An emerging role for tissue plasticity in developmental precision.

Reproducible tissue morphology is a fundamental feature of embryonic development. To ensure such robustness during tissue morphogenesis, inherent noise in biological processes must be buffered. While redundant genes, parallel signaling pathways and intricate network topologies are known to reduce noise, over the last few years, mechanical properties of tissues have been shown to play a vital role. Here, taking the example of somite shape changes, I will discuss how tissues are highly plastic in their ability to change shapes leading to increased precision and reproducibility.

Proteogenomics produces comprehensive and highly accurate protein-coding gene annotation in a complete genome assembly of Malassezia sympodialis.

Complete and accurate genome assembly and annotation is a crucial foundation for comparative and functional genomics. Despite this, few complete eukaryotic genomes are available, and genome annotation remains a major challenge. Here, we present a complete genome assembly of the skin commensal yeast Malassezia sympodialis and demonstrate how proteogenomics can substantially improve gene annotation. Through long-read DNA sequencing, we obtained a gap-free genome assembly for M. sympodialis (ATCC 42132), comprising eight nuclear and one mitochondrial chromosome. We also sequenced and assembled four M. sympodialis clinical isolates, and showed their value for understanding Malassezia reproduction by confirming four alternative allele combinations at the two mating-type loci. Importantly, we demonstrated how proteomics data could be readily integrated with transcriptomics data in standard annotation tools. This increased the number of annotated protein-coding genes by 14% (from 3612 to 4113), compared to using transcriptomics evidence alone. Manual curation further increased the number of protein-coding genes by 9% (to 4493). All of these genes have RNA-seq evidence and 87% were confirmed by proteomics. The M. sympodialis genome assembly and annotation presented here is at a quality yet achieved only for a few eukaryotic organisms, and constitutes an important reference for future host-microbe interaction studies.

Repeat-Associated Fission Yeast-Like Regional Centromeres in the Ascomycetous Budding Yeast Candida tropicalis.

The centromere, on which kinetochore proteins assemble, ensures precise chromosome segregation. Centromeres are largely specified by the histone H3 variant CENP-A (also known as Cse4 in yeasts). Structurally, centromere DNA sequences are highly diverse in nature. However, the evolutionary consequence of these structural diversities on de novo CENP-A chromatin formation remains elusive. Here, we report the identification of centromeres, as the binding sites of four evolutionarily conserved kinetochore proteins, in the human pathogenic budding yeast Candida tropicalis. Each of the seven centromeres comprises a 2 to 5 kb non-repetitive mid core flanked by 2 to 5 kb inverted repeats. The repeat-associated centromeres of C. tropicalis all share a high degree of sequence conservation with each other and are strikingly diverged from the unique and mostly non-repetitive centromeres of related Candida species--Candida albicans, Candida dubliniensis, and Candida lusitaniae. Using a plasmid-based assay, we further demonstrate that pericentric inverted repeats and the underlying DNA sequence provide a structural determinant in CENP-A recruitment in C. tropicalis, as opposed to epigenetically regulated CENP-A loading at centromeres in C. albicans. Thus, the centromere structure and its influence on de novo CENP-A recruitment has been significantly rewired in closely related Candida species. Strikingly, the centromere structural properties along with role of pericentric repeats in de novo CENP-A loading in C. tropicalis are more reminiscent to those of the distantly related fission yeast Schizosaccharomyces pombe. Taken together, we demonstrate, for the first time, fission yeast-like repeat-associated centromeres in an ascomycetous budding yeast.

Emergence of a left-right symmetric body plan in vertebrate embryos.

