Monoclonal antibody against the poly-γ-d-glutamic acid capsule of Bacillus anthracis protects mice from enhanced lethal toxin activity due to capsule and anthrax spore challenge

Background

The poly-γ-d-glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis, protects bacilli from immune surveillance and allows its unimpeded growth in the host. Recently, the importance of the PGA in the pathogenesis of anthrax infection has been reported. The PGA capsule is associated with lethal toxin (LT) in the blood of experimentally infected animals and enhances the cytotoxicity of LT.

Methods

To investigate the role of anti-PGA Abs on progression of anthrax infection, two mouse anti-PGA mAbs with K_d values of 0.8 μM and 2.6 μM respectively were produced and in silico three dimensional (3D) models of mAbs with their cognitive PGA antigen complex were analyzed.

Results

Anti-PGA mAbs specifically bound encapsulated B. anthracis H9401 and showed opsonophagocytosis activity against the bacteria with complement. The enhancement effect of PGA on LT-mediated cytotoxicity was confirmed ex vivo using mouse bone marrow-derived macrophages and was effectively inhibited by anti-PGA mAb. Passive immunization of mAb completely protected mice from PGA-enhanced LT toxicity and partially rescued mice from anthrax spore challenges. 3D structure models of these mAbs and PGA complex support specific interactions between CDR and cognitive PGA. These results indicate that mouse mAb against PGA capsule prevents the progress of anthrax disease not only by eliminating the vegetative form of encapsulated B. anthracis but also by inhibiting the enhanced cytotoxic activity of LT by PGA through specific binding with PGA capsule antigen.

General significance

Our results suggest a potential role for PGA antibodies in preventing and treating anthrax infection.     

Comparison of the Efficacy of Entecavir and Tenofovir in Nucleos(T)ide Analogue-Experienced Chronic Hepatitis B Patients

The efficacy of entecavir (ETV) and tenofovir (TDF) for the treatment of nucleos(t)ide analogue (NA)-experienced chronic hepatitis B (CHB) patients has been little studied. Here, we compare the efficacy of both ETV and TDF in NA-experienced CHB patients without detectable genotypic resistance. This retrospective cohort study included consecutive NA-experienced patients who had neither current nor previous genotypic resistance and had received ETV or TDF for at least 6 months. Overall, 202 patients (146 patients in the ETV group and 56 in the TDF group) were analyzed. The cumulative probabilities of complete virologic suppression (CVS) at month 12 were 76.1% in the ETV group and 95.0% in the TDF group (P<0.001), respectively. The TDF-treated group achieved CVS more rapidly than the ETV group for both Hepatitis B e antigen (HBeAg)-negative and -positive patients (P = 0.006 and < 0.001, respectively), and for those with both low (< 2,000 IU/mL) and high (≥ 2,000 IU/mL) HBV DNA levels (P = 0.01 and 0.002, respectively). TDF group had an increased probability of achieving CVS (hazard ratio, 2.242; 95% confidence interval, 1.587–3.165; P = 0.001), after adjustment for HBV DNA level, the presence of HBeAg, and a history of CVS during prior treatment. During the treatment period, 23 patients (15.8%) in the ETV group developed virologic breakthrough, compared to none in the TDF group. The cumulative probabilities of developing virologic breakthrough and ETV-resistance at month 24 were 9.7% and 5.3%, respectively. In conclusion, TDF is preferable to ETV for achieving CVS in NA-experienced CHB patients without genotypic resistance.     

Identification and characterization of a putative cyclic nucleotide-gated channel, CNG-1, in C. elegans

Cyclic nucleotide-gated (CNG) channels encoded by the tax-4 and tax-2 genes are required for chemosensing and thermosensing in the nematode C. elegans. We identified a gene in the C. elegans genome, which we designated cng-1, that is highly homologous to tax-4. Partial CNG-1 protein tagged with green fluorescent protein was expressed in several sensory neurons of the amphid. We created a deletion mutant of cng-1, cng-1 (jh111), to investigate its in vivo function. The mutant worms had no detectable abnormalities in terms of their basic behavior or morphology. Whereas tax-4 and tax-2 mutants failed to respond to water-soluble or volatile chemical attractants, the cng-1 null mutant exhibited normal chemotaxis to such chemicals and a tax-4;cng-1 double mutant had a similar phenotype to tax-4 single mutants. Interestingly, cng-1 and tax-4 had a synergistic effect on brood size.     

