasma modified and control poly(D,L-lactide) films. RESULTS: It was found that the hydrophilicity and surface energy of the materials have been increased after plasma treatment. Cell culture results showed that ammonia plasma treatment could promote the cell attachment and cells growth. After 4 days culture, the cells on the plasma treated films were 2-folds quantitatively compared with that of the control films. CONCLUSION: Ammonia plasma treatment can improve the cell affinity to poly(D,L-lactide).

Synthesis and degradation of a tri-component copolymer derived from glycolide, L-lactide, and epsilon-caprolactone.

A series of tri-component copolymers was synthesized by ring opening copolymerization of cyclic lactones, i.e. glycolide, L-lactide, and caprolactone, using stannous octoate as a catalyst. Various techniques, including FT-IR, 1H NMR, DSC, X-ray diffraction, tensile strength, and contact angle measurements, were used to elucidate structural characteristics, thermal behavior, mechanical properties, and hydrophilicity of the resulting copolymers. Data showed that the properties of these copolymers could be modulated by adjusting the composition of the copolymers. The DSC and X-ray analysis demonstrated amorphous structures for most of the PGLC copolyesters. The degradation behavior of these PGLC copolymers had been studied in vitro, i.e. in 0.10 M pH 7.4 phosphate buffer solution (PBS). The degradation was monitored by intrinsic viscosity and weight loss measurements. SEM and GPC were also used to monitor the morphology and molecular weight change during degradation. The PGLC copolymers were shown to have variable degradation rates, and most of them could disappear within a few months due to their amorphous structure and low glass transition temperature.

Preliminary study of oral polylactide microcapsulated insulin in vitro and in vivo.

AIM: Although the oral route for insulin delivery is the most convenient, directly administered oral insulin is degraded by proteolytic enzymes in the gastrointestinal (GI) tract. Polylactide was prepared in order to microcapsulate the insulin to avoid the enzymes in the GI. The physical characteristics and therapeutic possibilities of polylactide microcapsulated insulin (PLA-MCI) were studied in vivo and in vitro. METHODS: PLA-MCI was prepared by the two-step method of emulsion and solvent extraction. Its morphologic character was observed by scanning electron microscopy (SEM). The insulin release profile was determined in vitro by insulin measurement and in vivo by blood glucose measurement after the force-feeding of 66 diabetic rats. RESULTS: When the microcapsule was spherical in shape (diameter 1.5-2.0 microm) the entrapment efficiency of insulin was 90% and the loading rate was 10% (W/W). The PLA-MCI (which contained 3.0 units of insulin/mg of PLA) had peak release rates of 65-74% over 6-8 h in phosphate buffer. The same dose of PLA-MCI (insulin 2.5 mg) led to decreased responses (from 28% to 68% of control blood glucose levels) in the level of blood glucose in 32 rats which had not fasted after they had been force-fed. When 1.2, 1.8, 2.2 and 3.0 mg of insulin + PLA-MCI was administered to eight diabetic rats, their blood glucose levels decreased by 28%, 36%, 54% and 78%, respectively. CONCLUSIONS: PLA microcapsules are capable of protecting insulin from degradation by the proteolytic enzymes in the GI and of alleviating hyperglycaemia for a prolonged period of time in diabetic rats. It may therefore be considered as a new carrier for oral insulin.

A method for the preparation of polymeric nanocapsules without stabilizer.

In this communication poly(L-lactide) (PLLA) nanocapsules containing bovine serum albumin (BSA) were prepared by means of a modified W/O/W double emulsion technology. A mixture of glycerin and water was used instead of the traditional stabilizer system in the preparation of polymeric nanocapsules. The preliminary results showed that the high viscosity of the mixture and the hydroxyl group of the glycerin were helpful to the formation of the nanocapsules. The prepared nanocapsules had a similar spherical form. By comparison of different polymers of poly(L-lactide) and polycaprolactone-poly(ethylene oxide) block copolymer (PCE), it was found that the entrapment efficiency of the BSA was strongly dependent on the hydrophilicity of the polymer. A lower entrapment efficiency of BSA and nanocapsules with smaller size were obtained when the relative hydrophilic PCE polymer was used as the entrapping material.

A new stabilizer used in microencapsulation.

In this communication, a new microencapsulation method is reported to entrap solid drug powder in an aqueous system. A hydrophobically modified, random polyacrylamide derivative was used as a stabilizer: with a hydrophilic back bone and hydrophobic side chain, it showed good dispersing and stabilizing effects in the preparation of microcapsules. The preparation of streptomycin microcapsules, using poly(lactide) and poly(caprolactone), showed the successful entrapment of streptomycin powder which is readily soluble in water (solubility larger than 20 mg/ml). In addition, a low concentration of stabilizer (0.25%) is used and a short preparation process is also an advantage of the method.

Synthesis and characterization of polycaprolactone/poly(ethylene oxide)/polylactide tri-componet copolymers.

Polycaprolactone/poly(ethylene oxide)/polylactide tri-component copolymers (PCEL) with different compositions were synthesized by copolymerization of epsilon-caprolactone and L-lactide in the presence of poly(ethylene glycol) using stannous octoate as a catalyst. The copolymers were purified and characterized by various analytical techniques such as GPC, FT-IR, 1H NMR, 13C NMR, DSC, and X-ray diffractometry. It was evidenced that these copolymers were pure tri-component compounds which exhibited partially random chain structures, and possessed good mechanical properties and variable biodegradability.