External bilateral symmetry is a prevalent feature in vertebrates, which emerges during early embryonic development. To begin with, vertebrate embryos are largely radially symmetric before transitioning to bilaterally symmetry, after which, morphogenesis of various bilateral tissues (e.g somites, otic vesicle, limb bud), and structures (e.g palate, jaw) ensue. While a significant amount of work has probed the mechanisms behind symmetry breaking in the left-right axis leading to asymmetric positioning of internal organs, little is known about how bilateral tissues emerge at the same time with the same shape and size and at the same position on the two sides of the embryo. By discussing emergence of symmetry in many bilateral tissues and structures across vertebrate model systems, we highlight that understanding symmetry establishment is largely an open field, which will provide deep insights into fundamental problems in developmental biology for decades to come.

Functional plasticity in chromosome-microtubule coupling on the evolutionary time scale.

The Dam1 complex is essential for mitotic progression across evolutionarily divergent fungi. Upon analyzing amino acid (aa) sequences of Dad2, a Dam1 complex subunit, we identified a conserved 10-aa-long Dad2 signature sequence (DSS). An arginine residue (R126) in the DSS is essential for viability in Saccharomyces cerevisiae that possesses point centromeres. The corresponding arginine residues are functionally important but not essential for viability in Candida albicans and Cryptococcus neoformans; both carry several kilobases long regional centromeres. The purified recombinant Dam1 complex containing either Dad2ΔDSS or Dad2R126A failed to bind microtubules (MTs) or form any visible rings like the WT complex. Intriguingly, functional analysis revealed that the requirement of the conserved arginine residue for chromosome biorientation and mitotic progression reduced with increasing centromere length. We propose that plasticity of the invariant arginine of Dad2 in organisms with regional centromeres is achieved by conditional elevation of the kinetochore protein(s) to enable multiple kinetochore MTs to bind to each chromosome. The capacity of a chromosome to bind multiple kinetochore MTs may mask the deleterious effects of such lethal mutations.

Brain imaging in lung cancer staging: A real-world, multi-centre study of prevalence of brain metastases, impact on treatment and re-modelling of the NICE health economic analysis.

INTRODUCTION: In 2019, the National Institute for Health and Care Excellence (NICE) updated their recommendations with respect to brain imaging in the staging of non-small cell lung cancer (NSCLC) based on an analytic cost-effectiveness model using published data and modelling assumptions from committee experts. In this study, we aimed to re-run this model using real-world multi-centre UK data. MATERIALS AND METHODS: Retrospective data was collected on consecutive patients with radically treatable clinical stage II and III lung cancer from eleven acute NHS Trusts during the calendar year 01/01/2018 to 31/12/2018. Following a written application to the NICE lung cancer guideline committee, we were granted access to the NG122 brain imaging economic model for the purpose of updating the input parameters in line with the real-world findings from this study. RESULTS: A total of 444 patients had data for analysis. The combined prevalence of occult brain metastases was 6.2% (10/165) in stage II and 6% (17/283) in stage III, compared to 9.5% and 9.3% used in the NICE economic model. 30% of patients with clinical stage III NSCLC and occult BMs on pre-treatment imaging went onto complete the planned curative intent treatment of extracranial disease, 60% completed SRS to the brain and 30% completed WBRT. This compares to 0%, 10% and 0% in the NICE assumptions. The health economic analysis concluded that brain imaging was no longer cost-effective in stage II disease (ICERs £50,023-£115,785) whilst brain imaging remained cost-effective for stage III patients (ICERs 17,000-£22,173), with MRI being the most cost-effective strategy. CONCLUSION: This re-running of the NICE health economic model with real-world data strongly supports the NICE guideline recommendation for brain imaging prior to curative-intent treatment in stage III lung cancer but questions the cost-effectiveness of CT brain imaging prior to curative-intent treatment in stage II lung cancer.

Atypical Protein Kinase C Promotes its own Asymmetric Localisation by Phosphorylating Cdc42 in Polarising Cells.

