Engineering Lignin Nanomicroparticles for the Antiphotolysis and Controlled Release of the Plant Growth Regulator Abscisic Acid.

Lignin is the most abundant aromatic biopolymer in nature and is a major byproduct from the paper industry. The unlocking of lignin's potential for high-value applications has gained increasing attention in recent years. In this study, alkali lignin (AL), with a rigid conjugated structure and amphiphilic property, was used as a sustainable and eco-friendly encapsulation material for the protection and controlled release of photosensitive abscisic acid (ABA), an important and widely used plant growth regulator. Cetyltrimethylammonium bromide (CTAB) was used to induce the formation of AL-CTAB nanomicroparticles by self-assembly. The size and morphology of AL-CTAB particles were modified by changing the AL concentration and the dispersion agent. AL (0.3 M) dissolved in tetrahydrofuran could form a uniform size (300 nm) of particles with a regular spherical structure. Subsequently, ABA was loaded on the prepared nanomicroparticles to synthesize the capsule formulation of ABA@AL-CTAB. The controlled-release behavior and the antiphotolysis performance as well as the thermal stability of ABA@AL-CTAB were proved to be superior. Lasting inhibition of Arabidopsis and rice seed germination by ABA@AL-CTAB under light irradiations implied protection of ABA from photolysis. In addition, ABA@AL-CTAB could effectively regulate plant stomata, thereby increasing plant drought resistance. Overall, lignin is suitable for the preparation of agrochemical formulations with excellent controlled release and antiphotolysis performances.

Development of a sensitive monoclonal antibody-based enzyme-linked immunosorbent assay for the antimalaria active ingredient artemisinin in the Chinese herb Artemisia annua L.

Artemisinin is an endoperoxide sesquiterpene lactone isolated from the Chinese medicinal plant Artemisia annua L. It has been widely used in South-East Asia and Africa as an effective drug against sensitive and multidrug-resistant Plasmodium falciparum malaria. A monoclonal antibody (mAb), designated as 3H2, was generated with artesunate-bovine serum albumin conjugate as the immunogen. mAb 3H2 was used to develop a highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (icELISA) for artemisinin. The concentration of analyte producing 50% of inhibition (IC(50)) and the working range of the icELISA were 1.3 and 0.2-5.8 ng/mL, respectively. The mAb 3H2 recognized the artemisinin analogs artesunate, dihydroartemisinin, and artemether with cross-reactivity of 650%, 57%, and 3%, respectively, but negligibly recognized deoxyartemisinin and the artemisinin precursors arteannuin B and artemisinic acid. The average recoveries of artemisinin fortified in A. annua samples at concentrations from 156 to 5,000 microg/g determined by icELISA ranged from 91% to 98%. The icELISA was applied for the determination of artemisinin in different wild A. annua samples and the results were confirmed by high-performance liquid chromatography (HPLC) analysis. The correlation coefficient (R(2)) between the two assays was larger than 0.99, demonstrating a good agreement between the icELISA and HPLC results. This ELISA is suitable for quality assurance of A. annua L. materials.

Vaccine Potential and Diversity of the Putative Cell Binding Factor (CBF, NMB0345/NEIS1825) Protein of Neisseria meningitidis.

The cbf gene from Neisseria meningitidis strain MC58 encoding the putative Cell Binding Factor (CBF, NMB0345/NEIS1825) protein was cloned into the pRSETA system and a ~36-kDa recombinant (r)CBF protein expressed in Escherichia coli and purified by metal affinity chromatography. High titres of rCBF antibodies were induced in mice following immunization with rCBF-saline, rCBF-Al(OH)3, rCBF-Liposomes or rCBF-Zwittergent (Zw) 3-14 micelles, both with and without incorporated monophosphoryl lipid A (MPLA) adjuvant. Anti-rCBF sera reacted in western blots of meningococcal lysates with a single protein band of molecular mass ~29.5 kDa, indicative of mature CBF protein, but did not react with a lysate of a Δnmb0345 mutant (CBF-), demonstrating specificity of the murine immune responses. CBF protein was produced by all strains of meningococci studied thus far and the protein was present on the surface of MC58 (CBF+) bacteria, but absent on Δnmb0345 mutant (CBF-) bacteria, as judged by FACS reactivity of anti-rCBF sera. Analysis of the NEIS1825 amino acid sequences from 6644 N. meningitidis isolates with defined Alleles in the pubmlst.org/Neisseria database showed that there were 141 ST types represented and there were 136 different allelic loci encoding 49 non-redundant protein sequences. Only 6/6644 (<0.1%) of N. meningitidis isolates lacked the nmb0345 gene. Amongst serogroup B isolates worldwide, ~68% and ~20% expressed CBF encoded by Allele 1 and 18 respectively, with the proteins sharing >99% amino acid identity. Murine antisera to rCBF in Zw 3-14 micelles + MPLA induced significant serum bactericidal activity (SBA) against homologous Allele 1 and heterologous Allele 18 strains, using both baby rabbit serum complement and human serum complement (h)SBA assays, but did not kill strains expressing heterologous protein encoded by Alelle 2 or 3. Furthermore, variable bactericidal activity was induced by murine antisera against different meningococcal strains in the hSBA assay, which may correlate with variable surface exposure of CBF. Regardless, the attributes of amino acid sequence conservation and protein expression amongst different strains and the ability to induce cross-strain bactericidal antibodies indicates that rCBF could be a potential meningococcal vaccine antigen and merits further testing.

