The road to drug resistance in Mycobacterium tuberculosis

Sequencing of serial isolates of extensively drug-resistant tuberculosis highlights how drug resistance develops within a single patient and reveals unexpected levels of pathogen diversity

Electrical weed control in sugar beet - A comparison of pre-emergence methods

Im Zuckerrübenanbau können schon geringe Unkrautdichten zu enormen Ertragseinbußen führen. Diese wurden in der Vergangenheit durch den Einsatz von chemischen Herbiziden effektiv begrenzt. Mit Blick auf den zunehmenden Wegfall verfügbarer chemischer Wirkstoffe, wachsender Unkrautresistenzen, einen nachhaltigeren Bodenschutz und die Einhaltung anspruchsvollerer Ernährungssicherungsstandards müssen neue Managementansätze für einen moderneren Pflanzenschutz umgesetzt werden. Aus diesem Grund wurde die elektrische Unkrautbekämpfung im Vorauflauf in der Zuckerrübe, im Mulchsaatverfahren angebaut, in chemische und mechanische Unkrautbekämpfungsstrategien integriert. Es wurde ein randomisiertes Blockfeldversuchsdesign mit dreimal replizierten Versuchsplots und acht Varianten angewendet, wobei einzelne Plots (100 m Länge) jeweils in 50 m getrennt, mit und ohne Glyphosat als Vorsaatanwendung behandelt wurden. Das Electroherb™ der Firma Zasso wurde mit 3 und 5 km h‑1 Fahrgeschwindigkeit und 72 kW elektrischer Nennleistung in einer 3 m Flächenbehandlung angewendet. Der Unkrautdichte nach der Vorauflauf-Applikation setzte sich hauptsächlich aus Ausfallgetreide und zweikeimblättrigen Unkrautpflanzen zusammen und betrug 5 bis 23 Pflanzen 10 m-2, während ohne Glyphosatanwendung 5 bis 97 Pflanzen 10 m-2 gezählt wurden. Der Vergleich der Wirkungsgrade zeigte, dass die Verunkrautung am besten mit der Electroherb™ Technologie kontrolliert wurde, so dass in den Parzellen ohne Glyphosatanwendung deutlich geringere Unkrautzahlen (von 5 bis 11 Pflanzen 10 m-2) vor dem Auflaufen der Zuckerrüben zurückblieben. Mechanisches Eggen auf der anderen Seite zeigte eine geringe Effizienz bei der Reduzierung von Ausfallgetreide; die durchschnittliche Unkrautdichte betrug hier 72 Pflanzen 10 m-2. Die Ergebnisse zum Zuckerrübenertrag und -qualität werden in den Datensatz integriert und erlauben so umfassende Schlussfolgerungen zur Wirtschaftlichkeit des Electroherb™ Verfahrens zu schlussfolgern. Unsere Ergebnisse zeigen, dass die Integration von elektrischen Unkrautbekämpfungsmethoden in bestehende Strategien den Einsatz von synthetisch-chemischen Herbiziden reduzieren kann und auch einen wesentlichen Beitrag zur Vermeidung von Herbizidresistenzen leisten kann.Competition between sugar beet and weeds can result in tremendous yield losses, which were confined by the use of chemical herbicides in the past. In view to address soil conservation, a reducing number of available chemical herbicides, the need to prevent weed resistances and exacting food security standards, new management approaches must be implemented into modern crop protection. In this context, electrical weed control for pre-emergence in sugar beet, cultivated in mulch sowing to prevent erosion and to improve water infiltration after heavy rainfall, was integrated in chemical and mechanical weed control strategies. A randomized complete block field trial design with three times replicated plots and eight variants was applied, whereby individual plots (100 m length in total) were separated in 50 m, with and without glyphosate as pre-sowing application. The Zasso Electroherb™ was applied with 3 and 5 km h‑1 speed and 72 kW nominal electrical power in a 3 m area application. Weed infestation after the pre-emergence weed control, mainly volunteer grain and dicotyledonous weed plants, with glyphosate averaged densities of 5 to 23 plants 10 m-2, whereas without glyphosate application 5 to 97 plants 10 m-2 were counted, respectively. Weed control efficiency indicated that the weed community was controlled best by Electroherb™, leaving in the plots without glyphosate application significantly lower weed numbers (of 5 to 11 plants 10 m-2) behind. Mechanical harrowing on the other side showed here minor efficiency in reducing the volunteer grain weed; averaging weed densities of 72 plants 10 m-2. Results on sugar beet yields and quality are pending but will be included in the data set for comprehensive conclusions. Our results indicate that the integration of electrical weed control methods into existing strategies can reduce the use of synthetic-chemical herbicides and can also make a significant contribution to the prevention of ongoing and future herbicide resistances