Atypical protein kinase C (aPKC) is a major regulator of cell polarity. Acting in conjunction with Par6, Par3 and the small GTPase Cdc42, aPKC becomes asymmetrically localised and drives the polarisation of cells. aPKC activity is crucial for its own asymmetric localisation, suggesting a hitherto unknown feedback mechanism contributing to polarisation. Here we show in C. elegans zygotes that the feedback relies on CDC-42 phosphorylation at serine 71 by aPKC, which in turn results in aPKC dissociation from CDC-42. The dissociated aPKC then associates with PAR-3 clusters, which are transported anteriorly by actomyosin-based cortical flow. Moreover, the turnover of aPKC-mediated CDC-42 phosphorylation regulates the organisation of the actomyosin cortex that drives aPKC asymmetry. Given the widespread role of aPKC and Cdc42 in cell polarity, this form of self-regulation of aPKC may be vital for the robust polarisation of many cell types.

Crystal structures of KPC-2 ß-lactamase in complex with 3-nitrophenyl boronic acid and the penam sulfone PSR-3-226.

Class A carbapenemases are a major threat to the potency of carbapenem antibiotics. A widespread carbapenemase, KPC-2, is not easily inhibited by ß-lactamase inhibitors (i.e., clavulanic acid, sulbactam, and tazobactam). To explore different mechanisms of inhibition of KPC-2, we determined the crystal structures of KPC-2 with two ß-lactamase inhibitors that follow different inactivation pathways and kinetics. The first complex is that of a small boronic acid compound, 3-nitrophenyl boronic acid (3-NPBA), bound to KPC-2 with 1.62-Å resolution. 3-NPBA demonstrated a K(m) value of 1.0 ± 0.1 µM (mean ± standard error) for KPC-2 and blocks the active site by making a reversible covalent interaction with the catalytic S70 residue. The two boron hydroxyl atoms of 3-NPBA are positioned in the oxyanion hole and the deacylation water pocket, respectively. In addition, the aromatic ring of 3-NPBA provides an edge-to-face interaction with W105 in the active site. The structure of KPC-2 with the penam sulfone PSR-3-226 was determined at 1.26-Å resolution. PSR-3-226 displayed a K(m) value of 3.8 ± 0.4 µM for KPC-2, and the inactivation rate constant (k(inact)) was 0.034 ± 0.003 s(-1). When covalently bound to S70, PSR-3-226 forms a trans-enamine intermediate in the KPC-2 active site. The predominant active site interactions are generated via the carbonyl oxygen, which resides in the oxyanion hole, and the carboxyl moiety of PSR-3-226, which interacts with N132, N170, and E166. 3-NPBA and PSR-3-226 are the first ß-lactamase inhibitors to be trapped as an acyl-enzyme complex with KPC-2. The structural and inhibitory insights gained here could aid in the design of potent KPC-2 inhibitors.

Modifications of the C6-substituent of penicillin sulfones with the goal of improving inhibitor recognition and efficacy.

In order to evaluate the importance of a hydrogen-bond donating substituent in the design of ß-lactamase inhibitors, a series of C6-substituted penicillin sulfones, lacking a C2' substituent, and having an sp(3) hybridized C6, was prepared and evaluated against a representative classes A and C ß-lactamases. It was found that a C6 hydrogen-bond donor is necessary for good inhibitory activity, but that this feature alone is not sufficient in this series of C6ß-substituted penicillin sulfones. Other factors which may impact the potency of the inhibitor include the steric bulk of the C6 substituent (e.g., methicillin sulfone) which may hinder recognition in the class A ß-lactamases, and also high similarity to the natural substrates (e.g., penicillin G sulfone) which may render the prospective inhibitor a good substrate of both classes of enzyme. The best inhibitors had non-directional hydrogen-bonding substituents, such as hydroxymethyl, which may allow sufficient conformational flexibility of the acyl-enzyme for abstraction of the C6 proton by E166 (class A), thus promoting isomerization to the ß-aminoacrylate as a stabilized acyl-enzyme.

