Preparation and in Vitro Evaluation of pH Low Insertion Peptide-Modified Liposomes Encapsulating siRNA / 中国药学杂志

OBJECTIVE: Therapeutic nucleic acids(siRNA etc.) have unique advantages such as high specificity, safety, and target diversity in the treatment of diseases. However, the naked siRNAs are easily degraded by nucleases(RNase), have short half-lives and low transfection efficiency, which limit their therapeutic application. To construct a mildly acidic microenvironment sensitive liposomal nanocarrier for the efficient localization and delivery of siRNA(small interfering RNA) at the tissue, cell and even organelle levels. METHODS: Blank liposomes were prepared by thin-film hydration using dioleoyl phosphatidylethanolamine(DOPE) and cholesteryl hemisuccinate(CHEMS). The siRNA was compressed with the amphiphilic material SA-R8 to obtain SA-R8/siRNA, which was then incubated with blank liposomes to prepare liposomes encapsulating siRNA. The terminally functionalized phospholipid(DSPE-PEG2000-MAL) was linked to the low pH insertion peptide(pHLIP). The product was then incubated with liposomes encapsulating siRNA to construct pHLIP-modified liposomes encapsulating siRNA. The particle size and distribution of liposomes were characterized by dynamic light scattering principle. The cellular uptake, intracellular transport and distribution were monitored by flow cytometry and confocal laser-scanning technique. RESULTS: The RESULTS showed that the average size of the prepared liposomes encapsulating siRNA was between 150 and 190 nm. The cell uptake of siRNA at pH 6.5 was significantly higher than that of pH 7.4. And the siRNA internalized was well localized in the cytoplasm. CONCLUSION: This carrier shows strong pH sensitivity and can significantly increase the cell uptake of siRNA in the tumor acidic environment.

Survival of microencapsulated Lactobacillus casei (prepared by vibration technology) in fruit juice during cold storage

Background: Foods including probiotics are considered "functional foods." As an alternative to dairy products, we investigated the behavior of Lactobacillus casei when exposed to low-pH fruit juice. Juices of fruits such as pineapple, raspberry, and orange were assessed. Free and microencapsulated forms of L. casei were compared, and the viability of the probiotic was evaluated under storage at 4°C for 28 d. Microbiological analyses were carried out to ensure a safe and healthy product for consumers who look for foods with probiotics from sources other than dairy. Results: Low pH affected L. casei survival during storage depending on the type of fruit juice. In the case of pineapple juice, some microcapsules were broken, but microcapsules recovered at the end of the storage period had 100% viability (2.3 × 107 CFU/g spheres). In the case of orange juice, more than 91% viability (5.5 × 106 CFU/g spheres) was found. In raspberry juice, viability decreased rapidly, disappearing at the end of the storage period, which was caused by the absorption of high concentrations of anthocyanin inside microcapsules more than low pH. Conclusion: Low pH affected the survival of L. casei under refrigeration; even when they were microencapsulated, acidic conditions impacted their viability. Although pH affects viability, its value is very sensitive and will depend on the type of fruit juice and its composition. Some fruit juices contain compounds used as substrates for Lactobacillus and other compounds with antimicrobial effects.

Improvement of hydrogen yield of ethanol-producing Escherichia coli recombinants in acidic conditions

Background: An effective single culture with high glycerol consumption and hydrogen and ethanol coproduction yield is still in demand. A locally isolated glycerol-consuming Escherichia coli SS1 was found to produce lower hydrogen levels under optimized ethanol production conditions. Molecular approach was proposed to improve the hydrogen yield of E. coli SS1 while maintaining the ethanol yield, particularly in acidic conditions. Therefore, the effect of an additional copy of the native hydrogenase gene hycE and recombinant clostridial hydrogenase gene hydA on hydrogen production by E. coli SS1 at low pH was investigated. Results: Recombinant E. coli with an additional copy of hycE or clostridial hydA was used for fermentation using 10 g/L (108.7 mmol/L) of glycerol with an initial pH of 5.8. The recombinant E. coli with hycE and recombinant E. coli with hydA showed 41% and 20% higher hydrogen yield than wild-type SS1 (0.46 ± 0.01 mol/mol glycerol), respectively. The ethanol yield of recombinant E. coli with hycE (0.50 ± 0.02 mol/mol glycerol) was approximately 30% lower than that of wild-type SS1, whereas the ethanol yield of recombinant E. coli with hydA (0.68 ± 0.09 mol/mol glycerol) was comparable to that of wild-type SS1. Conclusions: Insertion of either hycE or hydA can improve the hydrogen yield with an initial pH of 5.8. The recombinant E. coli with hydA could retain ethanol yield despite high hydrogen production, suggesting that clostridial hydA has an advantage over the hycE gene in hydrogen and ethanol coproduction under acidic conditions. This study could serve as a useful guidance for the future development of an effective strain coproducing hydrogen and ethanol.

