Chaperone proteins as single component reagents to assess antibody nonspecificity

Early stage assays that evaluate monoclonal antibody drug-like properties serve as valuable tools for selection of lead candidates. One liability for clinical development, off-target reactivity, is often assessed by binding to a mixture or panel of noncognate proteins. While robust, these mixes are often ill-defined, and can suffer from issues such as lot-to-lot variability. In this study, we discovered in immunoprecipitation experiments that certain chaperones are present in one of these mixtures;we then explored the use of recombinant chaperone proteins as well-characterized agents to predict antibody nonspecificity. Antibody binding to the heat shock proteins HSP70, HSP90, or trigger factor all served as predictors of cross-interaction propensity, with HSP90 providing the greatest ability to predict antibody clearance rates in mouse. Individual chaperone binding correlates surprisingly closely with binding to complex cell extracts, with the exception of a few “false negatives” (assuming a complex cell extract as the “true” value). As defined reagents, these chaperone reagents present advantages for high throughput assays of nonspecificity.     

The midgut microbiota plays an essential role in sand fly vector competence for Leishmania major

For many arthropod vectors, the diverse bacteria and fungi that inhabit the gut can negatively impact pathogen colonization. Our attempts to exploit antibiotic treatment of colonized Phlebotomus duboscqi sand flies in order to improve their vector competency for Leishmania major resulted instead in flies that were refractory to the development of transmissible infections due to the inability of the parasite to survive and to colonize the anterior midgut with infective, metacyclic stage promastigotes. The parasite survival and development defect could be overcome by feeding the flies on different symbiont bacteria but not by feeding them on bacterial supernatants or replete medium. The inhibitory effect of the dysbiosis was moderated by lowering the concentration of sucrose (<30% w/v) used in the sugar feeds to maintain the colony. Exposure of promastigotes to 30% sucrose was lethal to the parasite in vitro. Confocal imaging revealed that the killing in vivo was confined to promastigotes that had migrated to the anterior plug region, corresponding to the highest concentrations of sucrose. The data suggest that sucrose utilization by the microbiota is essential to promote the appropriate osmotic conditions required for the survival of infective stage promastigotes in vivo.     

Totarol inhibits bacterial cytokinesis by perturbing the assembly dynamics of FtsZ

Totarol, a diterpenoid phenol, has been shown to inhibit the proliferation of several pathogenic Gram-positive bacteria including Mycobacterium tuberculosis. In this study, totarol was found to inhibit the proliferation of Bacillus subtilis cells with a minimum inhibitory concentration of 2 microM. It did not detectably perturb the membrane structure of B. subtilis; it strongly induced the filamentation in B. subtilis cells, suggesting that it inhibits bacterial cytokinesis. Totarol (1.5 microM) reduced the frequency of the Z-ring occurrence per micrometer of the bacterial cell length but did not affect the nucleoid frequency, suggesting that it blocks cytokinesis by inhibiting the formation of the Z-ring. The assembly dynamics of FtsZ is thought to play an important role in the formation and functioning of the Z-ring, a machine that engineers cytokinesis in bacteria. Since totarol was shown to inhibit the proliferation of M. tuberculosis, we examined the effects of totarol on the assembly dynamics of M. tuberculosis FtsZ (MtbFtsZ) in vitro. Totarol decreased the assembly of MtbFtsZ protofilaments and potently suppressed the GTPase activity of MtbFtsZ. It bound to MtbFtsZ with a dissociation constant of 11 +/- 2.3 microM. It increased the fluorescence intensity of the MtbFtsZ-1-anilinonaphthalene-8-sulfonic acid complex and inhibited the fluorescence intensity of N-(1-pyrene)maleimide-labeled MtbFtsZ, suggesting that totarol induces conformational changes in MtbFtsZ. The results indicated that totarol can perturb the assembly dynamics of FtsZ protofilaments in the Z-ring. Totarol exhibited extremely weak inhibitory effects on HeLa cell proliferation. It did not affect microtubule organization in HeLa cells. The results suggest that totarol inhibits bacterial proliferation by targeting FtsZ and it may be useful as a lead compound to develop an effective antitubercular drug.     

