Hydrolysis of Clavulanate by Mycobacterium tuberculosis β-Lactamase BlaC Harboring a Canonical SDN Motif

Combinations of β-lactams with clavulanate are currently being investigated for tuberculosis treatment. Since Mycobacterium tuberculosis produces a broad spectrum β-lactamase, BlaC, the success of this approach could be compromised by the emergence of clavulanate-resistant variants, as observed for inhibitor-resistant TEM variants in enterobacteria. Previous analyses based on site-directed mutagenesis of BlaC have led to the conclusion that this risk was limited. Here, we used a different approach based on determination of the crystal structure of β-lactamase Bla_MAb of Mycobacterium abscessus, which efficiently hydrolyzes clavulanate. Comparison of Bla_MAb and BlaC allowed for structure-assisted site-directed mutagenesis of BlaC and identification of the G¹³²N substitution that was sufficient to switch the interaction of BlaC with clavulanate from irreversible inactivation to efficient hydrolysis. The substitution, which restored the canonical SDN motif (SDG→SDN), allowed for efficient hydrolysis of clavulanate, with a more than 10⁴-fold increase in k_cat (0.41 s⁻¹), without affecting the hydrolysis of other β-lactams. Mass spectrometry revealed that acylation of BlaC and of its G¹³²N variant by clavulanate follows similar paths, involving sequential formation of two acylenzymes. Decarboxylation of the first acylenzyme results in a stable secondary acylenzyme in BlaC, whereas hydrolysis occurs in the G¹³²N variant. The SDN/SDG polymorphism defines two mycobacterial lineages comprising rapidly and slowly growing species, respectively. Together, these results suggest that the efficacy of β-lactam–clavulanate combinations may be limited by the emergence of resistance. β-Lactams active without clavulanate, such as faropenem, should be prioritized for the development of new therapies.     

Negative selection on human genes underlying inborn errors depends on disease outcome and both the mode and mechanism of inheritance

Genetic variants underlying life-threatening diseases, being unlikely to be transmitted to the next generation, are gradually and selectively eliminated from the population through negative selection. We study the determinants of this evolutionary process in human genes underlying monogenic diseases by comparing various negative selection scores and an integrative approach, CoNeS, at 366 loci underlying inborn errors of immunity (IEI). We find that genes underlying autosomal dominant (AD) or X-linked IEI have stronger negative selection scores than those underlying autosomal recessive (AR) IEI, whose scores are not different from those of genes not known to be disease causing. Nevertheless, genes underlying AR IEI that are lethal before reproductive maturity with complete penetrance have stronger negative selection scores than other genes underlying AR IEI. We also show that genes underlying AD IEI by loss of function have stronger negative selection scores than genes underlying AD IEI by gain of function, while genes underlying AD IEI by haploinsufficiency are under stronger negative selection than other genes underlying AD IEI. These results are replicated in 1,140 genes underlying inborn errors of neurodevelopment. Finally, we propose a supervised classifier, SCoNeS, which predicts better than state-of-the-art approaches whether a gene is more likely to underlie an AD or AR disease. The clinical outcomes of monogenic inborn errors, together with their mode and mechanisms of inheritance, determine the levels of negative selection at their corresponding loci. Integrating scores of negative selection may facilitate the prioritization of candidate genes and variants in patients suspected to carry an inborn error.

Negative (or purifying) selection is the natural process by which deleterious alleles are selectively purged from the population (1). In diploid species, the strength of negative selection at a given locus is predicted to increase with decreasing fitness and increasing dominance of the genetic variants controlling traits: Variation causing early death in the heterozygous state are the least likely to be transmitted to the next generation, as their carriers have fewer offspring than noncarriers (2). Human genetic variants that cause severe diseases are, thus, expected to be the primary targets of negative selection, particularly for diseases affecting heterozygous individuals. In humans, several studies have ranked protein-coding genes according to their levels of negative selection (3–5). Nevertheless, the extent to which negative selection affects human disease-causing genes, and the factors determining its strength, remain largely unknown, particularly because our knowledge of the severity, mode, and mechanism of inheritance of the corresponding human diseases remains incomplete (3, 6–8).The strength of negative selection at a given gene has been traditionally approximated by comparing the coding sequence of the gene in a given species with that of one or several closely related species; it depends on the proportion of amino acid changes that have accumulated during evolution (9–11). With the advent of high-throughput sequencing, intraspecies metrics have been developed, based on the comparison of the probability of predicted loss-of-function (pLOF) mutations for a gene under a random model with the frequency of pLOF mutations observed in population databases (5, 12, 13), which capture the species-specific evolution of genes. Using an interspecies-based method and a hand-curated version of the Online Mendelian Inheritance in Man (hOMIM) database, a previous study elegantly showed that most human genes for which mutations cause highly penetrant diseases, including autosomal dominant (AD) diseases in particular, evolve under stronger negative selection than genes associated with complex disorders (6). However, other studies based on OMIM genes have reported conflicting results (3, 14–17), probably due to the incompleteness and heterogeneity of the datasets used. Moreover, no study has yet addressed this problem with intraspecies metrics, even though it has been suggested that the choice of the reference species for interspecies metrics contributes to discrepancies across studies (6).We aimed to improve the identification of the drivers of negative selection acting on human disease-causing genes, by developing a negative selection score combining several informative intraspecies and interspecies statistics, focusing on inborn errors of immunity (IEI). IEI, previously known as primary immunodeficiencies (18), are genetic diseases that disrupt the development or function of human immunity. They form a large and expanding group of genetic diseases that has been widely studied, and they are well characterized physiologically (immunologically) and phenotypically (clinically) (19–21). IEI are often symptomatic in early childhood, and at least until the turn of the 20th century and the introduction of antibiotics, most individuals with IEI probably died before reaching reproductive maturity. Accordingly, IEI genes have probably been under strong negative selection from the dawn of humankind until very recently. In this study, we investigated whether the severity of IEI and their mode and mechanism of inheritance have left signatures of negative selection of various intensities in the corresponding human genes. Furthermore, we validated our model on genes underlying inborn errors of neurodevelopment (IEND), another group of well-characterized severe genetic diseases.     

