Meprin A and meprin α generate biologically functional IL-1β from pro-IL-1β

The present study demonstrates that both oligomeric metalloendopeptidase meprin A purified from kidney cortex and recombinant meprin α are capable of generating biologically active IL-1β from its precursor pro-IL-1β. Amino-acid sequencing analysis reveals that meprin A and meprin α cleave pro-IL-1β at the His¹¹⁵-Asp¹¹⁶ bond, which is one amino acid N-terminal to the caspase-1 cleavage site and five amino acids C-terminal to the meprin β site. The biological activity of the pro-IL-1β cleaved product produced by meprin A, determined by proliferative response of helper T-cells, was 3-fold higher to that of the IL-1β product produced by meprin β or caspase-1. In a mouse model of sepsis induced by cecal ligation puncture that results in elevated levels of serum IL-1β, meprin inhibitor actinonin significantly reduces levels of serum IL-1β. Meprin A and meprin α may therefore play a critical role in the production of active IL-1β during inflammation and tissue injury.     

Nitrooxyethylation reverses the healing-suppressant effect of Ibuprofen

Nonsteroidal antiinflammatory drugs like ibuprofen impede tissue repair by virtue of retarding inflammation. The present study was undertaken to explore if linking of nitrooxyethyl ester to ibuprofen reverses its healing-depressant propensity. Nitrooxyethyl ester of ibuprofen (NOE-Ibu) was synthesized in our laboratory through a well-established synthetic pathway. NOE-Ibu was screened for its influence on collagenation, wound contraction and epithelialization phases of healing, and scar size of healed wound in three wound models, namely, incision, dead space, and excision wounds. Besides, its influence on the oxidative stress (levels of GSH and TBARS) was also determined in 10-day-old granulation tissue. NOE-Ibu was further screened for its antiinflammatory activity in rat paw edema model. NOE-Ibu promoted collagenation (increase in breaking strength, granulation weight, and collagen content), wound contraction and epithelialization phases of healing. NOE-Ibu also showed a significant antioxidant effect in 10-day-old granulation tissue as compared to ibuprofen. Results vindicate that the esterification of ibuprofen with nitrooxyethyl group reverses the healing-suppressant effect of ibuprofen. The compound also showed equipotent antiinflammatory activity as ibuprofen.     

A Mycobacterium tuberculosis Sigma Factor Network Responds to Cell-Envelope Damage by the Promising Anti-Mycobacterial Thioridazine

Background

Novel therapeutics are urgently needed to control tuberculosis (TB). Thioridazine (THZ) is a candidate for the therapy of multidrug and extensively drug-resistant TB.

Methodology/Principal Findings

We studied the impact of THZ on Mycobacterium tuberculosis (Mtb) by analyzing gene expression profiles after treatment at the minimal inhibitory (1x MIC) or highly inhibitory (4x MIC) concentrations between 1–6 hours. THZ modulated the expression of genes encoding membrane proteins, efflux pumps, oxido-reductases and enzymes involved in fatty acid metabolism and aerobic respiration. The Rv3160c-Rv3161c operon, a multi-drug transporter and the Rv3614c/3615c/3616c regulon, were highly induced in response to THZ. A significantly high number of Mtb genes co-expressed with σ^B (the σ^B regulon) was turned on by THZ treatment. σ^B has recently been shown to protect Mtb from envelope-damage. We hypothesized that THZ damages the Mtb cell-envelope, turning on the expression of the σ^B regulon. Consistent with this hypothesis, we present electron-microscopy data which shows that THZ modulates cell-envelope integrity. Moreover, the Mtb mutants in σ^H and σ^E, two alternate stress response sigma factors that induce the expression of σ^B, exhibited higher sensitivity to THZ, indicating that the presence and expression of σ^B allows Mtb to resist the impact of THZ. Conditional induction of σ^B levels increased the survival of Mtb in the presence of THZ.