Understanding the enablers and barriers to implementing smoke-free NHS sites across acute care trusts in Greater Manchester: results of a hospital staff survey.

INTRODUCTION: The current study aims to identify enablers and barriers to implementing smoke-free NHS hospital grounds through a hospital staff survey. METHODS: Staff members from eight acute care NHS trusts in Greater Manchester were invited to complete a 15-minute web-based questionnaire. RESULTS: Five-hundred and eighty-eight participants completed the questionnaire. Nineteen per cent (114/588) of respondents were current smokers and 10% (61/588) were currently vaping. Sixty per cent (68/114) smoked at work and 66% (40/61) vaped at work. Sixty-seven per cent (314/468) supported dedicated on-site tobacco addiction treatment services for hospital staff with specific support for drop-in clinics and free pharmacotherapy for staff. Sixty-one per cent (290/477) and 67% (318/477) strongly agreed / agreed that patients/visitors and staff, respectively, should not smoke on hospital grounds. Seventeen per cent (83/484) had received training in very brief advice. Thirty-five per cent (190/547) felt vaping was less harmful than smoking, 19% (92/472) felt exhaled vapour was likely to be safe to bystanders, 36% (172/475) would support vaping-friendly hospital grounds and 31% (37/120) felt confident in discussing vaping. DISCUSSION: Enablers to a smoke-free NHS site include dedicated tobacco addiction services for staff and empowering staff through appropriate training to support smokers on the hospital grounds. Barriers include the lack of awareness and support for the harm reduction benefits of vaping.

Performance monitoring of EBUS for the staging and diagnosis of lung cancer: auditing the Greater Manchester EBUS service against new national standards.

INTRODUCTION: Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a pivotal test in lung cancer staging and diagnosis, mandating robust audit and performance monitoring of EBUS services. We present the first regional cancer alliance EBUS performance audit against the new National EBUS specification. METHODS: Across the five EBUS centres in the Greater Manchester Cancer Alliance, data are recorded at the point of procedure, when pathological results are available and at 6 months postprocedure to review any further pathological sampling (eg, at surgical resection) and the outcome of clinical-radiological follow-up. Outcomes across all five centres were compared with national standards for all lung cancer EBUS procedures from 01 January 2017 to 31 December 2018. RESULTS: 1899 lung cancer staging or diagnostic EBUS procedures were performed across the five centres during the study period; 1309 staging EBUS procedures and 590 diagnostic EBUS procedures. Major complications were seen in six cases (<1%). All five trusts demonstrated performance above that set national standards in key metrics for both staging and diagnostic EBUS, however the provision of adequate tissue for predictive marker testing was below national standards at one trust. Across Greater Manchester, 72% and 64% of patients had their EBUS procedure performed within 7 days of referral in 2017 and 2018, respectively. Only one out of five trusts met the national targets of >85% of procedures performed within 7 days of referral. CONCLUSION: The National EBUS service specification is an important framework to drive the quality of EBUS services across the UK. Our data provide assurance of appropriate performance and safety while also highlighting specific areas for attention that can be addressed with the support of the cancer alliance.

Health economic analysis for the 'CURE Project' pilot: a hospital-based tobacco dependency treatment service in Greater Manchester.