The change of regular semen parameters in infertile patients with simple low-pH semen before and after symptomatic treatment / 国际检验医学杂志

Objective To study the change of regular semen parameters in infertile patients with simple low-pH semen before and after symptomatic treatment.Methods A total of 100 male infertility patients were recruited in the study,and were divided into two groups according to the pH range:patients group Ⅰ(n=40)and patients group Ⅱ(n=60);The two patients groups were tea-ted for low pH semen.When the first and second course of treatment were finished,semen pH values and conventional semen pa-rameters were tested and compared with those before treatment and control group.Results When the first course of treatment were finished,semen pH value of 75%(30/40)patients in group Ⅰ reached normal range,compared with the control group,the semen parameters were statistically different(P 0.05);Semen pH value of 90%(54/60)patients in group Ⅱ reached normal range,compared with the control group,the semen parameters and pH values were not statistically different(P >0.05 ). Conclusion The semen quality improved after the symptomatic treatment for low semen pH value and its an effective therapeutic approach for infertile patients with simple low-pH semen.

A low-pH medium in vitro or the environment within a macrophage decreases the transcriptional levels of fimA, fimZ and lrp in Salmonella enterica serovar Typhimurium.

Many Salmonella Typhimurium isolates produce type 1 fimbriae and exhibit fimbrial phase variation in vitro. Static broth culture favours the production of fimbriae, while solid agar medium inhibits the generation of these appendages. Little information is available regarding whether S. Typhimurium continues to produce type 1 fimbriae during in vivo growth. We used a type 1 fimbrial phase-variable strain S. Typhimurium LB5010 and its derivatives to infect RAW 264.7 macrophages. Following entry into macrophages, S. Typhimurium LB5010 gradually decreased the transcript levels of fimbrial subunit gene fimA, positive regulatory gene fimZ, and global regulatory gene lrp. A similar decrease in transcript levels was detected by RT-PCRwhen the pH of static brothmediumwas shifted frompH 7 to amore acidic pH 4. A fimA-deleted strain continued to multiply within macrophages as did the parental strain. An lrp deletion strain was unimpaired for in vitro growth at pH 7 or pH 4, while a strain harboring an lrp-containing plasmid exhibited impaired in vitro growth at pH 4. We propose that acidic medium, which resembles one aspect of the intracellular environment in a macrophage, inhibits type 1 fimbrial production by down-regulation of the expression of lrp, fimZ and fimA.

Alterations in ambient salinity and pH lead to modulation of developmental gene expression in Microhyla ornata (Duméril and Bibron) and Xenopus laevis (Daudin).

Naturally fertilized Microhyla ornata and Xenopus laevis embryos at dorsal lip of blastopore stage were exposed to 0.3, and 0.6% sodium chloride for high salinity treatment and dilute hydrochloric acid and sodium hydroxide for treatment with low and high pH, respectively. After treatment for different durations, embryos were studied morphologically and using in situ hybridization with selected genes important for normal development and for coping with environmental stress. Altered salinity and pH caused defects in axis formation and neural tube closure, delay in hatching, abnormal swimming of tadpoles and even developmental arrest. This was accompanied by significant decrease in the expression of selected development-regulating genes like goosecoid (required for gastrulation movements), brachyury (mesodermal marker gene), noggin (involved in neural induction), NCAM (required for neural cell adhesion) and MyoD (essential for muscle development), and considerable increase in the transcription of stress response genes hsp30 and hsp70. Altering the expression of embryonic genes could be one of the mechanisms through which environmental factors influence development of amphibian embryos.