Quality assurance for the diagnostics of viral diseases to enhance the emergency preparedness in Europe

The threat posed by emerging and re-emerging communicable diseases and, more recently, by the intentional release of infectious agents in a susceptible population, has been receiving considerable attention at the national and international levels. Public health efforts to strengthen disease detection, surveillance and control have been intensified. However, clinicians and clinical microbiology laboratories play an important role in the early detection of disease, the identification of the putative agent, and notification of the appropriate authorities. To be effective in this role, laboratories must be specially prepared to handle viral agents safely, and need, among other things, the appropriate rapid and sensitive diagnostic tests. In 1998 the European Network for Diagnostics of 'Imported' Viral Diseases (ENIVD) was established. ENIVD presently comprises, as permanent members, 44 expert laboratories in 21 European Union (EU) member states and 4 non-EU countries and is one of the networks on infectious diseases funded by the European Commission. ENIVD fulfils many of the important tasks required for the surveillance and control of imported, rare and emerging viral infections such as the exchange of expertise and the organisation of external quality assurance (EQA) programmes, both of which are needed to improve diagnostics. Here, we summarise the data generated by recent EQA activities focussed on the diagnostics of infections with hantavirus, dengue virus, filovirus, Lassa virus, orthopox virus and the SARS-coronavirus (SARS-CoV). These were carried out between 1999 and 2004 and involved 93 laboratories from 41 countries, including laboratories from additional countries outside of Europe. Particularly the EU-candidate countries and Eastern neighbouring countries will be invited to join the network in the near future. A public website is available at http://www.enivd.de.     

Participants’ perceptions of an aphasia-friendly occupational therapy home program

Background: Upper extremity impairments post-stroke impact participation in valued occupations. Home programs are used to extend the frequency and amount of upper extremity practice for people post-stroke, but novel ways of increasing adherence to these programs is needed to ensure intense repetitive practice.

Objectives: The purpose of this process evaluation, was to examine the perceptions of people with aphasia as it relates to modifications to home programs that could influence participation, specifically listening to music and aphasia-friendly adaptations of written materials.

Methods: Seven people with chronic aphasia completed an upper extremity home program that included the use of music and aphasia-friendly modified materials (ClinicalTrials.gov ID:2016/06/18). After the home program, participants completed semi-structured interviews to understand their perceptions of the program and the modifications.

Results: Five themes were identified including (1) music, (2) activities, (3) instructions, (4) logistics, and (5) progress. Most participants perceived music as helpful, but a few reported it being distracting. Participants found the variety of activities helpful as well as specific characteristics of the instructions such as photographs and images. Some participants expressed elements of the home program to be barriers to practice such as using the activity monitors and logbook, which required assistance at times. Finally, participants noted their own progress resulting from the program and liked that they could adjust the intensity of the program as they progressed.

Conclusions: Overall participants in this study with aphasia perceived the modifications to home programs including aphasia friendly written instructions and music to facilitate successful home practice.     

Single embryo transfer in preimplantation genetic diagnosis cycles for women <36 years does not reduce delivery rate

BACKGROUND: The Belgian legislation imposes single embryo transfer (SET) on women of <36 years in their first treatment cycle to avoid multiple pregnancies. The aim of this study is to assess the impact of this legislation on the outcome of preimplantation genetic diagnosis (PGD) for inherited diseases in young women undergoing SET. METHODS: A retrospective analysis of PGD cycles for monogenic disorders and translocations in women <36 years on their first treatment cycle. Two groups of patients were defined according to the implementation of the Belgian legislation: (i) double embryo transfer (DET), January 2001-June 2003 (ii) SET, July 2003-June 2005. The primary and secondary outcome measures were delivery per embryo transfer and multiple pregnancy rates, respectively. A subgroup analysis for monogenic disorders and translocations was performed. RESULTS: 62 cycles were included in the DET group and 73 cycles in the SET group. The mean age, number of cumulus-oocyte complexes, number of fertilized oocytes, number of biopsied and cryopreserved embryos were comparable between both groups. There was no significant difference in the delivery rates between the DET and the SET groups (33.9% versus 27.4%, respectively). Multiple pregnancies were avoided when SET was performed. When monogenic disorders and chromosomal translocations were separately evaluated, no significant difference in the delivery rate after SET was observed. CONCLUSIONS: The implementation of a SET policy in young women undergoing PGD for monogenic disorders and translocations enables a significant reduction of multiple pregnancies without significantly affecting the delivery rate.