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Many strains from the Bacillus subtilis species complex exert strong plant growth-promoting activities. However, their efficacy in relevant conditions is variable, due in part to their inability to establish a strong interaction with roots in stressful environmental conditions. Adaptative laboratory evolution (ALE) is a powerful tool to generate novel strains with traits of interest. Many Bacillus evolved isolates, stemming from ALE performed with plants, possess a stronger root colonization capacity. An in-depth analysis of these isolates also allowed the identification of key features influencing the interaction with plant roots. However, many variables can influence the outcome of these assays, and thus, caution should be taken when designing ALE destined to generate better root colonizers., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Horizontal gene transfer (HGT) by integrative and conjugative elements (ICEs) is an important mechanism in the spread of antibiotic resistance genes. However, little is known about the spatiotemporal dynamic of ICE propagation in bacterial biofilms, which are multicellular structures ubiquitous in natural and clinical environments. We report here that a high level of biofilm matrix production favors ICE Bs1 acquisition. Also, using a fluorescently marked ICE Bs1 , we observed that conjugation appears restricted to clusters of bacteria in a close neighborhood in which a high level of ICE Bs1 transfer occurs. These conjugative clusters are heterogeneously distributed in the biofilm, forming close to the air-biofilm interface. Importantly, we established that transconjugant cells are the main contributors to ICE Bs1 propagation in biofilms. Our findings provide a novel spatiotemporal understanding of ICEs propagation in biofilms, which should have an important role in our understanding of horizontal gene transfer in relevant settings. IMPORTANCE The transfer of mobile genetic elements between bacteria is the main cause of the spread of antibiotic resistance genes. While biofilms are the predominant bacterial lifestyle both in the environment and in clinical settings, their impact on the propagation of mobile genetic elements is still poorly understood. In this study, we examined the spatiotemporal propagation of the well-characterized ICE Bs1 . Using the Gram-positive Bacillus subtilis, we observed that the main actors of ICE Bs1 propagation in biofilms are the newly formed transconjugants that allow rapid transfer of ICE Bs1 to new recipients. Our study provides a better understanding of the spatiotemporal dynamic of conjugative transfer in biofilms.

Cutaneous leishmaniasis (CL) affects the lives of 0.7-1 million people every year causing lesions that take months to heal. These lesions can result in disfiguring scars with psychological, social and economic consequences. Antimonials are the first line of therapy for CL, however the treatment is lengthy and linked to significant toxicities; further, its efficacy is variable and resistant parasites are emerging. Shorter or lower dose antimonial treatment regimens, which would decrease the risk of adverse events and improve patient compliance, have shown reduced efficacy and further increase the risk emergence of antimonial-resistant strains. The progression of lesions in CL is partly determined by the immune response it elicits, and previous studies showed that administration of immunomodulatory type D CpG ODNs, magnifies the immune response to Leishmania and reduces lesion severity in nonhuman primates (NHP) challenged with Leishmania major or Leishmania amazonensis. Here we explored whether the addition of a single dose of immunomodulating CpG ODN D35 augments the efficacy of a short-course, low-dose pentavalent antimonial treatment regimen. Results show that macaques treated with D35 plus 5mg/kg sodium stibogluconate (SbV) for 10 days had smaller lesions and reduced time to re-epithelization after infection with Leishmania major. No toxicities were evident during the studies, even at doses of D35 10 times higher than those used in treatment. Critically, pentavalent antimonial treatment did not modify the ability of D35 to induce type I IFNs. The findings support the efficacy of D35 as adjuvant therapy for shorter, low dose pentavalent antimonial treatment., Competing Interests: Daniela Verthelyi is named as an inventor on a patent covering D35 filed on April, 1999, US 7,960,356 B2 under the Serial number 60/128,898. WO2000/061151. The patent is owned by the US government and licensed to DNDi.