Effect of cola on surface microhardness and marginal integrity of resin modified glass ionomer and compomer restoration: An in vitro study.

This study was carried out to evaluate the surface microhardness and marginal integrity of a resin modified glass ionomer and a compomer after immersing them in a cola soft drink for variable periods of time. The study was conducted in two parts. Forty two standard class V cavities were prepared on extracted human premolars, restored with Dyract AP and Fuji II LC and evaluated for marginal integrity. Forty two circular discs (CDs) were prepared with Dyract AP and Fuji II LC to test microhardness. These specimens were immersed in a cola drink under low, medium and high immersion regimes. Marginal integrity was analyzed using stereomicroscope and surface microhardness was evaluated on the circular discs using a microhardness tester. Statistical analysis revealed that the marginal integrity was least affected in low and medium immersion regimes and highly affected under high immersion regime. Surface microhardness was least affected in low immersion regime, while, medium and high immersion regimes showed appreciable softening of both restorative materials evaluated.

An alternative method for evaluation of resistance to low pH and bile salts in probiotic characterization of Lactobacillus reuteri strains: a technical note

A considerable amount of attention has been focused on the use of lactobacilli as probiotics. The use of microorganisms as probiotics must meet certain criteria, among these; the resistance to low pH and bile salts is of great importance. Different procedures have been designed to evaluate tolerance to gastrointestinal transit. Since resistance to low pH and bile salts is critical in the evaluation of probiotic strains, the aim of this study was to compare resistance to bile salts for 20 strains of porcine Lactobacillus reuteri that had been isolated from healthy pigs, using two different procedures based on exposure to low pH. Statistical analysis revealed no difference in resistance of the strains to bile salts whether pH was low or not. The use of low pH prior to exposure to bile salts therefore more closely represents physiological conditions that should be used for the evaluation of potential porcine probiotic lactobacilli. Further studies using digestive enzymes are required to evaluate resistance to gastrointestinal transit, since it is an important factor in the evaluation of strains with potential probiotic activity.

Existe un interés creciente en el uso de lactobacilos como probióticos. El uso de microorganismos como probióticos, requiere que éstos cubran ciertos requisitos, dentro de los cuales la resistencia a pH ácido y a sales biliares, se consideran de suma importancia. Actualmente existen distintos procedimientos que permiten evaluar la tolerancia al tránsito gastrointestinal. Debido a que la resistencia a pH ácido y a sales biliares es una condición crítica dentro de la evaluación de cepas probióticas, la finalidad del presente trabajo consistió en comparar la resistencia a sales biliares de 20 cepas de Lactobacillus reuteri aisladas de cerdos sanos, mediante dos procedimientos diferentes basados en la exposición a pH ácido. El análisis estadístico no mostró diferencias en la resistencia de las cepas a sales biliares cuando se realizó una exposición previa a pH ácido. Así, la exposición a pH ácido previo a la incubación con sales biliares, asemeja más a las condiciones fisiológicas que el microorganismo encontrará de manera natural y es una buena alternativa en la evaluación probiótica de lactobacilos de origen porcino. Se requieren estudios que incluyan enzimas digestivas, con el fin de evaluar la resistencia al tránsito gastrointestinal, dado que es uno de los factores más importantes en la evaluación de cepas con potencial actividad probiótica.

A Study on Cultivation of Acid-tolerant Methanogenic Granular Sludge under Low pH Values and its Characteristics / 微生物学通报

Two lab-scale expanded granular sludge bed reactors were operated using granular sludge and river sediments as seed sludge respectively, through gradually lowering down the inner pH values, acid-tolerant methanogenic granular sludge with good methanogenic activity were acquired and formed. Two EGSB reactors could be operated steadily under the condition of pH 5.8～6.2, and their volumetric loading rates were about 5.5～7.5 kg COD/(m3/d), the COD removal efficiencies were about 90%. The granular sludges taken from two reactors could maintain relatively higher methanogenic activity under low pH, and the relative activities of the granular could be 51.78% and 55.6% of the value with the condition of pH 7.0 when pH was 5.5. Studies on the diameter distribution, settling velocities, concentrations of different metal elements and microbiological characteristics of these acid-tolerant granular sludges were also conducted.