Evaluation of shelf life and rock phosphate solubilization of Burkholderia sp. in nutrient-amended clay, rice bran and rock phosphate-based granular formulation

Five phosphate-solubilizing bacteria (PSB) used in this study were isolated based on their ability to solubilize tricalcium phosphate (TCP) in Pikovskaya’s medium. Among the tested bacterial strains Burkholderia sp. strain CBPB-HIM showed the highest solubilization (363 μg of soluble P ml⁻¹) activity at 48 h of incubation. Further, this strain has been selected to assess its shelf life in nutrient-amended and -unamended clay, rice bran and rock phosphate (RP) pellet-based granular formulation. The results showed that the maximum viability of bacterium was observed in clay and rice bran (1:1) + 10% RP pellets than clay-RP pellets, irrespective of tested storage temperatures. Further, clay and rice bran (1:1) + 10% RP pellets amended with 1% glucose supported the higher number of cells compared to glycerol-amended and nutrient-unamended pellets. In this carrier solubilization of Morocco rock phosphate (MRP) by Burkholderia sp. strain CBPB-HIM was also investigated. The maximum of water and bicarbonate extractable P (206 and 245 μg P g⁻¹ of pellet respectively) was recorded in clay and rice bran (1:1) + 10% RP pellets amended with 1% glucose and glycerol respectively on day 5 of incubation. Therefore, this study proved the possibility of developing granular inoculant technology combining clay, rice bran and RP as substrates with phosphate-solubilizing Burkholderia.     

Microarray multiplex assay for the simultaneous detection and discrimination of hepatitis B, hepatitis C, and human immunodeficiency type-1 viruses in human blood samples

Hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type-1 (HIV-1) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health worldwide. We developed a microarray multiplex assay for the simultaneous detection and discrimination of these three viruses. The microarray consists of 16 oligonucleotide probes, immobilized on a silylated glass slide. Amplicons from multiplex PCR were labeled with Cy-5 and hybridized to the microarray. The assay detected 1 International Unit (IU), 10 IU, 20 IU of HBV, HCV, and HIV-1, respectively, in a single multiplex reaction. The assay also detected and discriminated the presence of two or three of these viruses in a single sample. Our data represent a proof-of-concept for the possible use of highly sensitive multiplex microarray assay to screen and confirm the presence of these viruses in blood donors and patients.     

Subunit dependence of Na channel slow inactivation and open channel block in cerebellar neurons

Purkinje and cerebellar nuclear neurons both have Na currents with resurgent kinetics. Previous observations, however, suggest that their Na channels differ in their susceptibility to entering long-lived inactivated states. To compare fast inactivation, slow inactivation, and open-channel block, we recorded voltage-clamped, tetrodotoxin-sensitive Na currents in Purkinje and nuclear neurons acutely isolated from mouse cerebellum. In nuclear neurons, recovery from all inactivated states was slower, and open-channel unblock was less voltage-dependent than in Purkinje cells. To test whether specific subunits contributed to this differential stability of inactivation, experiments were repeated in Na(V)1.6-null (med) mice. In med Purkinje cells, recovery times were prolonged and the voltage dependence of open-channel block was reduced relative to control cells, suggesting that availability of Na(V)1.6 is quickly restored at negative potentials. In med nuclear cells, however, currents were unchanged, suggesting that Na(V)1.6 contributes little to wild-type nuclear cells. Extracellular Na(+) prevented slow inactivation more effectively in Purkinje than in nuclear neurons, consistent with a resilience of Na(V)1.6 to slow inactivation. The tendency of nuclear Na channels to inactivate produced a low availability during trains of spike-like depolarization. Hyperpolarizations that approximated synaptic inhibition effectively recovered channels, suggesting that increases in Na channel availability promote rebound firing after inhibition.     

