Photonic bandgap microcombs at 1064 nm.

Microresonator frequency combs and their design versatility have revolutionized research areas from data communication to exoplanet searches. While microcombs in the 1550 nm band are well documented, there is interest in using microcombs in other bands. Here, we demonstrate the formation and spectral control of normal-dispersion dark soliton microcombs at 1064 nm. We generate 200 GHz repetition rate microcombs by inducing a photonic bandgap of the microresonator mode for the pump laser with a photonic crystal. We perform the experiments with normal-dispersion microresonators made from Ta2O5 and explore unique soliton pulse shapes and operating behaviors. By adjusting the resonator dispersion through its nanostructured geometry, we demonstrate control over the spectral bandwidth of these combs, and we employ numerical modeling to understand their existence range. Our results highlight how photonic design enables microcomb spectra tailoring across wide wavelength ranges, offering potential in bioimaging, spectroscopy, and photonic-atomic quantum technologies.

Ancestral aneuploidy and stable chromosomal duplication resulting in differential genome structure and gene expression control in trypanosomatid parasites.

Aneuploidy is widely observed in both unicellular and multicellular eukaryotes, usually associated with adaptation to stress conditions. Chromosomal duplication stability is a tradeoff between the fitness cost of having unbalanced gene copies and the potential fitness gained from increased dosage of specific advantageous genes. Trypanosomatids, a family of protozoans that include species that cause neglected tropical diseases, are a relevant group to study aneuploidies. Their life cycle has several stressors that could select for different patterns of chromosomal duplications and/or losses, and their nearly universal use of polycistronic transcription increases their reliance on gene expansion/contraction, as well as post-transcriptional control as mechanisms for gene expression regulation. By evaluating the data from 866 isolates covering seven trypanosomatid genera, we have revealed that aneuploidy tolerance is an ancestral characteristic of trypanosomatids but has a reduced occurrence in a specific monophyletic clade that has undergone large genomic reorganization and chromosomal fusions. We have also identified an ancient chromosomal duplication that was maintained across these parasite's speciation, named collectively as the trypanosomatid ancestral supernumerary chromosome (TASC). TASC has most genes in the same coding strand, is expressed as a disomic chromosome (even having four copies), and has increased potential for functional variation, but it purges highly deleterious mutations more efficiently than other chromosomes. The evidence of stringent control over gene expression in this chromosome suggests that these parasites have adapted to mitigate the fitness cost associated with this ancient chromosomal duplication.

Threshold and Laser Conversion in Nanostructured-Resonator Parametric Oscillators.

We explore optical parametric oscillation (OPO) in nanophotonic resonators, enabling arbitrary, nonlinear phase matching and nearly lossless control of energy conversion. Such pristine OPO laser converters are determined by nonlinear light-matter interactions, making them both technologically flexible and broadly reconfigurable. We utilize a nanostructured inner-wall modulation in the resonator to achieve universal phase matching for OPO-laser conversion, but coherent backscattering also induces a counterpropagating pump laser. This depletes the intraresonator optical power in either direction, increasing the OPO threshold power and limiting laser-conversion efficiency, the ratio of optical power in target signal and idler frequencies to the pump. We develop an analytical model of this system that emphasizes an understanding of optimal laser-conversion and threshold behaviors, and we use the model to guide experiments with nanostructured-resonator OPO laser-conversion circuits, fully integrated on chip and unlimited by group-velocity dispersion. Our Letter demonstrates the fundamental connection between OPO laser-conversion efficiency and the resonator coupling rate, subject to the relative phase and power of counterpropagating pump fields. We achieve (40±4) mW of on-chip power, corresponding to (41±4)% conversion efficiency, and discover a path toward near-unity OPO laser-conversion efficiency.

The dynamic subcellular localisation of Rad1 is cell cycle dependent in Leishmania major.

The subcellular localisation of Rad1, a subunit of the Leishmania major 9-1-1 complex, remains unexplored. Herein, we reveal that Rad1 localises predominantly to the nucleus. Upon hydroxyurea treatment, the diffuse nuclear localisation of Rad1 becomes more punctate, suggesting that Rad1 is responsive to replication stress. Moreover, Rad1 localisation correlates with cell cycle progression. In the majority of G1 to early S-phase cells, Rad1 localises predominantly to the nucleus. As cells progress from late-S phase to mitosis, Rad1 relocalizes to both the nucleus and the cytoplasm in ∼90 % of cells. This pattern of distribution is different from Rad9 and Hus1, which remain nuclear throughout the cell cycle, suggesting Leishmania Rad1 may regulate 9-1-1 activities and/or perform relevant functions outside the 9-1-1 complex.