The fitness costs of drug resistance mutations in Mycobacteria

MSc., Faculty of Science, University of the Witwatersrand, 2011The increasing emergence of drug-resistant pathogens poses a major threat to public health. Although influenced by multiple factors, resistance is often associated with mutations in drug target-encoding or associated genes. The potential fitness cost of such resistance mutations is, in turn, a key determinant of the spread of drug-resistant strains. Rifampicin (RIF) is a frontline anti-tuberculosis agent that targets the rpoB-encoded β-subunit of the DNA-dependent RNA polymerase (RNAP). RIF resistance (RIFR) maps primarily to mutations in rpoB that might be expected to affect transcription and so the ability of the organism to cause disease. Accordingly, numerous studies have assessed the impact of RIFR on key fitness indicators in pathogens including Mycobacterium tuberculosis (MTB). In contrast, the specific consequences of RIFR for bacterial physiology remain poorly understood. Notably, previous studies of the effects of RIFR-associated rpoB mutations on mycobacterial physiology have been conducted using strains generated by RIF exposure, without accounting for the potential impact of second-site mutations that may compensate for fitness costs or contribute to drug resistance. In this study, site-directed mutagenesis and allelic exchange were employed to generate a panel of M. smegmatis (MSM) strains containing clinically-relevant RIFR-associated point mutations. Importantly, this methodology enables the introduction of rpoB mutations into defined strain backgrounds in the complete absence of RIF. Using this approach, we constructed “RIF naive” MSM rpoB mutant strains carrying either an S531L or H526Y mutation. The resulting mutants were 100-fold less susceptible to RIF than the isogenic, parental strain. Notably, the inclusion of selected efflux inhibitors in susceptibility assays had little impact on mutant susceptibility to RIF. In contrast, restoration of the wild-type allele returned the observed susceptibility to parental levels, thereby providing strong evidence of the sufficiency of a single rpoB mutation for clinical RIFR in mycobacteria. Competitive growth assays utilizing the S531L mutant and the parental strain exposed a growth defect for the S531L mutant. However, discriminating between wild-type and mutant rpoB strains proved a significant technical challenge, again highlighting the difficulties associated with inferring in vivo fitness from in vitro assays conducted under a limited number of different conditions. In summary, our results suggest the benefit of a deeper exploration of the physiological and fitness implications of RIFR-associated mutations. In addition, in coupling a system which enables an evaluation of the physiological consequences of drug resistance-associated mutations with evolutionary analyses, we provide preliminary evidence of the benefits of a multipronged approach to elucidating the physiological implications of drug resistance in MTB

In Vitro Development of Human iPSC-Derived Functional Neuronal Networks on Laser-Fabricated 3D Scaffolds