Some findings of FADD knockdown in inhibition of HIV-1 replication in Jurkat cells and PBMCs

Fas-associated protein with death domain (FADD) is a key adaptor molecule transmitting the death signal mediated by death receptors, and it is also required for T cell proliferation. A recent study indicated that FADD is able to affect HIV-1 production, but the mechanism is not known. Using the susceptible Jurkat cell line and peripheral blood mononuclear cells, we studied the effects of FADD on HIV-1 production. TaqMan RT-PCR was used to quantify HIV-1 viral RNA copies, and Western blot analysis was used to detect protein expression. FADD knockdown decreased HIV-1 replication and inactivated caspase-3 activity in the cells and blocked CD4 translocation to the lipid rafts of the plasma membrane. Reduced expression of FADD suppressed TCR signaling through downregulation of TCR, CD3, and Zap-70 in response to HIV-1 infection and blocked the trafficking of TCR, CD3, CD28, and Zap-70 to lipid rafts, leading to reduced activation of NF-κB and NFAT, which are required for HIV-1 replication. FADD knockdown diminished caspase-8 migration to lipid rafts and its expression in response to HIV-1 infection. These results indicate that FADD, as a host pro-apoptotic protein, plays important roles in regulating HIV-1 replication and production in several ways, and apoptotic pathway inhibition is able to decrease HIV-1 replication and production.     

Parametric Model of Combination Therapy for Non-Hodgkin Lymphoma

The development and clinical testing of drug combinations for the treatment of Non-Hodgkin Lymphoma (NHL) and other cancers has recently shown great promise. However, determining the optimum combination and its associated dosages for maximum efficacy and minimum side effects is still a challenge. This paper describes a parametric analysis of the dynamics of malignant B-cells and the effects of an anti-sense oligonucleotide targeted to BCL-2 (as-bcl-2), anti-CD-20 (rituximab) and their combination, for a SCID mouse human lymphoma xenograft model of NHL. Our parametric model is straightforward. Several mechanisms of malignant B-cell birth and death in the nodal micro-environment are simulated. Cell death is accelerated by hypoxia and starvation induced by tumor scale, by modification of anti-apoptosis with as-bcl-2, and by direct kill effects of rituximab (cell kill by cytotoxic immune cells is not included, due to the absence of an immune system in the corresponding experiments). We show that the cell population dynamics in the control animals are primarily determined by K*, the ratio of rate constants for malignant cell death, K_d, and cell birth, K_b. Tumor growth with independent treatments is reproduced by the model, and is used to predict their effect when administered in combination. Malignant cell lifetimes are derived to provide a quantitative comparison of the efficacy of these treatments. Future experimental and clinical applications of the model are discussed.     

Comparative tuberculosis (TB) prevention effectiveness in children of Bacillus Calmette-Guérin (BCG) vaccines from different sources, Kazakhstan

Background

Except during a 1-year period when BCG vaccine was not routinely administered, annual coverage of infants with Bacillus Calmette-Guérin (BCG) in Kazakhstan since 2002 has exceeded 95%. BCG preparations from different sources (Japan, Serbia, and Russia) or none were used exclusively in comparable 7-month time-frames, September through March, in 4 successive years beginning in 2002. Our objective was to assess relative effectiveness of BCG immunization.

Methods/Findings

We compared outcomes of birth cohorts from the 4 time-frames retrospectively. Three cohorts received vaccine from one of three manufacturers exclusively, and one cohort was not vaccinated. Cohorts were followed for 3 years for notifications of clinical TB and of culture-confirmed TB, and for 21 months for TB meningitis notifications. Prevention effectiveness based on relative risk of TB incidence was calculated for each vaccinated cohort compared to the non-vaccinated cohort.Although there were differences in prevention effectiveness observed among the three BCG vaccines, all were protective. The Japanese vaccine (currently used in Kazakhstan), the Serbian vaccine, and the Russian vaccine respectively were 69%, 43%, and 22% effective with respect to clinical TB notifications, and 92%, 82%, and 51% effective with respect to culture confirmed TB. All three vaccines were >70% effective with respect to TB meningitis.

Limitations

Potential limitations included considerations that 1) the methodology used was retrospective, 2) multiple risk factors could have varied between cohorts and affected prevention effectiveness measures, 3) most cases were clinically diagnosed, and TB culture-positive case numbers and TB meningitis case numbers were sparse, and 4) small variations in reported population TB burden could have affected relative risk of exposure for cohorts.

Conclusions/Significance

All three BCG vaccines evaluated were protective against TB, and prevention effectiveness varied by manufacturer. When setting national immunization policy, consideration should be given to prevention effectiveness of BCG preparations.     