AAK1-like: A putative pseudokinase with potential roles in cargo uptake in bloodstream form Trypanosoma brucei parasites.

Selection and internalization of cargo via clathrin-mediated endocytosis requires adaptor protein complexes. One complex, AP-2, acts during cargo selection at the plasma membrane. African trypanosomes lack all components of the AP-2 complex, except for a recently identified orthologue of the AP-2-associated protein kinase 1, AAK1. In characterized eukaryotes, AAK1 phosphorylates the µ2 subunit of the AP-2 complex to enhance cargo recognition and uptake into clathrin-coated vesicles. Here, we show that kinetoplastids encode not one, but two AAK1 orthologues: one (AAK1L2) is absent from salivarian trypanosomes, while the other (AAK1L1) lacks important kinase-specific residues in a range of trypanosomes. These AAK1L1 and AAK1L2 novelties reinforce suggestions of functional divergence in endocytic uptake within salivarian trypanosomes. Despite this, we show that AAK1L1 null mutant Trypanosoma brucei, while viable, display slowed proliferation, morphological abnormalities including swelling of the flagellar pocket, and altered cargo uptake. In summary, our data suggest an unconventional role for a putative pseudokinase during endocytosis and/or vesicular trafficking in T. brucei, independent of AP-2.

Life in plastic, it's fantastic! How Leishmania exploit genome instability to shape gene expression.

Leishmania are kinetoplastid pathogens that cause leishmaniasis, a debilitating and potentially life-threatening infection if untreated. Unusually, Leishmania regulate their gene expression largely post-transcriptionally due to the arrangement of their coding genes into polycistronic transcription units that may contain 100s of functionally unrelated genes. Yet, Leishmania are capable of rapid and responsive changes in gene expression to challenging environments, often instead correlating with dynamic changes in their genome composition, ranging from chromosome and gene copy number variations to the generation of extrachromosomal DNA and the accumulation of point mutations. Typically, such events indicate genome instability in other eukaryotes, coinciding with genetic abnormalities, but for Leishmania, exploiting these products of genome instability can provide selectable substrates to catalyse necessary gene expression changes by modifying gene copy number. Unorthodox DNA replication, DNA repair, replication stress factors and DNA repeats are recognised in Leishmania as contributors to this intrinsic instability, but how Leishmania regulate genome plasticity to enhance fitness whilst limiting toxic under- or over-expression of co-amplified and co-transcribed genes is unclear. Herein, we focus on fresh, and detailed insights that improve our understanding of genome plasticity in Leishmania. Furthermore, we discuss emerging models and factors that potentially circumvent regulatory issues arising from polycistronic transcription. Lastly, we highlight key gaps in our understanding of Leishmania genome plasticity and discuss future studies to define, in higher resolution, these complex regulatory interactions.

Emergence and adaptation of the cellular machinery directing antigenic variation in the African trypanosome.

Survival of the African trypanosome within its mammalian hosts, and hence transmission between hosts, relies upon antigenic variation, where stochastic changes in the composition of their protective variant-surface glycoprotein (VSG) coat thwart effective removal of the pathogen by adaptive immunity. Antigenic variation has evolved remarkable mechanistic complexity in Trypanosoma brucei, with switching of the VSG coat executed by either transcriptional or recombination reactions. In the former, a single T. brucei cell selectively transcribes one telomeric VSG transcription site, termed the expression site (ES), from a pool of around 15. Silencing of the active ES and activation of one previously silent ES can lead to a co-ordinated VSG coat switch. Outside the ESs, the T. brucei genome contains an enormous archive of silent VSG genes and pseudogenes, which can be recombined into the ES to execute a coat switch. Most such recombination involves gene conversion, including copying of a complete VSG and more complex reactions where novel 'mosaic' VSGs are formed as patchworks of sequences from several silent (pseudo)genes. Understanding of the cellular machinery that directs transcriptional and recombination VSG switching is growing rapidly and the emerging picture is of the use of proteins, complexes and pathways that are not limited to trypanosomes, but are shared across the wider grouping of kinetoplastids and beyond, suggesting co-option of widely used, core cellular reactions. We will review what is known about the machinery of antigenic variation and discuss if there remains the possibility of trypanosome adaptations, or even trypanosome-specific machineries, that might offer opportunities to impair this crucial parasite-survival process.

Single-cell transcriptomics reveals hidden information in trypanosomatids.

Dixenic parasites often encounter environmental extremes during the transition from vector to host. Preadapted transmission stages overcome these challenges to promote parasites' survival and ensure life cycle progression. Recently, Vigneron et al. and Briggs et al. used single-cell transcriptomics to investigate developmental stage specific gene expression patterns during parasite differentiation.