Neural progenitor cells generated from human induced pluripotent stem cells (hiPSCs) are the forefront of ″brain-on-chip″ investigations. Viable and functional hiPSC-derived neuronal networks are shaping powerful in vitro models for evaluating the normal and abnormal formation of cortical circuits, understanding the underlying disease mechanisms, and investigating the response to drugs. They therefore represent a desirable instrument for both the scientific community and the pharmacological industry. However, culture conditions required for the full functional maturation of individual neurons and networks are still unidentified. It has been recognized that three-dimensional (3D) culture conditions can better emulate in vivo neuronal tissue development compared to 2D cultures and thus provide a more desirable in vitro approach. In this paper, we present the design and implementation of a 3D scaffold platform that supports and promotes intricate neuronal network development. 3D scaffolds were produced through direct laser writing by two-photon polymerization (2PP), a high-resolution 3D laser microstructuring technology, using the biocompatible and nondegradable photoreactive resin Dental LT Clear (DClear). Neurons developed and interconnected on a 3D environment shaped by vertically stacked scaffold layers. The developed networks could support different cell types. Starting at the day 50 of 3D culture, neuronal progenitor cells could develop into cortical projection neurons (CNPs) of all six layers, different types of inhibitory neurons, and glia. Additionally and in contrast to 2D conditions, 3D scaffolds supported the long-term culturing of neuronal networks over the course of 120 days. Network health and functionality were probed through calcium imaging, which revealed a strong spontaneous neuronal activity that combined individual and collective events. Taken together, our results highlight advanced microstructured 3D scaffolds as a reliable platform for the 3D in vitro modeling of neuronal functions.publishedVersio

Novel approach to estimate tuberculosis transmission in primary care clinics in sub-Saharan Africa: protocol of a prospective study.

INTRODUCTION Tuberculosis (TB) transmission is difficult to measure, and its drivers are not well understood. The effectiveness of infection control measures at healthcare clinics and the most appropriate intervention strategies to interrupt transmission are unclear. We propose a novel approach using clinical, environmental and position-tracking data to study the risk of TB transmission at primary care clinics in TB and HIV high burden settings in sub-Saharan Africa. METHODS AND ANALYSIS We describe a novel and rapid study design to assess risk factors for airborne TB transmission at primary care clinics in high-burden settings. The study protocol combines a range of different measurements. We will collect anonymous data on the number of patients, waiting times and patient movements using video sensors. Also, we will collect acoustic sound recordings to determine the frequency and intensity of coughing. Environmental data will include indoor carbon dioxide levels (CO2 in parts per million) and relative humidity. We will also extract routinely collected clinical data from the clinic records. The number of Mycobacterium tuberculosis particles in the air will be ascertained from dried filter units using highly sensitive digital droplet PCR. We will calculate rebreathed air volume based on people density and CO2 levels and develop a mathematical model to estimate the risk of TB transmission. The mathematical model can then be used to estimate the effect of possible interventions such as separating patient flows or improving ventilation in reducing transmission. The feasibility of our approach was recently demonstrated in a pilot study in a primary care clinic in Cape Town, South Africa. ETHICS AND DISSEMINATION The study was approved by the University of Cape Town (HREC/REF no. 228/2019), the City of Cape Town (ID-8139) and the Ethics Committee of the Canton Bern (2019-02131), Switzerland. The results will be disseminated in international peer-reviewed journals

Tracing the String: BMN correspondence at Finite J^2/N

Employing the string bit formalism of hep-th/0209215, we identify the basis transformation that relates BMN operators in N=4 gauge theory to string states in the dual string field theory at finite g_2=J^2/N. In this basis, the supercharge truncates at linear order in g_2, and the mixing amplitude between 1 and 2-string states precisely matches with the (corrected) answer of hep-th/0206073 for the 3-string amplitude in light-cone string field theory. Supersymmetry then predicts the order g_2^2 contact term in the string bit Hamiltonian. The resulting leading order mass renormalization of string states agrees with the recently computed shift in conformal dimension of BMN operators in the gauge theory.Comment: 11 pages, 1 figur

Imatinib inhibits VEGF-independent angiogenesis by targeting neuropilin 1-dependent ABL1 activation in endothelial cells.