Knotting the NETs: Analyzing histone modifications in neutrophil extracellular traps

Neutrophil extracellular chromatin traps (NETs) are a recently described mechanism of innate immune responses to bacteria and fungi. Evidence indicates that NETs are induced by inflammation, that they contribute to diverse disease pathologies, and that they associate with bactericidal substances. Genomic DNA is released in NETs, leading to a cell death that has been labeled NETosis. Although NETosis clearly differs from apoptosis, the classical form of cell death, recent experiments indicate a connection between NETosis and autophagy. The regulated deployment of NETs may require covalent modification of histones, the basic DNA-binding proteins that organize chromatin in the cell''s nucleus and within NETs. Histone modification by peptidylarginine deiminase 4 (PAD4) is necessary for NET release. The functions of additional histone modifications, however, remain to be tested.Less than a decade since their discovery, neutrophil extracellular traps (NETs) remain in the headlines. Initially, interest focused on the structure of extracellular NET chromatin and its capacity to capture and damage bacteria. Soon, however, researchers began to see the implications of extracellular chromatin for the development of autoimmune diseases. One quintessential autoimmune disease, systemic lupus erythematosus (SLE), is known to arise together with autoantibodies to DNA and chromatin, although the immediate trigger for the production of these autoantibodies is unclear. A connection between NETs and autoimmunity was made by discovering that histones, a set of proteins that act as a structural harness for DNA in chromatin, are modified by peptidylarginine deiminase 4 (PAD4), an enzyme that converts arginines to citrullines. Researchers had long suspected that autoantigen modifications could provide the initial stimuli in autoimmunity because subtle alterations in a protein''s primary sequence can break tolerance. PAD4 is implicated in the development of rheumatoid arthritis (RA) because the most reliable clinical test for RA uses the detection of anti-citrulline antibodies in the sera of patients.In a sophisticated set of experiments reported in the previous issue of Arthritis Research & Therapy, Liu and colleagues [1] accomplished an extensive inventory of post-translational modifications in NET histones. The researchers induced NETs from human neutrophils, as well as two cell lines that assume neutrophil-like characteristics, and used a panel of 40 commercially available antisera to identify histone modifications that arise in parallel with NETs. Stimuli that were used to elicit NET release also induced histone H3 and H4 citrullination in human neutrophils and the EPRO cell line. However, other modifications such as histone H4 lysine 20 methylation and H4 lysine 16 acetylation showed inconsistent results in neutrophils versus the EPRO cells. To survey histone modifications, Liu and colleagues [1] confronted technical difficulties in that histone amino terminal tails contain the highest concentration of histone modifications yet are also highly susceptible to proteases secreted by activated neutrophils [2,3]. The histone tails act as flexible tethers that organize chromatin into higher-order structures. Interestingly, purified NETs failed to induce an immune response in mice, although a subset of SLE sera reacted strongly with citrullinated histone H3 [1]. Therefore, mechanisms that regulate histone modification deserve further attention.Neeli and colleagues [4] were the first to identify citrullinated histone H3 in NETs, a discovery that was confirmed by others [5]. Neeli and colleagues [4] provided a second important insight, namely that PAD4-citrullinated histone H3 is a reliable marker of inflammation. Thus, it became clear that the release of NETs is not an ''accident'' caused by a barrage of proteases and reactive oxygen species unleashed from neutrophils. Instead, production of NETs requires enzymatic activity and input from neutrophil surface receptors and the cytoskeleton [6]. By analyzing PAD4-deficient mice, Li and colleagues [7] demonstrated that PAD4 is essential for the production of NETs in response to bacterial infections. The regulation of PAD4 activity thus moved to the forefront of the research on NETs.It is now clear that NET release takes advantage of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase and the main granule proteases to trigger and construct the extended chromatin network [3,8]. In addition, myeloperoxidase is found in NETs after their release from the cells, and this enzyme and its products are the main components in NETs that kill bacteria [9]. In a notable study from the labs of Banchereau and Pascual [10], it was reported that SLE neutrophils are poised to undergo NETosis upon stimulation with anti-ribonucleo-protein autoantibodies and that NETs released by these neutrophils contain LL37 and HMGB-1, well-known stimulators of immune responses. In subsequent analyses using sera from patients with connective tissue disease, anti-citrullinated histone antibodies were observed in Felty''s syndrome, a rare disorder that shares serologic features with RA and SLE, whereas such autoantibodies were infrequent in SLE and RA [11]. These findings indicate that the process of NETosis is highly relevant to the development of human autoimmune responses, although a direct cause and effect may not connect the release of NETs to the production of autoantibodies.The detailed characterization of NET histone modifications, as accomplished by Liu and colleagues [1], invites speculations about the possible functions of these modifications. Several questions deserve further study: Will NET histone modifications, such as methylation, acetylation, and citrullination, be found to participate in gene regulation that sets the stage for NET release? Will the primary function of histone modifications turn out to be the decondensation of nuclear chromatin that is required for NETs expand to their optimal size and internal structure? Alternatively, NET histone modifications may serve non-traditional purposes. For example, certain modifications may anchor other NET components such as elastase, LL37, or myeloperoxidase to the chromatin meshwork. Unique modifications in NETs may attract phagocytes and stimulate them to ingest the trapped microorganisms. Other histone modifications may activate or dampen the inflammatory response by acting on innate pattern recognition receptors. The answers to these questions will, no doubt, keep research on NETs in leading immunology and microbiology journals for years to come.     