Reactivity of platina-β-diketones towards chelating nitrogen and sulfur donors: formation of acyl(hydrido)platinum(IV) and acyl(chloro)platinum(II) complexes

The platina-β-diketone [Pt₂{(COMe)₂H}₂(μ-Cl)₂] (1) was found to react with chelating N,N-ligands 2(R^′NCR)C₅H₄N (R/R^′=Ph/OH, H/Ph, Me/Ph) to form acyl(hydrido)platinum(IV) complexes [Pt(COMe)₂Cl(H){2-(R^′NCR)C₅H₄N}] (R/R^′=Ph/OH 2a; H/Ph 2b; Me/Ph (2c)). Reactions of complex 1 with chelating S,S- and N,S-donors (RS-CH₂-CH₂-SR, 2-(RSCH₂)C₅H₄N, R=Et, Ph, t-Bu) afforded acyl(chloro)platinum(II) complexes [Pt(COMe)Cl(RSCH₂CH₂SR)] (R=Et, 3a; Ph, 3b; t-Bu, 3c) and [Pt(COMe)Cl{2-(RSCH₂)C₅H₄N}] (R=Et, 4a; Ph, 4b; t-Bu, 4c), respectively. All complexes were fully characterized by microanalysis, IR and NMR (¹H, ¹³C) spectroscopy. Furthermore, molecular structures of complexes 3b and 4b were determined by single-crystal X-ray diffraction analyses revealing close to square-planar configuration. In complex 4b the acetyl ligand is trans to pyridine N atom (configuration index SP-4-2). The reactions are discussed in terms of consecutive oxidative addition and reductive elimination reactions.     

Nematicidal and Molecular Docking Investigation of Essential Oils from Pogostemon cablin Ecotypes against Meloidogyne incognita

Root-knot nematode, Meloidogyne incognita is one of the most destructive nematodes worldwide. Essential oils (EOs) are being extensively utilized as eco-benign bionematicides, although the precise mechanism of action remains unclear. Pogostemon cablin Benth. is well-known as “Patchouli”. It is native to South East Asia and known for ethno-pharmacological properties. In this study, chemical composition and potential nematicidal effect of EOs hydrodistilled from the leaves of P. cablin grown at three different locations in India were comprehensively investigated to correlate their mechanism of action for target specific binding affinities toward nematode proteins. Aromatic volatile Pogostemon essential oils (PEO) from Northern India (PEO-NI), Southern India (PEO-SI) and North Eastern India (PEO-NEI) were analyzed by Gas Chromatography-Mass Spectrometry (GC/MS) to characterize forty volatile compounds. Maximum thirty-three components were identified in PEO-NEI. Sesquiterpenes were predominant with higher content of α-guaiene (2.3–24.4 %), patchoulol (6.1–32.7 %) and α-bulnesene (5.9–27.1 %). Patchoulol was the major component in PEO-SI (32.7±1.2 %) and PEO-NEI (29.2±1.1 %), while α-guaiene in PEO-NI (24.4±1.2 %). In vitro nematicidal assay revealed significant nematicidal action (LC₅₀ 44.6–87.0 μg mL⁻¹) against juveniles of M. incognita within 24 h exposure. Mortality increases with increasing time to 48 h (LC₅₀ 33.6–71.6 μg mL⁻¹) and 72 h (LC₅₀ 27.7–61.2 μg mL⁻¹). Molecular modelling and in silico studies revealed multi-modal inhibitive action of α-bulnesene (−22 to −13 kJ mol⁻¹) and α-guaiene (−22 to −12 kJ mol⁻¹) against three target proteins namely, acetyl cholinesterase (AChE), odorant response gene-1 (ODR1), odorant response gene-3 (ODR3). Most preferable binding mechanism was observed against AChE due to pi-alkyl, pi-sigma, and hydrophobic interactions. Structure nematicidal activity relationship suggested the presence of hydroxy group for nematicidal activity is nonessential, rather highly depends on synergistic composition of sesquiterpene hydrocarbons.     

Calmodulin-mediated activation of Akt regulates survival of c-Myc-overexpressing mouse mammary carcinoma cells

c-Myc-overexpressing mammary epithelial cells are proapoptotic; their survival is strongly promoted by epidermal growth factor (EGF). We now demonstrate that EGF-induced Akt activation and survival in transgenic mouse mammary tumor virus-c-Myc mouse mammary carcinoma cells are both calcium/calmodulin-dependent. Akt activation is abolished by the phospholipase C-gamma inhibitor U-73122, by the intracellular calcium chelator BAPTA-AM, and by the specific calmodulin antagonist W-7. These results implicate calcium/calmodulin in the activation of Akt in these cells. In addition, Akt activation by serum and insulin is also inhibited by W-7. EGF-induced and calcium/calmodulin-mediated Akt activation occurs in both tumorigenic and non-tumorigenic mouse and human mammary epithelial cells, independent of their overexpression of c-Myc. These results imply that calcium/calmodulin may be a common regulator of Akt activation, irrespective of upstream receptor activator, mammalian species, and transformation status in mammary epithelial cells. However, only c-Myc-overexpressing mouse mammary carcinoma cells (but not normal mouse mammary epithelial cells) undergo apoptosis in the presence of the calmodulin antagonist W-7, indicating the vital selective role of calmodulin for survival of these cells. Calcium/calmodulin-regulated Akt activation is mediated directly by neither calmodulin kinases nor phosphatidylinositol 3-kinase (PI-3 kinase). Pharmacological inhibitors of calmodulin kinase kinase and calmodulin kinases II and III do not inhibit EGF-induced Akt activation, and calmodulin antagonist W-7 does not inhibit phosphotyrosine-associated PI-3 kinase activation. Akt is, however, co-immunoprecipitated with calmodulin in an EGF-dependent manner, which is inhibited by calmodulin antagonist W-7. We conclude that calmodulin may serve a vital regulatory function to direct the localization of Akt to the plasma membrane for its activation by PI-3 kinase.     