To enable new blood vessel growth, endothelial cells (ECs) express neuropilin 1 (NRP1), and NRP1 associates with the receptor tyrosine kinase VEGFR2 after binding the vascular endothelial growth factor A (VEGF) to enhance arteriogenesis. We report that NRP1 contributes to angiogenesis through a novel mechanism. In human and mouse ECs, the integrin ligand fibronectin (FN) stimulated actin remodeling and phosphorylation of the focal adhesion component paxillin (PXN) in a VEGF/VEGFR2-independent but NRP1-dependent manner. NRP1 formed a complex with ABL1 that was responsible for FN-dependent PXN activation and actin remodeling. This complex promoted EC motility in vitro and during angiogenesis on FN substrates in vivo. Accordingly, both physiological and pathological angiogenesis in the retina were inhibited by treatment with Imatinib, a small molecule inhibitor of ABL1 which is widely used to prevent the proliferation of tumor cells that express BCR-ABL fusion proteins. The finding that NRP1 regulates angiogenesis in a VEGF- and VEGFR2-independent fashion via ABL1 suggests that ABL1 inhibition provides a novel opportunity for anti-angiogenic therapy to complement VEGF or VEGFR2 blockade in eye disease or solid tumor growth

Evaluation of Nanopore sequencing for Mycobacterium tuberculosis drug susceptibility testing and outbreak investigation: a genomic analysis

Mycobacterium tuberculosis whole-genome sequencing (WGS) has been widely used for genotypic drug susceptibility testing (DST) and outbreak investigation. For both applications, Illumina technology is used by most public health laboratories; however, Nanopore technology developed by Oxford Nanopore Technologies has not been thoroughly evaluated. The aim of this study was to determine whether Nanopore sequencing data can provide equivalent information to Illumina for transmission clustering and genotypic DST for M tuberculosis

The Influence of HIV on the Evolution of Mycobacterium tuberculosis.

HIV significantly affects the immunological environment during tuberculosis coinfection, and therefore may influence the selective landscape upon which M. tuberculosis evolves. To test this hypothesis whole genome sequences were determined for 169 South African M. tuberculosis strains from HIV-1 coinfected and uninfected individuals and analyzed using two Bayesian codon-model based selection analysis approaches: FUBAR which was used to detect persistent positive and negative selection (selection respectively favoring and disfavoring nonsynonymous substitutions); and MEDS which was used to detect episodic directional selection specifically favoring nonsynonymous substitutions within HIV-1 infected individuals. Among the 25,251 polymorphic codon sites analyzed, FUBAR revealed that 189-fold more were detectably evolving under persistent negative selection than were evolving under persistent positive selection. Three specific codon sites within the genes celA2b, katG, and cyp138 were identified by MEDS as displaying significant evidence of evolving under directional selection influenced by HIV-1 coinfection. All three genes encode proteins that may indirectly interact with human proteins that, in turn, interact functionally with HIV proteins. Unexpectedly, epitope encoding regions were enriched for sites displaying weak evidence of directional selection influenced by HIV-1. Although the low degree of genetic diversity observed in our M. tuberculosis data set means that these results should be interpreted carefully, the effects of HIV-1 on epitope evolution in M. tuberculosis may have implications for the design of M. tuberculosis vaccines that are intended for use in populations with high HIV-1 infection rates

Correction for Kipkorir et al., "De Novo Cobalamin Biosynthesis, Transport, and Assimilation and Cobalamin-Mediated Regulation of Methionine Biosynthesis in Mycobacterium smegmatis"

Volume 203, no. 7, e00620-20, 2021, https://doi.org/10.1128/JB.00620-20. Page 1: This article was published on 8 March 2021 with Stephanie S. Dawes missing from the byline. The byline was updated in the version posted on 21 January 2022. The following corrections were also made in that version. Page 1, footnote box: The following footnote was added. “§Present address: Stephanie S. Dawes, Laboratory of Structural Biology, School of Biological Sciences and Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, New Zealand.” Page 14, Acknowledgments, line 3: “and Stephanie Dawes for providing the DmetE cobK::hyg mutant” was deleted. Page 14, Acknowledgments, lines 1 and 2 from bottom: “(to V.M. and D.F.W.)” was changed to “(to S.S.D., V.M., and D.F.W.).