INTRODUCTION: Treating tobacco dependency in patients admitted to acute care National Health Service (NHS) trusts is a key priority in the NHS 10-year plan. This paper sets out the results of a health economic analysis for 'The CURE Project' pilot; a new hospital-based tobacco dependency service. METHODS: A health economic analysis to understand the costs of the intervention (both for the inpatient service and postdischarge costs), the return on investment (ROI) and the cost per quality-adjusted life year (QALY) of the CURE Project pilot in Greater Manchester. ROI and cost per QALY were calculated using the European Study on Quantifying Utility of Investment in Protection from Tobacco and Greater Manchester Cost Benefit Analysis Tools. RESULTS: The total intervention costs for the inpatient service in the 6-month CURE pilot were £96 224 with a cost per patient who smokes of £40.21. The estimated average cost per patient who was discharged on pharmacotherapy was £97.40. The cost per quit (22% quit rate for smokers at 12 weeks post discharge) was £475. The gross financial ROI ratio was £2.12 return per £1 invested with a payback period of 4 years. The cashable financial ROI ratio was £1.06 return per £1 invested with a payback period of 10 years. The public value ROI ratio was £30.49 per £1 invested. The cost per QALY for this programme was £487. DISCUSSION: The CURE Project pilot has been shown to be exceptionally cost-effective with highly significant ROI in this health economic analysis. This supports the NHS priority to embed high-quality tobacco addiction treatment services in acute NHS trusts, and the CURE Project provides a blueprint and framework to achieve this.

Haematological malignancy and nosocomial transmission are associated with an increased risk of death from COVID-19: results of a multi-center UK cohort.

The COVID-19 pandemic has been a disruptive event for cancer patients, especially those with haematological malignancies (HM). They may experience a more severe clinical course due to impaired immune responses. This multi-center retrospective UK audit identified cancer patients who had SARS-CoV-2 infection between 1 March and 10 June 2020 and collected data pertaining to cancer history, COVID-19 presentation and outcomes. In total, 179 patients were identified with a median age of 72 (IQR 61, 81) and follow-up of 44 days (IQR 42, 45). Forty-one percent were female and the overall mortality was 37%. Twenty-nine percent had HM and of these, those treated with chemotherapy in the preceding 28 days to COVID-19 diagnosis had worse outcome compared with solid malignancy (SM): 62% versus 19% died [HR 8.33 (95% CI, 2.56-25), p < 0.001]. Definite or probable nosocomial SARS-CoV-2 transmission accounted for 16% of cases and was associated with increased risk of death (HR 2.47, 95% CI 1.43-4.29, p = 0.001). Patients with haematological malignancies and those who acquire nosocomial transmission are at increased risk of death. Therefore, there is an urgent need to reassess shielding advice, reinforce stringent infection control, and ensure regular patient and staff testing to prevent nosocomial transmission.

Design, synthesis, and crystal structures of 6-alkylidene-2'-substituted penicillanic acid sulfones as potent inhibitors of Acinetobacter baumannii OXA-24 carbapenemase.

Class D ß-lactamases represent a growing and diverse class of penicillin-inactivating enzymes that are usually resistant to commercial ß-lactamase inhibitors. As many such enzymes are found in multi-drug resistant (MDR) Acinetobacter baumannii and Pseudomonas aeruginosa, novel ß-lactamase inhibitors are urgently needed. Five unique 6-alkylidene-2'-substituted penicillanic acid sulfones (1-5) were synthesized and tested against OXA-24, a clinically important ß-lactamase that inactivates carbapenems and is found in A. baumannii. Based upon the roles Tyr112 and Met223 play in the OXA-24 ß-lactamase, we also engineered two variants (Tyr112Ala and Tyr112Ala,Met223Ala) to test the hypothesis that the hydrophobic tunnel formed by these residues influences inhibitor recognition. IC(50) values against OXA-24 and two OXA-24 ß-lactamase variants ranged from 10 ± 1 (4 vs WT) to 338 ± 20 nM (5 vs Tyr112Ala, Met223Ala). Compound 4 possessed the lowest K(i) (500 ± 80 nM vs WT), and 1 possessed the highest inactivation efficiency (k(inact)/K(i) = 0.21 ± 0.02 µM(-1) s(-1)). Electrospray ionization mass spectrometry revealed a single covalent adduct, suggesting the formation of an acyl-enzyme intermediate. X-ray structures of OXA-24 complexed to four inhibitors (2.0-2.6 Å) reveal the formation of stable bicyclic aromatic intermediates with their carbonyl oxygen in the oxyanion hole. These data provide the first structural evidence that 6-alkylidene-2'-substituted penicillin sulfones are effective mechanism-based inactivators of class D ß-lactamases. Their unique chemistry makes them developmental candidates. Mechanisms for class D hydrolysis and inhibition are discussed, and a pathway for the evolution of the BlaR1 sensor of Staphylococcus aureus to the class D ß-lactamases is proposed.