Expression and secretion of the protective antigen of Bacillus anthracis in Bacillus brevis

We used the Bacillus brevis-pNU212 system to develop a mass production system for the protective antigen (PA) of Bacillus anthracis. A moderately efficient expression-secretion system for PA was constructed by fusing the PA gene from B. anthracis with the B. brevis cell-wall protein signal-peptide encoding region of pNU212, and by introducing the recombinant plasmid, pNU212-mPA, into B. brevis 47-5Q. The clone producing PA secreted about 300 microg of recombinant PA (rPA) per ml of 5PY-erythromycin medium after 4 days incubation at 30 degrees C. The rPA was fractionated from the culture supernatant of B. brevis 47-5Q carrying pNU212-mPA using ammonium sulfate at 70% saturation followed by anion exchange chromatography on a Hitrap Q, a Hiload 16/60 Superdex 200 gel filtration column and a phenyl sepharose hydrophobic interaction column, yielding 70 mg rPA per liter of culture. The N-terminal sequence of the purified rPA was identical to that of native PA from B. anthracis. The purified rPA exhibited cytotoxicity towards J774A.1 cells when combined with lethal factor. The rPA formulated in either Rehydragel HPA or MPL-TDM-CWS adjuvant (Ribi-Trimix) elicited the expression of a large amount of anti-PA and neutralizing antibodies in guinea pigs and completely protected them against a 100 LD50 challenge with fully virulent B. anthracis spores.     

Rice Chlorina-1 and Chlorina-9 encode ChlD and ChlI subunits of Mg-chelatase, a key enzyme for chlorophyll synthesis and chloroplast development

Photosynthetic organisms exhibit a green color due to the accumulation of chlorophyll pigments in chloroplasts. Mg-protoporphyrin IX chelatase (Mg-chelatase) comprises three subunits (ChlH, ChlD and ChlI) and catalyzes the insertion of Mg²⁺ into protoporphyrin IX, the last common intermediate precursor in both chlorophyll and heme biosyntheses, to produce Mg-protoporphyrin IX (MgProto). Chlorophyll deficiency in higher plants results in chlorina (yellowish-green) phenotype. To date, 10 chlorina (chl) mutants have been isolated in rice, but the corresponding genes have not yet been identified. Rice Chl1 and Chl9 genes were mapped to chromosome 3 and isolated by map-based cloning. A missense mutation occurred in a highly conserved amino acid of ChlD in the chl1 mutant and ChlI in the chl9 mutant. Ultrastructural analyses have revealed that the grana are poorly stacked, resulting in the underdevelopment of chloroplasts. In the seedlings fed with aminolevulinate-dipyridyl in darkness, MgProto levels in the chl1 and chl9 mutants decreased up to 25% and 31% of that in wild-type, respectively, indicating that the Mg-chelatase activity is significantly reduced, causing the eventual decrease in chlorophyll synthesis. Furthermore, Northern blot analysis indicated that the nuclear genes encoding the three subunits of Mg-chelatase and LhcpII in chl1 mutant are expressed about 2-fold higher than those in WT, but are not altered in the chl9 mutant. This result indicates that the ChlD subunit participates in negative feedback regulation of plastid-to-nucleus in the expression of nuclear genes encoding chloroplast proteins, but not the ChlI subunit.Haitao Zhang and Jinjie Li contributed equally to this work     

CAPS markers using mitochondrial consensus primers for molecular identification of Panax species and Korean ginseng cultivars (Panax ginseng C. A. Meyer)

Cleaved amplified polymorphic sequence (CAPS) marker system using mitochondrial consensus primers was applied for molecular identification of Korean ginseng cultivars (Panax ginseng). Initially, a total of 34 primers were tested to six Korean ginseng cultivars and two foreign Panax species, P. quinquefolius and P. notoginseng. In the polymerase chain reaction (PCR) amplification results, four primers (mt7, mt11, mt13, and mt18) generated co-dominant polymorphic banding patterns discriminating the Korean ginseng cultivars from P. quinquefolius and P. notoginseng. In the CAPS analysis results, the majority of the cleaved PCR products also yielded additional latent polymorphisms between the Korean ginseng cultivars and two foreign Panax species. Specific latent CAPS polymorphisms for cultivar Gopoong and Chunpoong were detected from internal region amplified with mt9 primer by treating HinfI and Tsp509I endonucleases, respectively. Sequencing analysis revealed that the length of amplified region of Korean ginseng cultivars was 2,179 bp, and those of P. quinquefolius and P. notoginseng were 2,178 and 2,185 bp, respectively. Blast search revealed that the amplified region was a mitochondrial cytochrome oxidase subunit 2 (cox2) gene intron II region. Nineteen single nucleotide polymorphisms (SNP) including each specific SNP for Gopoong and Chunpoong, and three insertion and deletion (InDel) polymorphisms were detected by sequence alignment. The CAPS markers developed in this study, which are specific to Gopoong and Chunpoong, and between the Korean ginseng cultivars and two foreign Panax species, will serve as a practical and reliable tool for their identification, purity maintenance, and selection of candidate lines and cultivars.     