Group-velocity-dispersion engineering of tantala integrated photonics.

Designing integrated photonics, especially to leverage Kerr-nonlinear optics, requires accurate and precise knowledge of the refractive index across the visible to infrared spectral ranges. Tantala (Ta2O5) is an emerging material platform for integrated photonics and nanophotonics that offers broadband ultralow loss, moderately high nonlinearity, and advantages for scalable and heterogeneous integration. We present refractive index measurements on a thin film of tantala, and we explore the efficacy of this data for group-velocity-dispersion (GVD) engineering with waveguide and ring-resonator devices. In particular, the observed spectral extent of supercontinuum generation in fabricated waveguides and the wavelength dependence of free spectral range (FSR) in optical resonators provide a sensitive test of our integrated photonics design process. Our work opens up new design possibilities with tantala, including with octave-spanning soliton microcombs.

Enhanced Detection of Single Viruses On-Chip via Hydrodynamic Focusing.

Planar optofluidics provide a powerful tool for facilitating chip-scale light-matter interactions. Silicon-based liquid core waveguides have been shown to offer single molecule sensitivity for efficient detection of bioparticles. Recently, a PDMS based planar optofluidic platform was introduced that opens the way to rapid development and prototyping of unique structures, taking advantage of the positive attributes of silicon dioxide-based optofluidics and PDMS based microfluidics. Here, hydrodynamic focusing is integrated into a PDMS based optofluidic chip to enhance the detection of single H1N1 viruses on-chip. Chip-plane focusing is provided by a system of microfluidic channels to force the particles towards a region of high optical collection efficiency. Focusing is demonstrated and enhanced detection is quantified using fluorescent polystyrene beads where the coefficient of variation is found to decrease by a factor of 4 with the addition of hydrodynamic focusing. The mean signal amplitude of fluorescently tagged single H1N1 viruses is found to increase with the addition of focusing by a factor of 1.64.

Potent Antitrypanosomal Activities of 3-Aminosteroids against African Trypanosomes: Investigation of Cellular Effects and of Cross-Resistance with Existing Drugs.

Treatment of animal African trypanosomiasis (AAT) requires urgent need for safe, potent and affordable drugs and this has necessitated this study. We investigated the trypanocidal activities and mode of action of selected 3-aminosteroids against Trypanosoma brucei brucei. The in vitro activity of selected compounds of this series against T. congolense (Savannah-type, IL3000), T. b. brucei (bloodstream trypomastigote, Lister strain 427 wild-type (427WT)) and various multi-drug resistant cell lines was assessed using a resazurin-based cell viability assay. Studies on mode of antitrypanosomal activity of some selected 3-aminosteroids against Tbb 427WT were also carried out. The tested compounds mostly showed moderate-to-low in vitro activities and low selectivity to mammalian cells. Interestingly, a certain aminosteroid, holarrhetine (10, IC50 = 0.045 ± 0.03 µM), was 2 times more potent against T. congolense than the standard veterinary drug, diminazene aceturate, and 10 times more potent than the control trypanocide, pentamidine, and displayed an excellent in vitro selectivity index of 2130 over L6 myoblasts. All multi-drug resistant strains of T. b. brucei tested were not significantly cross-resistant with the purified compounds. The growth pattern of Tbb 427WT on long and limited exposure time revealed gradual but irrecoverable growth arrest at ≥ IC50 concentrations of 3-aminosteroids. Trypanocidal action was not associated with membrane permeabilization of trypanosome cells but instead with mitochondrial membrane depolarization, reduced adenosine triphosphate (ATP) levels and G2/M cell cycle arrest which appear to be the result of mitochondrial accumulation of the aminosteroids. These findings provided insights for further development of this new and promising class of trypanocide against African trypanosomes.

Multi-channel velocity multiplexing of single virus detection on an optofluidic chip.

Liquid-core waveguide-based optofluidic devices have proven to be valuable tools for analysis of biological samples in fluid. They have enabled single bioparticle sensitivity while maintaining in-plane detection via light-induced fluorescence. The incorporation of multi-spot excitation with multimode interference (MMI) waveguides has enabled spatially and spectrally multiplexed detection of single viruses on an oxide-based optofluidic platform. Here, we introduce a new way of MMI-based multiplexing where multiple analysis channels are placed within a single multi-spot pattern. This stacked channel design enables both velocity and spectral multiplexing of single particles. The principle is demonstrated with differentiated detection of single H3N2 and H1N1 viruses on a polydimethylsiloxane platform.