Ruthenium red-induced bundling of bacterial cell division protein, FtsZ

The assembly of FtsZ plays a major role in bacterial cell division, and it is thought that the assembly dynamics of FtsZ is a finely regulated process. Here, we show that ruthenium red is able to modulate FtsZ assembly in vitro. In contrast to the inhibitory effects of ruthenium red on microtubule polymerization, we found that a substoichiometric concentration of ruthenium red strongly increased the light-scattering signal of FtsZ assembly. Further, sedimentable polymer mass was increased by 1.5- and 2-fold in the presence of 2 and 10 microm ruthenium red, respectively. In addition, ruthenium red strongly reduced the GTPase activity and prevented dilution-induced disassembly of FtsZ polymers. Electron microscopic analysis showed that 4-10 microm of ruthenium red produced thick bundles of FtsZ polymers. The significant increase in the light-scattering signal and pelletable polymer mass in the presence of ruthenium red seemed to be due to the bundling of FtsZ protofilaments into larger polymers rather than the actual increase in the level of polymeric FtsZ. Furthermore, ruthenium red was found to copolymerize with FtsZ, and the copolymerization of substoichiometric amounts of ruthenium red with FtsZ polymers promoted cooperative assembly of FtsZ that produced large bundles. Calcium inhibited the binding of ruthenium red to FtsZ. However, a concentration of calcium 1000-fold higher than that of ruthenium red was required to produce similar effects on FtsZ assembly. Ruthenium red strongly modulated FtsZ polymerization, suggesting the presence of an important regulatory site on FtsZ and suggesting that a natural ligand, which mimics the action of ruthenium red, may regulate the assembly of FtsZ in bacteria.     

In silico structural,functional and phylogenetic analysis of <Emphasis Type="Italic">Klebsiella</Emphasis> phytases

Phytic acid or phytate (myo-inositol hexakisphosphate) is the principal storage indigestible form of phosphorus in different crops. It is considered as an antinutrient in human as well as animal (including fish, poultry, pig, chicken etc.) diet due to its chelating behavior of certain essential divalent minerals (Fe²⁺, Mg²⁺, Zn²⁺, Ca²⁺ etc.). The unabsorbed, indigested form of phosphorus also causes phosphate pollution in the soil by animal wastes. Phytate degrading enzymes like phytases (myo-inositol hexakisphosphate phosphohydrolase) in this regard can be very useful and also economically feasible to reduce the risk of phosphate pollution and increase the nutrient value in animal feeds at the same time. The Klebsiella phytases are suitable to use in the food industries of plant origin for their excellent thermal stability and high pH tolerance. From the present in silico investigation, it was found that Klebsiella phytases were 46–47 kDa molecular weight protein of histidine phosphatase superfamily having thermostability and alkalinity nature. This thermostability can be achieved due to possession of higher percentage of α helices and β sheets at the same time; the presence of higher aliphatic indices (range in between 88 and 91) etc. Interestingly, a strong correlation was found to be pertinent from phylogenetic studies of proteins with their cDNA among both species and strain level. Hence, the present study would be beneficial for future researchers (3D model available in Protein Model Database with acc. no.: PM0080562) to meet the demand of agricultural and industrial production of bacterial phytases particularly for agricultural farming.     

Liquid biopsy: A new avenue in pathology

Liquid biopsy is a relatively new entity. This non‐invasive technique provides real‐time information about a tumour. The liquid biopsy contains circulating tumour cells, cell‐free DNA and exosomes. The main indications for liquid biopsy include early diagnosis, screening, detection of minimal residual disease, designing personalised treatment and predicting biological behaviour of the tumour. In this review, we discuss various aspects of liquid biopsy and compare it with conventional biopsy.