Conclusions/Significance

THZ targets different pathways and can thus be used as a multi-target inhibitor itself as well as provide strategies for multi-target drug development for combination chemotherapy. Our results show that the Mtb sigma factor network comprising of σ^H, σ^E and σ^B plays a crucial role in protecting the pathogen against cell-envelope damage.     

Microdissection approaches in tuberculosis research

Mycobacterium tuberculosis, a globally significant pathogen, results in active or latent tuberculosis. The granuloma is the characteristic lesion that offers insight into host–pathogen interactions in these distinct states. Microdissection provides a way to isolate and consequently investigate specific tissue sections. We review various techniques available and in use.     

Quality control of integral membrane proteins

Integral membrane proteins (IMPs) are essential components of the plasma and organellar membranes of the eukaryotic cell. Non-native IMPs, which can arise as a result of mutations, errors during biosynthesis or cellular stress, can disrupt these membranes and potentially lead to cell death. To protect against this outcome, the cell possesses quality control (QC) systems that detect and dispose of non-native IMPs from cellular membranes. Recent studies suggest that recognition of non-native IMPs by the QC machinery is correlated with the thermodynamic stability of these proteins. Consistent with this, small molecules known as chemical and pharmacological chaperones have been identified that stabilize non-native IMPs and enable them to evade QC. These findings have far-reaching implications for treating human diseases caused by defective IMPs.     

Longitudinal patterns in carbon and nitrogen fluxes and stream metabolism along an urban watershed continuum

An urban watershed continuum framework hypothesizes that there are coupled changes in (1) carbon and nitrogen cycling, (2) groundwater-surface water interactions, and (3) ecosystem metabolism along broader hydrologic flowpaths. It expands our understanding of urban streams beyond a reach scale. We evaluated this framework by analyzing longitudinal patterns in: C and N concentrations and mass balances, groundwater-surface interactions, and stream metabolism and carbon quality from headwaters to larger order streams. 52 monitoring sites were sampled seasonally and monthly along the Gwynns Falls watershed, which drains 170 km² of the Baltimore Long-Term Ecological Research site. Regarding our first hypothesis of coupled C and N cycles, there were significant inverse linear relationships between nitrate and dissolved organic carbon (DOC) and nitrogen longitudinally (P < 0.05). Regarding our second hypothesis of coupled groundwater-surface water interactions, groundwater seepage and leaky piped infrastructure contributed significant inputs of water and N to stream reaches based on mass balance and chloride/fluoride tracer data. Regarding our third hypothesis of coupled ecosystem metabolism and carbon quality, stream metabolism increased downstream and showed potential to enhance DOC lability (e.g., ~4 times higher mean monthly primary production in urban streams than forest streams). DOC lability also increased with distance downstream and watershed urbanization based on protein and humic-like fractions, with major implications for ecosystem metabolism, biological oxygen demand, and CO₂ production and alkalinity. Overall, our results showed significant in-stream retention and release (0–100 %) of watershed C and N loads over the scale of kilometers, seldom considered when evaluating monitoring, management, and restoration effectiveness. Given dynamic transport and retention across evolving spatial scales, there is a strong need to longitudinally and synoptically expand studies of hydrologic and biogeochemical processes beyond a stream reach scale along the urban watershed continuum.     

In Vitro Evaluation of a Soluble Leishmania Promastigote Surface Antigen as a Potential Vaccine Candidate against Human Leishmaniasis