Signal-to-noise Enhancement in Optical Detection of Single Viruses with Multi-spot Excitation.

We present fluorescence detection of single H1N1 viruses with enhanced signal to noise ratio (SNR) achieved by multi-spot excitation in liquid-core anti-resonant reflecting optical waveguides (ARROWs). Solid-core Y-splitting ARROW waveguides are fabricated orthogonal to the liquid-core section of the chip, creating multiple excitation spots for the analyte. We derive expressions for the SNR increase after signal processing, and analyze its dependence on signal levels and spot number. Very good agreement between theoretical calculations and experimental results is found. SNR enhancements up to 5x104 are demonstrated.

In Vitro Activity of Retapamulin and Antimicrobial Susceptibility Patterns in a Longitudinal Collection of Methicillin-Resistant Staphylococcus aureus Isolates from a Veterans Affairs Medical Center.

Mupirocin is a topical antimicrobial used to decolonize patients who carry methicillin-resistant Staphylococcus aureus (MRSA), and the topical agent retapamulin may be a potential alternative therapy. The goal of this study was to determine the in vitro activity of retapamulin as well as a panel of 15 antimicrobial agents, including mupirocin, for 403 MRSA isolates collected longitudinally from a naive population at the Veterans Affairs Puget Sound Health Care System. The MICs for retapamulin had a unimodal distribution, ranging from 0.008 to 0.5 µg/ml. One isolate had an MIC of >16 µg/ml, was also resistant to clindamycin and erythromycin, and was recovered from the nares of a patient undergoing hemodialysis. Twenty-four isolates (6%) and 11 isolates (3%) demonstrated low-level resistance (MICs of 8 to 64 µg/ml) and high-level resistance (MICs of ≥ 512 µg/ml), respectively, to mupirocin. Isolates were recovered from 10 patients both before and after mupirocin therapy. Of those, isolates from 2 patients demonstrated MIC changes postmupirocin therapy; in both cases, however, strain typing demonstrated that the pre- and postmupirocin strains were different. A total of 386 isolates (96%) had vancomycin MICs of ≤ 1.0 µg/ml; 340 isolates (84%) were resistant to levofloxacin, 18 isolates (4.5%) were resistant to trimethoprim-sulfamethoxazole, and 135 isolates (33%) had elevated MICs of 4 µg/ml for linezolid. The baseline levels of resistance were low for mupirocin (9%) and even lower for retapamulin (0.25%) Although the use of mupirocin is currently the standard therapy for decolonization practices, the activity of retapamulin warrants its consideration as an alternative therapy in MRSA decolonization regimens.

Atomic cooling via AC Stark shift.

We theoretically investigate atomic cooling using a spatially varying AC Stark shift to compensate for the changing Doppler shift of an unchirped cooling beam. An integrated approach using waveguide-based atom photonics is ideal to achieve the required spatial tailoring of the AC Stark beam intensity. We present two examples of the design procedure to cool sodium atoms in hollow-core antiresonant reflecting optical waveguides over tens of centimeters resulting in final velocities comparable to a Zeeman slower. The methods presented here are applicable to other experimental arrangements and atomic species.

Multiple genotypic changes in hypersusceptible strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients do not always correlate with the phenotype.

OBJECTIVES: Although Pseudomonas aeruginosa from cystic fibrosis patients are well known for their antibiotic resistance, isolates that are highly susceptible to multiple drug classes have also been encountered. In this study, hypersusceptible P. aeruginosa isolates were analysed for changes in intrinsic resistance mechanisms to explain the observed phenotype. METHODS: P. aeruginosa strains PA30 and PA431 were isolated from the sputa of cystic fibrosis patients and susceptibilities were determined by agar dilution. Isolates were genetically unrelated by PFGE analysis. Expression of efflux pumps, porins, a chromosomal cephalosporinase and a gene, glmS, previously implicated in hypersusceptibility were evaluated by real-time RT-PCR, outer membrane protein analysis and beta-lactamase hydrolysis assays. RESULTS: PA30 was hypersusceptible to beta-lactams, fluoroquinolones and antimetabolites, with MICs at least 4-fold lower than those for the prototype strain PAO1, while PA431 was hypersusceptible to beta-lactams and antimetabolites. Both isolates overproduced the porin OprF but showed down-regulation in the production of the carbapenem channel OprD despite carbapenem hypersusceptibility. PA30 had decreased expression of the mexAB-oprM pump involved with intrinsic antibiotic resistance but overexpressed the mexCD-oprJ and mexEF-oprN efflux systems normally associated with acquired resistance. PA431 showed down-regulation of oprM, the last gene in the mexAB-oprM operon, but overexpressed the mexXY pump. The ampC beta-lactamase was weakly inducible in strain PA30, corresponding to cefoxitin hypersusceptibility. CONCLUSIONS: The changes in expression of several intrinsic mechanisms in the hypersusceptible strains did not correlate with the observed phenotype. These data highlight the complex interactions of resistance mechanisms in P. aeruginosa and their roles in drug susceptibility.