[Effects of histamine on proliferation, apoptosis and differentiation of human keratinocytes].

OBJECTIVE: To investigate the effects of histamine on the proliferation, apoptosis and differentiation of human keratinocytes (HKC) and the mechanisms. METHODS: The effect of histamine on the growth of HKC in vitro was examined by MTT assay and trypan blue exclusion assay. Cell cycle analysis and early apoptosis analysis by double staining with Annexin V-FITC and PI were carried out by flow cytometry. DNA ladder assay was performed for the detection of cell apoptosis. HKC free calcium concentration ([Ca(2+)](i)) was measured by laser scanning confocal microscopy in combination with calcium fluorescence probe Fluo-3/AM. HKC differentiation markers keratin 10 (K10) and involucrin was detected by streptavidinbiotin complex immunocytochemical assay. RESULTS: Histamine at high concentration inhibited the proliferation of HKC with cell viability ratio of 65.6% at 1 x 10(-4) mol/L, while histamine at low concentrations promoted proliferation of HKC with the cell viability ratio of 130.7% at 1 x 10(-8) mol/L. Histamine at 1 x 10(-4) mol/L altered cell cycle distribution of HKC with an increase in G0/G1-phase cell population to 30.97%, a decrease in S-phase population to 73.81% and inhibition of G1/S switching. Histamine at 1 x 10(-4) mol/L induced obvious apoptosis of HKC with early apoptosis ratio of 18.64% as compared with the control (5.60%, P<0.05). Histamine 1 x 10(-4) mol/L induced an increase of HKC [Ca(2+)](i) up to 58.9% and cimetidine (an H(2) receptor antagonist) decreased HKC [Ca(2+)](i) down to 25.4%. Histamine at this concentration down-regulated the expressions of K10 and involucrin of HKC but these changes were not significantly different from those of the control (P>0.05). CONCLUSIONS: Histamine at high concentrations inhibits cell cycle progress of HKC, mediates cell apoptosis and induces the increase of [Ca(2+)](i), which might be a partial explanation for growth arrest of HKC elicited by histamine. Histamine may regulate epidermal tissue turnover under physiological conditions, whereas under pathological circumstances of the skin as in trauma and inflammation, histamine at high concentrations may inhibit the regeneration of epidermis and the differentiation of HKC.

Immunization with recombinant Chaperonin60 (Chp60) outer membrane protein induces a bactericidal antibody response against Neisseria meningitidis.

Sera from individuals colonized with Neisseria meningitidis and from patients with meningococcal disease contain antibodies specific for the neisserial heat-shock/chaperonin (Chp)60 protein. In this study, immunization of mice with recombinant (r)Chp60 in saline; adsorbed to aluminium hydroxide; in liposomes and detergent micelles, with and without the adjuvant MonoPhosphoryl Lipid A (MPLA), induced high and similar (p>0.05) levels of antibodies that recognized Chp60 in outer membranes (OM). FACS analysis and immuno-fluorescence experiments demonstrated that Chp60 was surface-expressed on meningococci. By western blotting, murine anti-rChp60 sera recognized a protein of Mr 60kDa in meningococcal cell lysates. However, cross-reactivity with human HSP60 protein was also observed. By comparing translated protein sequences of strains, 40 different alleles were found in meningococci in the Bacterial Isolate Genome Sequence database with an additional 5 new alleles found in our selection of 13 other strains from colonized individuals and patients. Comparison of the non-redundant translated amino acid sequences from all the strains revealed ≥97% identity between meningococcal Chp60 proteins, and in our 13 strains the protein was expressed to high and similar levels. Bactericidal antibodies (median reciprocal titres of 32-64) against the homologous strain MC58 were induced by immunization with rChp60 in liposomes, detergent micelles and on Al(OH)3. Bactericidal activity was influenced by the addition of MPLA and the delivery formulation used. Moreover, the biological activity of anti-Chp60 antisera did not extend significantly to heterologous meningococcal strains. Thus, in order to provide broad coverage, vaccines based on Chp60 would require multiple proteins and specific bactericidal epitope identification.

Improved biological effects of uniconazole using porous hollow silica nanoparticles as carriers.