Change in body size of juvenile marbled sole <Emphasis Type="Italic">Pseudopleuronectes yokohamae</Emphasis> after preservation in ethanol

We evaluated the extent of shrinkage in body size of juvenile marbled sole Pseudopleuronectes yokohamae after preservation in 70% ethanol for 6, 12, 24, or 48 h, or 4 weeks. Standard length (SL, range of the analyzed specimens: 22.82–53.01 mm) decreased by 5.6% after 12 h and body weight (BW 0.174–2.964 g) decreased 27.8% after 24 h preservation in 70% ethanol. There was no further decrease in SL or BW after 12 or 24 h of preservation, respectively. The original body size could be estimated based on the size after preservation in 70% ethanol for 4 weeks using the following equations: SL _original = 1.05 SL _preserved + 0.37 and BW _original = 1.36 BW _preserved + 0.04. The condition factor calculated using the body size of preserved individuals was 16.0 and 15.8% lower than that calculated from the original body size and the body size back-calculated using the linear regression equations described above, respectively. Thus, our data suggest that the shrinkage of body size due to preservation in ethanol can cause errors in estimation of the condition factor. Our results can be used to improve the accuracy of estimating size-related biological parameters based on juvenile marbled sole that are preserved in ethanol.     

Protein tyrosine phosphatase profiling analysis of HIB-1B cells during brown adipogenesis

A number of evidence have been accumulated that the regulation of reversible tyrosine phosphorylation, which can be regulated by the combinatorial activity of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), plays crucial roles in various biological processes including differentiation. There are a total of 107 PTP genes in the human genome, collectively referred to as the "PTPome." In this study, we performed PTP profiling analysis of the HIB-1B cell line, a brown preadipocyte cell line, during brown adipogenesis. Through RT-PCR and real-time PCR, several PTPs showing differential expression pattern during brown adipogenesis were identified. In the case of PTP-RE, it was shown to decrease significantly until 4 days after brown adipogenic differentiation, followed by a dramatic increase at 6 days. The overexpression of PTP-RE led to decreased brown adipogenic differentiation via reducing the tyrosine phosphorylation of the insulin receptor, indicating that PTP-RE functions as a negative regulator at the early stage of brown adipogenesis.     

Human papillomavirus (HPV) type distribution in Korean women: a meta-analysis

The aim of the present study is to estimate the overall prevalence and type distribution of human papillomavirus (HPV) in Korean women, through literature review and meta-analysis. We searched published data for the period between 1995 and 2007 using the following inclusion criteria; (1) studies using type-specific HPV tests, (2) data from Korean female, (3) with cytologic or pathologic results, (4) having more than 20 cases for each subgroup classified by cytologic results, and (5) HPV detection including types 16, 18, and at least one other type. In total, 18 studies (13,842 cases) published up to April 2007 were identified and selected. Adjusted overall HPV prevalence was 23.9% (95% CI: 23.8-24.1%) in women with normal cytology and 95.8% (95% CI: 95.4-96.2%) in women with cervical cancer. Type 16 was predominant regardless of cervical disease status, and type 58 occupied a significantly larger proportion in high-grade cervical intraepithelial lesions and cervical cancer in Korean women. HPV types 58, 33, and 52 together accounted for about 20% of infections in cervical cancer and high-grade intraepithelial lesions. After introduction of HPV prophylactic vaccines, extended protection, especially against types 58, 33, and 52, will be an important issue for cervical cancer prevention in Korea. The future dominant genotypes will require follow-up epidemiological studies with a large-scale, multicentered, and prospective design.     

Enhanced COD and nitrogen removals for the treatment of swine wastewater by combining submerged membrane bioreactor (MBR) and anaerobic upflow bed filter (AUBF) reactor

In order to enhance performances of organics removal and nitrification for the treatment of swine wastewater containing high concentration of organic solids and nitrogen than conventional biological nitrogen removal process, a submerged membrane bioreactor (MBR) was followed by an anaerobic upflow bed filter (AUBF) reactor in this research (AUBF–MBR process). The AUBF reactor is a hybrid reactor, which is the combination of an anoxic filter for denitrification and upflow anaerobic sludge blanket (UASB) for acid fermentation. In the AUBF–MBR process, it showed a considerable enhancement of the effluent quality in terms of COD removal and nitrification. The submerged MBR could maintain more than 14,000 mg VSS/L of the biomass concentration. Total nitrogen (T-N) removal efficiency represented 60% when internal recycle ratio was three times of flow-rate (Q), although the nitrification occurred completely. Although the volatile fatty acids produced in AUBF reactor can enhance denitrification rate, but the AUBF–MBR process showed reduction of overall removal efficiency of the nitrogen due to the reduction of carbon source by methane production in the AUBF reactor compared to that of theoretical nitrogen removal efficiency.

Long-term operation of the submerged MBR showed that the throughputs of the submerged MBR were respectively 74, 63, and 31 days at 10, 15, and 30 L/m² h (LMH) of permeate flux. Resistance to filtration by rejected solid is the primary cause of fouling, however the priority of cake resistance (R_c) and fouling resistance (R_f) with respect to filtration phenomenon was different according to the amount of permeate flux. The submerged MBR, here, achieved a steady-state flux of 15 LMH at 0.4 atm. of trans-membrane pressure (TMP) but the flux can be enhanced in the future because shear force by tangential flow will be greater when multi-layer sheets of membrane were used.