PSA (Promastigote Surface Antigen) belongs to a family of membrane-bound and secreted proteins present in several Leishmania (L.) species. PSA is recognized by human Th1 cells and provides a high degree of protection in vaccinated mice. We evaluated humoral and cellular immune responses induced by a L. amazonensis PSA protein (LaPSA-38S) produced in a L. tarentolae expression system. This was done in individuals cured of cutaneous leishmaniasis due to L. major (CCLm) or L. braziliensis (CCLb) or visceral leishmaniasis due to L. donovani (CVLd) and in healthy individuals. Healthy individuals were subdivided into immune (HHR-Lm and HHR-Li: Healthy High Responders living in an endemic area for L. major or L. infantum infection) or non immune/naive individuals (HLR: Healthy Low Responders), depending on whether they produce high or low levels of IFN-γ in response to Leishmania soluble antigen. Low levels of total IgG antibodies to LaPSA-38S were detected in sera from the studied groups. Interestingly, LaPSA-38S induced specific and significant levels of IFN-γ, granzyme B and IL-10 in CCLm, HHR-Lm and HHR-Li groups, with HHR-Li group producing TNF-α in more. No significant cytokine response was observed in individuals immune to L. braziliensis or L. donovani infection. Phenotypic analysis showed a significant increase in CD4+ T cells producing IFN-γ after LaPSA-38S stimulation, in CCLm. A high positive correlation was observed between the percentage of IFN-γ-producing CD4+ T cells and the released IFN-γ. We showed that the LaPSA-38S protein was able to induce a mixed Th1 and Th2/Treg cytokine response in individuals with immunity to L. major or L. infantum infection indicating that it may be exploited as a vaccine candidate. We also showed, to our knowledge for the first time, the capacity of Leishmania PSA protein to induce granzyme B production in humans with immunity to L. major and L. infantum infection.     

The Mycobacterium tuberculosis Rv2745c Plays an Important Role in Responding to Redox Stress

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death from an infectious disease worldwide. Over the course of its life cycle in vivo, Mtb is exposed to a plethora of environmental stress conditions. Temporal regulation of genes involved in sensing and responding to such conditions is therefore crucial for Mtb to establish an infection. The Rv2745c (clgR) gene encodes a Clp protease gene regulator that is induced in response to a variety of stress conditions and potentially plays a role in Mtb pathogenesis. Our isogenic mutant, Mtb:ΔRv2745c, is significantly more sensitive to in vitro redox stress generated by diamide, relative to wild-type Mtb as well as to a complemented strain. Together with the fact that the expression of Rv2745c is strongly induced in response to redox stress, these results strongly implicate a role for ClgR in the management of intraphagosomal redox stress. Additionally, we observed that redox stress led to the dysregulation of the expression of the σ^H/σ^E regulon in the isogenic mutant, Mtb:ΔRv2745c. Furthermore, induction of clgR in Mtb and Mtb:ΔRv2745c (comp) did not lead to Clp protease induction, indicating that clgR has additional functions that need to be elucidated. Our data, when taken together with that obtained by other groups, indicates that ClgR plays diverse roles in multiple regulatory networks in response to different stress conditions. In addition to redox stress, the expression of Rv2745c correlates with the expression of genes involved in sulfate assimilation as well as in response to hypoxia and reaeration. Clearly, the Mtb Rv2745c-encoded ClgR performs different functions during stress response and is important for the pathogenicity of Mtb in-vivo, regardless of its induction of the Clp proteolytic pathway.     

Purification of a moderate thermotolerant Bacillus coagulans BTS1 lipase and its properties in a hydro-gel system

An alkaline thermotolerant lipase of Bacillus coagulans BTS1 was successively purified by ammonium sulfate precipitation and DEAE anion exchange chromatography. The purified lipase immobilized in alginate beads showed an optimal activity at pH 7.5 and 55 degrees C. A pH of 5.0 or 10.0 completely quenched the activity of immobilized lipase. The alginate-bound lipase retained its activity following exposure to most of the organic solvents including amines, alkanes and alcohols. Chloride salt of Al3+, Co2+, Mg2+ and NH4+ modulated the lipase activity of alginate-immobilized enzyme. The alginate entrapped lipase showed a preferentially high activity towards p-nitrophenyl palmitate (C: 16) and activity of matrix increased following exposure to SDS. Moreover, the immobilized lipase retained more than 50% of its activity after 3rd cycle of reuse.