Different designs of kinase-phosphatase interactions and phosphatase sequestration shapes the robustness and signal flow in the MAPK cascade

Background

In developmental biology, there has been a recent focus on the robustness of morphogen gradients as possible providers of positional information. It was shown that functional morphogen gradients present strong biophysical constraints and lack of robustness to noise. Here we explore how the details of the mechanism which underlies the generation of a morphogen gradient can influence those properties.

Results

We contrast three gradient-generating mechanisms, (i) a source-decay mechanism; and (ii) a unidirectional transport mechanism; and (iii) a so-called reflux-loop mechanism. Focusing on the dynamics of the phytohormone auxin in the root, we show that only the reflux-loop mechanism can generate a gradient that would be adequate to supply functional positional information for the Arabidopsis root, for biophysically reasonable kinetic parameters.

Conclusions

We argue that traits that differ in spatial and temporal time-scales can impose complex selective pressures on the mechanism of morphogen gradient formation used for the development of the particular organism.     

Oscillations in MAPK cascade triggered by two distinct designs of coupled positive and negative feedback loops

ABSTRACT: BACKGROUND: Feedback loops, both positive and negative are embedded in the Mitogen Activated Protein Kinase (MAPK) cascade. In the three layer MAPK cascade, both feedback loops originate from the terminal layer and their sites of action are either of the two upstream layers. Recent studies have shown that the cascade uses coupled positive and negative feedback loops in generating oscillations. Two plausible designs of coupled positive and negative feedback loops can be elucidated from the literature; in one design the positive feedback precedes the negative feedback in the direction of signal flow and vice-versa in another. But it remains unexplored how the two designs contribute towards triggering oscillations in MAPK cascade. Thus it is also not known how amplitude, frequency, robustness or nature (analogous/digital) of the oscillations would be shaped by these two designs. RESULTS: We built two models of MAPK cascade that exhibited oscillations as function of two underlying designs of coupled positive and negative feedback loops. Frequency, amplitude and nature (digital/analogous) of oscillations were found to be differentially determined by each design. It was observed that the positive feedback emerging from an oscillating MAPK cascade and functional in an external signal processing module can trigger oscillations in the target module, provided that the target module satisfy certain parametric requirements. The augmentation of the two models was done to incorporate the nuclear-cytoplasmic shuttling of cascade components followed by induction of a nuclear phosphatase. It revealed that the fate of oscillations in the MAPK cascade is governed by the feedback designs. Oscillations were unaffected due to nuclear compartmentalization owing to one design but were completely abolished in the other case. CONCLUSION: The MAPK cascade can utilize two distinct designs of coupled positive and negative feedback loops to trigger oscillations. The amplitude, frequency and robustness of the oscillations in presence or absence of nuclear compartmentalization were differentially determined by two designs of coupled positive and negative feedback loops. A positive feedback from an oscillating MAPK cascade was shown to induce oscillations in an external signal processing module, uncovering a novel regulatory aspect of MAPK signal processing.