Stability of therapeutic proteins (TPs) is a critical quality attribute that impacts both safety and efficacy of the drug. Size stability is routinely performed during and after biomanufacturing. Dynamic light scattering (DLS) is a commonly used technique to characterize hydrodynamic size of the TPs. Herein, we have developed a novel method to evaluate in-use and thermal stress stability of TPs using algorithm-driven high-throughput DLS. Five marketed TPs were tested under the guidance of customized algorithms. The TPs were evaluated at relevant temperature conditions as well as under dilution and thermal stress for size stability. We found that the TPs were stable under the in-use conditions tested; however, sample loss due to evaporation can lead to large protein aggregates. A combined assessment of autocorrelation function and photos of sample well could be useful in formulation screening. Dilution of TPs also has an impact on the hydrodynamic size. Thermal stress experiments showed the importance of using different data processing methods to access size distribution. Polydispersity index was useful in evaluating sample heterogeneity. Herein, we show that algorithm-driven high-throughput DLS can provide additional supportive information during and after biomanufacturing and the potential to be used in a quality control environment., (Copyright © 2018. Published by Elsevier Inc.)

Isis 3521 and G3139 are 20- and 18-mer phosphorothioate oligonucleotides, respectively, targeted to the protein kinase C (PKC)-alpha and bcl-2 mRNAs. Treatment of T24 bladder and PC3 prostate carcinoma cells with full-length and 3'-truncation mutants of Isis 3521 causes down-regulation of PKC-alpha protein and mRNA. However, at the level of a 15-mer and shorter, down-regulation of mRNA expression is no longer observed. Further, no diminution in cellular viability, as measured by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide assay, in response to increasing concentrations of paclitaxel, can be observed for these shorter oligomers. These observations not only indicate that PKC-alpha protein expression can be down-regulated by both RNase H-dependent and -independent mechanisms but also that down-regulation of PKC-alpha is insufficient by itself to "chemosensitize" cells. G3139, which down-regulates bcl-2 protein and mRNA expression, also down-regulates PKC-alpha protein and mRNA expression but not that of PKC-betaI, -epsilon, or -zeta. However, the down-regulation of PKC-alpha and bcl-2 are not linked. When the carrier Eufectin 5 is employed, only bcl-2 is down-regulated in both T24 and PC3 cells at 50 nM oligonucleotide concentration. At 100 nM, both bcl-2 and PKC-alpha expression are down-regulated, and only at this concentration can "chemosensitization" to paclitaxel and carboplatin be observed. In contrast, the down-regulation of bcl-2 seems to be linked with that of RelA (p65). However, this too is also not sufficient for chemosensitization, even though it leads to the loss of expression of genes under the putative control of nuclear factor-kappaB and to detachment of the cells from plastic surfaces. These results underscore the complexity of the intracellular requirements for the initiation of chemosensitization to anti-neoplastic agents.

This report emphasizes the interfacial chemistry that is required to ensure proper attachment of oligonucleotides onto the surface of microarrays. For example, strategies for the covalent attachment of pre-synthesized oligonucleotides to glass slides, gold films, polyacrylamide gel pads, polypyrrole films, and optical fibers are surveyed in an attempt to better define the parameters for optimal formation and detection of DNA hybrids. These parameters include among others, the nature and length of the linkers attaching oligonucleotides to the arrays, and the surface density of oligonucleotides required for unhindered hybridization with DNA targets. Sensitive detection methods such as the use of light-scattering techniques, molecular beacons, surface plasmon resonance, attenuated total internal reflection-FTIR, and the evanescent field excitation of fluorescence from surface-bound fluorophores have been developed to study the kinetics and specificity of hybridization events. Finally, the synthesis of oligonucleotides directly on glass surfaces and polypropylene sheets has been investigated to enable DNA sequencing by hybridization and achieve oligonucleotide densities of ca. 10(6) sequences per cm(2) on DNA chips.

The phosphoramidite approach has had a major impact on the synthesis of oligonucleotides. This unit describes parameters that affect the performance of this method for preparing oligodeoxyribonucleotides, as well as a number of compatible strategies. Milestones that led to the discovery of the approach are chronologically reported. Alternate strategies are also described to underscore the versatility by which these synthons can be obtained. Mechanisms of deoxyribonucleoside phosphoramidite activation, factors affecting condensation, and deprotection strategies are discussed.

The detailed preparation of deoxyribonucleoside phosphoramidites bearing a 4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butyl group for P(III) protection is presented. The use of this group circumvents nucleobase alkylation during oligonucleotide deprotection. Two syntheses of phosphoramidites starting from either a phosphordichloridite precursor or a bis-(N,N-diisopropylamino)chlorophosphine intermediate are described for the phosphinylation of suitably protected deoxyribonucleosides.