Penicillin sulfone inhibitors of class D beta-lactamases.

OXA beta-lactamases are largely responsible for beta-lactam resistance in Acinetobacter spp. and Pseudomonas aeruginosa, two of the most difficult-to-treat nosocomial pathogens. In general, the beta-lactamase inhibitors used in clinical practice (clavulanic acid, sulbactam, and tazobactam) demonstrate poor activity against class D beta-lactamases. To overcome this challenge, we explored the abilities of beta-lactamase inhibitors of the C-2- and C-3-substituted penicillin and cephalosporin sulfone families against OXA-1, extended-spectrum (OXA-10, OXA-14, and OXA-17), and carbapenemase-type (OXA-24/40) class D beta-lactamases. Three C-2-substituted penicillin sulfone compounds (JDB/LN-1-255, JDB/LN-III-26, and JDB/ASR-II-292) showed low K(i) values for the OXA-1 beta-lactamase (0.70 +/- 0.14 --> 1.60 +/- 0.30 microM) and demonstrated significant K(i) improvements compared to the C-3-substituted cephalosporin sulfone (JDB/DVR-II-214), tazobactam, and clavulanic acid. The C-2-substituted penicillin sulfones JDB/ASR-II-292 and JDB/LN-1-255 also demonstrated low K(i)s for the OXA-10, -14, -17, and -24/40 beta-lactamases (0.20 +/- 0.04 --> 17 +/- 4 microM). Furthermore, JDB/LN-1-255 displayed stoichiometric inactivation of OXA-1 (the turnover number, i.e., the partitioning of the initial enzyme inhibitor complex between hydrolysis and enzyme inactivation [t(n)] = 0) and t(n)s ranging from 5 to 8 for the other OXA enzymes. Using mass spectroscopy to study the intermediates in the inactivation pathway, we determined that JDB/LN-1-255 inhibited OXA beta-lactamases by forming covalent adducts that do not fragment. On the basis of the substrate and inhibitor kinetics of OXA-1, we constructed a model showing that the C-3 carboxylate of JDB/LN-1-255 interacts with Ser115 and Thr213, the R-2 group at C-2 fits between the space created by the long B9 and B10 beta strands, and stabilizing hydrophobic interactions are formed between the pyridyl ring of JDB/LN-1-255 and Val116 and Leu161. By exploiting conserved structural and mechanistic features, JDB/LN-1-255 is a promising lead compound in the quest for effective inhibitors of OXA-type beta-lactamases.

CPAP management of COVID-19 respiratory failure: a first quantitative analysis from an inpatient service evaluation.