BACKGROUND: The aim of this work is to prepare a controlled-release formulation of uniconazole using porous hollow silica nanoparticles (PHSNs) as carrier, and to investigate the biological effects on rice growth. RESULTS: PHSNs with a shell thickness of ~15 nm and a particle size of 80-100 nm were synthesised through a sol-gel route using nanosized calcium carbonate particles as templates. Simple immersing (SI) and supercritical fluid drug loading (SFDL) technologies were employed to load uniconazole into PHSNs with loading efficiencies of ~22 and ~26% respectively. The prepared uniconazole-loaded PHSNs (UCZ-PHSNs) by SI and SFDL both demonstrated sustained release properties, and the latter showed better controlled release ability with a slower release rate. Compared with free uniconazole, UCZ-PHSNs exhibited a weaker growth retardation effect in the early stage but more significant retardation ability in later stages for agar-cultured rice seedlings. For the rice that grew in clay, UCZ-PHSNs demonstrated a weaker plant height retardation effect than free uniconazole at the early jointing stage by foliar spraying, but exhibited a stronger retardation capacity than free uniconazole by being applied into soil before seedling transplantation. CONCLUSION: The results indicated that the prepared UCZ-PHSNs possessed good controlled-release properties and had improved retardation effects on rice growth. It is recommended that UCZ-PHSNs be applied into soil before seedling transplantation rather than administered by foliar spraying at the early jointing stage.

Treatment outcome of superficial cerebral abscess: an analysis of two surgical methods.

BACKGROUND: The purpose of the study is to compare the two surgical methods (burr hole and craniotomy) used as treatment for superficial cerebral abscess and its outcome in terms of radiological clearance on brain CT, improvement of neurological status, the need for repeated surgery, and survival and morbidity at three months after surgery. This report is a retrospective case review of the patients who were treated surgically for superficial cerebral abscess in Hospital Kuala Lumpur (HKL) and Hospital Sultanah Aminah (HSA) over a period of four years (2004 to 2007). METHODS: Fifty-one cases were included in this study: 64.7% of patients were male and 35.5% were female. Most of the patients were Malay (70.6%); 28 patients (54.9%) had undergone craniotomy and excision of abscess, and the rest had undergone burr hole aspiration as their first surgical treatment. RESULTS: This study reveals that patients who had undergone craniotomy and excision of abscess showed a significantly earlier improvement in neurological function, better radiological clearance and lower rate of re-surgery as compared to the burr hole aspiration group (P<0.05). However, with respect to neurological improvement at 3 months, morbidity and mortality, there is no significant difference between the two surgical methods. CONCLUSION: The significance of these findings can only be confirmed by a prospective randomised series. Further study will be required to assess the cost effectiveness, intensive care needs, and possibility of shorter antibiotic usage as compared to burr hole aspiration.

[Effects of bradykinin on the proliferation, apoptosis and differentiation of human keratinocytes].

OBJECTIVE: To investigate the effects of bradykinin (BK) on the proliferation, apoptosis and differentiation of human keratinocyte (HKC) and the underlying mechanisms. METHODS: HKCs were cultured together with 1 x 10(-4) - 1 x 10(-9) mol/L of BK. With methyl thiotetrazole (MTT) and trypan blue staining it was shown that the BK in dose of 1 x 10(-4) mol/L possessed most powerful inhibitory effect, and the survival rate of HKC was 69.3%. Therefore, BK was employed in the dose of 1 x 10(-4) mol/L in the following studies. When the growth of HKCs reached the logarithmic phase, BK in the concentration of 1 x 10(-4) mol/L was added, and it was categorized as the test group (E). HKCs without BK served as the control group (C). The cell cycle and apoptosis were detected by flow cytometry after being cultured for 24 and 48 hours. The change in intracellular calcium [Ca(2+)](i) was determined by means of laser scanning confocal microscopy with calcium fluorescence probe Fluo-3/AM technique. The expression of HKC differentiation labeling protein keratin10 (K10) and involucrin were detected with Strept Avidin-Biotin Complex (SABC) immunocytochemical assay. RESULTS: The cell ratio in G0/G1 phase in E group increased by 34.57% while in S phase decreased by 58.91% in reference to that in C group. The G1/S phase switching of HKCs was obviously inhibited by BK, and apoptosis was stimulated (apoptotic rate of 15.34% in E group vs 5.60% in C group, P < 0.05). The [Ca(2+)](i) increased transiently in HKCs by 163.0% in E group after 3 minutes of BK activation and decreased thereafter in reference to that in C group. The K10 expression in HKC was down-regulated in E group with positive cell rate of 2.20%, which was lower than that of C group (6.89%, P < 0.05). CONCLUSION: The cell cycle process of HKC could be inhibited by high concentration of BK with increased apoptosis and an increase in [Ca(2+)](i), which might be the mechanism of inhibition of growth of HKC in vitro. Furthermore, the epithelial regeneration and HKC differentiation can also be inhibited by BK.