In vitro studies with DQ-113 and comparison fluoroquinolones to determine propensities to select resistance in gram-positive cocci.

DQ-113 was compared in vitro to sitafloxacin, moxifloxacin, levofloxacin, and ciprofloxacin for potential to select mutational resistance in multiresistant staphylococci, pneumococci, and enterococci. Its ability to select less-susceptible mutants varied according to species, being lowest with staphylococci, intermediate with pneumococci, and greatest with enterococci.

In vitro development of resistance to DX-619 and other quinolones in enterococci.

OBJECTIVES: To investigate the molecular events involved in the development of quinolone resistance in enterococci. METHODS: Clinical isolates of Enterococcus faecium and Enterococcus faecalis were exposed to inhibitory and subinhibitory concentrations of DX-619, ciprofloxacin, levofloxacin, gatifloxacin and moxifloxacin. Mutational frequencies were calculated and susceptibility changes were determined. The quinolone resistance determining regions (QRDRs) of gyrA and parC in less-susceptible mutants were amplified by PCR and sequenced. RESULTS: Single-step mutants of E. faecalis and E. faecium were selected with all drugs. There were no differences in the frequencies of mutant selection among drugs, with frequencies ranging from 10(-5) to 10(-8). All single-step mutants were inhibited by 0.03-1 mg/L DX-619, 0.25-8 mg/L moxifloxacin, 0.5-8 mg/L gatifloxacin, 1-16 mg/L levofloxacin and 1-32 mg/L ciprofloxacin. No QRDR changes were observed in single-step mutants. Less-susceptible mutants selected after five passages on agar containing subinhibitory quinolone concentrations were inhibited by 0.12-8 mg/L DX-619, 1-64 mg/L moxifloxacin, 2-64 mg/L gatifloxacin and 2-128 mg/L levofloxacin and ciprofloxacin. QRDR changes were detected in only 9 of the 20 fifth-passage mutants, suggesting that mutations outside the purported QRDRs and/or other resistance mechanisms were also involved. CONCLUSION: The relatively high frequencies at which single-step mutants were selected with all drugs indicate that caution is necessary if quinolones are to be considered for monotherapy of serious enterococcal infections. DX-619, the most potent quinolone, may have potential as an anti-enterococcal agent if sufficient concentrations can be safely attained in vivo.

Prevalence of newer beta-lactamases in gram-negative clinical isolates collected in the United States from 2001 to 2002.

Newer beta-lactamases such as extended-spectrum beta-lactamases (ESBLs), transferable AmpC beta-lactamases, and carbapenemases are associated with laboratory testing problems of false susceptibility that can lead to inappropriate therapy for infected patients. Because there appears to be a lack of awareness of these enzymes, a study was conducted during 2001 to 2002 in which 6,421 consecutive, nonduplicate clinical isolates of aerobically growing gram-negative bacilli from patients at 42 intensive care unit (ICU) and 21 non-ICU sites across the United States were tested on-site for antibiotic susceptibility. From these isolates, 746 screen-positive isolates (11.6%) were referred to a research facility and investigated to determine the prevalence of ESBLs in all gram-negative isolates, transferable AmpC beta-lactamases in Klebsiella pneumoniae, and carbapenemases in Enterobacteriaceae. The investigations involved phenotypic tests, isoelectric focusing, beta-lactamase inhibitor studies, spectrophotometric assays, induction assays, and molecular analyses. ESBLs were detected only in Enterobacteriaceae (4.9% of all Enterobacteriaceae) and were found in species other than those currently recommended for ESBL testing by the CLSI (formerly NCCLS). These isolates occurred at 74% of the ICU sites and 43% of the non-ICU sites. Transferable AmpC beta-lactamases were detected in 3.3% of K. pneumoniae isolates and at 16 of the 63 sites (25%) with no difference between ICU and non-ICU sites. Three sites submitted isolates that produced class A carbapenemases. No class B or D carbapenemases were detected. In conclusion, organisms producing ESBLs and transferable AmpC beta-lactamases were widespread. Clinical laboratories must be able to detect important beta-lactamases to ensure optimal patient care and infection control.