OBJECTIVE: To evaluate the role of continuous positive air pressure (CPAP) in the management of respiratory failure associated with COVID-19 infection. Early clinical management with limited use of CPAP (3% of patients) was compared with a later clinical management strategy which had a higher proportion of CPAP use (15%). DESIGN: Retrospective case-controlled service evaluation for a single UK National Health Service (NHS) Trust during March-June 2020 designed and conducted solely to estimate the effects of current care. SETTING: The acute inpatient unit in Wrightington, Wigan and Leigh Teaching Hospitals NHS Foundation Trust, a medium-sized English NHS Trust. PARTICIPANTS: 206 patients with antigen confirmed COVID-19 disease and severe acute respiratory syndrome admitted between 17 March 2020 and 3 April 2020 for the early group (controls), and between 10 April 2020 and 11 May 2020 for the late group (cases). Follow-up for all cases was until 11 June by which time all patients had a final outcome of death or discharge. Both groups were composed of 103 patients. Cases and controls were matched by age and sex. OUTCOME MEASURE: The outcome measure was the proportion of patients surviving at time t (time from the positive result of COVID-19 test to discharge/death date). The predictors were CPAP intervention, intubation, residence in care homes and comorbidities (renal, pulmonary, cardiac, hypertension and diabetes). A stratified Cox proportional hazard for clustered data (via generalised estimating equations) and model selection algorithms were employed to identify the effect of CPAP on patients' survival and the effect on gas exchange as measured by alveolar arterial (A-a) gradient and timing of CPAP treatment on CPAP patients' survival. RESULTS: CPAP was found to be significantly (HR 0.38, 95% CI 0.36 to 0.40) associated with lower risk of death in patients with hospital stay equal to, or below 7 days. However, for longer hospitalisation CPAP was found to be associated with increased risk of death (HR 1.72, 95% CI 1.40 to 2.12). When CPAP was initiated within 4 days of hospital admission, the survival probability was above 73% (95% CI 53% to 99%). In addition, lower A-a gradient was associated with lower risk of death in CPAP patients (HR 1.011, 95% CI 1.010 to 1.013). The selected model (best fit) was stratified by sex and clustered by case/control groups. The predictors were age, intubation, hypertension and the residency from care homes, which were found to be statistically significantly associated with patient's death/discharge. CONCLUSIONS: CPAP is a simple and cost-effective intervention. It has been established for care of other respiratory disorders but not for COVID-19 respiratory failure. This evaluation establishes that CPAP as a potentially viable treatment option for this group of patients during the first days of hospital admission. As yet there is limited availability of quantitative research on CPAP use for COVID-19. Whist this work is hampered by both the relatively small sample size and retrospective design (which reduced the ability to control potential confounders), it represents evidence of the significant benefit of early CPAP intervention. This evaluation should stimulate further research questions and larger study designs on the potential benefit of CPAP for COVID-19 infections. Globally, this potentially beneficial low cost and low intensity therapy could have added significance economically for healthcare provision in less developed countries.

Loss of centromere function drives karyotype evolution in closely related Malassezia species.

Genomic rearrangements associated with speciation often result in variation in chromosome number among closely related species. Malassezia species show variable karyotypes ranging between six and nine chromosomes. Here, we experimentally identified all eight centromeres in M. sympodialis as 3-5-kb long kinetochore-bound regions that span an AT-rich core and are depleted of the canonical histone H3. Centromeres of similar sequence features were identified as CENP-A-rich regions in Malassezia furfur, which has seven chromosomes, and histone H3 depleted regions in Malassezia slooffiae and Malassezia globosa with nine chromosomes each. Analysis of synteny conservation across centromeres with newly generated chromosome-level genome assemblies suggests two distinct mechanisms of chromosome number reduction from an inferred nine-chromosome ancestral state: (a) chromosome breakage followed by loss of centromere DNA and (b) centromere inactivation accompanied by changes in DNA sequence following chromosome-chromosome fusion. We propose that AT-rich centromeres drive karyotype diversity in the Malassezia species complex through breakage and inactivation.

Millions of yeast, bacteria and other microbes live in or on the human body. A type of yeast known as Malassezia is one of the most abundantmicrobes living on our skin. Generally, Malassezia do not cause symptoms in humans but are associated with dandruff, dermatitis and other skin conditions in susceptible individuals. They have also been found in the human gut, where they exacerbate Crohn's disease and pancreatic cancer. There are 18 closely related species of Malassezia and all have an unusually small amount of genetic material compared with other types of yeast. In yeast, like in humans, the genetic material is divided among several chromosomes. The number of chromosomes in different Malassezia species varies between six and nine. A region of each chromosome known as the centromere is responsible for ensuring that the equal numbers of chromosomes are passed on to their offspring. This means that any defects in centromeres can lead to the daughter yeast cells inheriting unequal numbers of chromosomes. Changes in chromosome number can drive the evolution of new species, but it remains unclear if and how centromere loss may have contributed to the evolution of Malassezia species. Sankaranarayanan et al. have now used biochemical, molecular genetic, and comparative genomic approaches to study the chromosomes of Malassezia species. The experiments revealed that nine Malassezia species had centromeres that shared common features such as being rich in adenine and thymine nucleotides, two of the building blocks of DNA. Sankaranarayanan et al. propose that these adenines and thymines make the centromeres more fragile leading to occasional breaks. This may have contributed to the loss of centromeres in some Malassezia cells and helped new species to evolve with fewer chromosomes. A better understanding of how Malassezia organize their genetic material should enable in-depth studies of how these yeasts interact with their human hosts and how they contribute to skin disease, cancer, Crohn's disease and other health conditions. More broadly, these findings may help scientists to better understand how changes in chromosomes cause new species to evolve.

Approaches to the simultaneous inactivation of metallo- and serine-beta-lactamases.

A series of cephalosporin-derived reverse hydroxamates and oximes were prepared and evaluated as inhibitors of representative metallo- and serine-beta-lactamases. The reverse hydroxamates showed submicromolar inhibition of the GIM-1 metallo-beta-lactamase. With respect to interactions with the classes A, C, and D serine beta-lactamases, as judged by their correspondingly low K(m) values, the reverse hydroxamates were recognized in a manner similar to the non-hydroxylated N-H amide side chains of the natural substrates of these enzymes. This indicates that, with respect to recognition in the active site of the serine beta-lactamases, the OC-NR-OH functionality can function as a structural isostere of the OC-NR-H group, with the N-O-H group presumably replacing the amide N-H group as a hydrogen bond donor to the appropriate backbone carbonyl oxygen of the protein. The reverse hydroxamates, however, displayed k(cat) values up to three orders of magnitude lower than the natural substrates, thus indicating substantial slowing of the hydrolytic action of these serine beta-lactamases. Although the degree of inactivation is not yet enough to be clinically useful, these initial results are promising. The substitution of the amide N-H bond by N-OH may represent a useful strategy for the inhibition of other serine hydrolases.

Morphogenetic degeneracies in the actomyosin cortex.

One of the great challenges in biology is to understand the mechanisms by which morphogenetic processes arise from molecular activities. We investigated this problem in the context of actomyosin-based cortical flow in C. elegans zygotes, where large-scale flows emerge from the collective action of actomyosin filaments and actin binding proteins (ABPs). Large-scale flow dynamics can be captured by active gel theory by considering force balances and conservation laws in the actomyosin cortex. However, which molecular activities contribute to flow dynamics and large-scale physical properties such as viscosity and active torque is largely unknown. By performing a candidate RNAi screen of ABPs and actomyosin regulators we demonstrate that perturbing distinct molecular processes can lead to similar flow phenotypes. This is indicative for a 'morphogenetic degeneracy' where multiple molecular processes contribute to the same large-scale physical property. We speculate that morphogenetic degeneracies contribute to the robustness of bulk biological matter in development.

Controlling contractile instabilities in the actomyosin cortex.

The actomyosin cell cortex is an active contractile material for driving cell- and tissue morphogenesis. The cortex has a tendency to form a pattern of myosin foci, which is a signature of potentially unstable behavior. How a system that is prone to such instabilities can rveliably drive morphogenesis remains an outstanding question. Here, we report that in the Caenorhabditis elegans zygote, feedback between active RhoA and myosin induces a contractile instability in the cortex. We discover that an independent RhoA pacemaking oscillator controls this instability, generating a pulsatory pattern of myosin foci and preventing the collapse of cortical material into a few dynamic contracting regions. Our work reveals how contractile instabilities that are natural to occur in mechanically active media can be biochemically controlled to robustly drive morphogenetic events.