Adherence, Sexual Behavior and Sexually Transmitted Infections in a New Zealand Prospective PrEP Cohort: 12 Months Follow-up and Ethnic Disparities.

Inequities in pre-exposure prophylaxis (PrEP) experiences will impede HIV epidemic elimination among gay and bisexual men (GBM). Ethnicity is a strong marker of inequity in the United States, but evidence from other countries is lacking. We investigated experiences on-PrEP to 12 months follow-up in a prospective cohort of 150 GBM in Auckland, New Zealand with an equity quota of 50% non-Europeans. Retention at 12 months was 85.9%, lower among Maori/Pacific (75.6%) than non-Maori/Pacific participants (90.1%). Missed pills increased over time and were higher among Maori/Pacific. PrEP breaks increased, by 12 months 35.7% of Maori/Pacific and 15.7% of non-Maori/Pacific participants had done so. Condomless receptive anal intercourse partners were stable over time. STIs were common but chlamydia declined; 12-month incidence was 8.7% for syphilis, 36.0% gonorrhoea, 46.0% chlamydia, 44.7% rectal STI, 64.0% any STI. Structural interventions and delivery innovations are needed to ensure ethnic minority GBM gain equal benefit from PrEP.Clinical trial number ACTRN12616001387415.

Inhibition of Erwinia amylovora by Bacillus nakamurai.

A variety of potential inhibitors were tested for the first time for the suppression of Erwinia amylovora, the causal agent of fire blight in apples and pears. Strain variability was evident in susceptibility to inhibitors among five independently isolated virulent strains of E. amylovora. However, most strains were susceptible to culture supernatants from strains of Bacillus spp., and particularly to the recently described species B. nakamurai. Minimal inhibitory concentrations (MICs) were 5-20% (vol/vol) of culture supernatant from B. nakamurai against all five strains of E. amylovora. Although Bacillus species have been previously reported to produce lipopeptide inhibitors of E. amylovora, matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF MS) and column chromatography indicated that the inhibitor from B. nakamurai was not a lipopeptide, but rather a novel inhibitor.

Baseline characteristics of gay and bisexual men in a HIV pre-exposure prophylaxis demonstration project with equity quotas in Auckland, New Zealand.

Background In New Zealand, pre-exposure prophylaxis (PrEP) should target gay and bisexual men (GBM), and equity is an important principle. Baseline characteristics of GBM offered PrEP in a demonstration project with an enrolment quota of 50% non-Europeans are described. METHODS: An open-label, single-arm treatment evaluation study design ('NZPrEP') was used. The settings were four publicly funded sexual health clinics in Auckland in 2017. The study population was 150 GBM recruited from clinics, community sources and social media. Participants self-completed an online questionnaire about PrEP awareness, attitudes and sexual risk behaviour in the last 3 months. Baseline characteristics are described and examined to determine whether these were associated with PrEP initiation status (self-referral vs doctor/nurse recommendation). RESULTS: In total, 150 GBM of whom half (52%) were non-European, including 21.3% Maori, 19.3% Asian and 8.7% Pacific, were enrolled into the study. Two-thirds (65.3%) self-referred for PrEP and one-third (34.7%) were recommended PrEP by the doctor/nurse. Participants reported a high number of male condomless receptive anal intercourse partners (MenAICLR) (median 3, range 0-50), with 10% reporting 10 or more MenAICLR and 45.3% reporting group sex. In the previous year, 65.3% had a sexually transmissible infection (STI); 18% had rectal chlamydia or gonorrhoea at enrolment. Almost half (47.7%) had recently used drugs with sex, including 8.1% who used methamphetamine. Participants recommended PrEP had lower education, lived less centrally and had a higher STI prevalence than PrEP self-referrers, but their risk behaviour was similar. CONCLUSIONS: Early PrEP adopters in New Zealand have high HIV risk. Demonstration projects should consider equity mechanisms so that minorities can participate meaningfully.

Microbial-derived products as potential new antimicrobials.

Due to the continuing global concerns involving antibiotic resistance, there is a need for scientific forums to assess advancements in the development of antimicrobials and their alternatives that might reduce development and spread of antibiotic resistance among bacterial pathogens. The objectives of the 2nd International Symposium on Alternatives to Antibiotics were to highlight promising research results and novel technologies that can provide alternatives to antibiotics for use in animal health and production, assess challenges associated with their authorization and commercialization for use, and provide actionable strategies to support their development. The session on microbial-derived products was directed at presenting novel technologies that included exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials, probiotics development via fecal microbiome transplants among monogastric production animals such as chickens and mining microbial sources such as bacteria or yeast to identify new antimicrobial compounds. Other research has included continuing development of antimicrobial peptides such as newly discovered bacteriocins as alternatives to antibiotics, use of bacteriophages accompanied by development of unique lytic proteins with specific cell-wall binding domains and novel approaches such as microbial-ecology guided discovery of anti-biofilm compounds discovered in marine environments. The symposium was held at the Headquarters of the World Organisation for Animal Health (OIE) in Paris, France during 12-15 December 2016.

Inactivation of virginiamycin by Aureobasidium pullulans.

OBJECTIVE: To test the inactivation of the antibiotic, virginiamycin, by laccase-induced culture supernatants of Aureobasidium pullulans. RESULTS: Fourteen strains of A. pullulans from phylogenetic clade 7 were tested for laccase production. Three laccase-producing strains from this group and three previously identified strains from clade 5 were compared for inactivation of virginiamycin. Laccase-induced culture supernatants from clade 7 strains were more effective at inactivation of virginiamycin, particularly at 50 °C. Clade 7 strain NRRL Y-2567 inactivated 6 µg virginiamycin/ml within 24 h. HPLC analyses indicated that virginiamycin was degraded by A. pullulans. CONCLUSIONS: A. pullulans has the potential for the bioremediation of virginiamycin-contaminated materials, such as distiller's dry grains with solubles (DDGS) animal feed produced from corn-based fuel ethanol production.

Novel Feruloyl Esterase from Lactobacillus fermentum NRRL B-1932 and Analysis of the Recombinant Enzyme Produced in Escherichia coli.

UNLABELLED: A total of 33 Lactobacillus strains were screened for feruloyl esterase (FE) activity using agar plates containing ethyl ferulate as the sole carbon source, and Lactobacillus fermentum NRRL B-1932 demonstrated the strongest FE activity among a dozen species showing a clearing zone on the opaque plate containing ethyl ferulate. FE activities were monitored using high-performance liquid chromatography with an acetonitrile-trifluoroacetic acid gradient. To produce sufficient purified FE from L. fermentum strain NRRL B-1932 (LfFE), the cDNA encoding LfFE (Lffae) was amplified and cloned by using available closely related genome sequences and overexpressed in Escherichia coli A 29.6-kDa LfFE protein was detected from the protein extract of E. coli BL21(pLysS) carrying pET28bLffae upon IPTG (isopropyl-ß-d-thiogalactopyranoside) induction. The recombinant LfFE containing a polyhistidine tag was purified by nickel-nitrilotriacetic acid affinity resin. The purified LfFE showed strong activities against several artificial substrates, including p-nitrophenyl acetate and 4-methylumbelliferyl p-trimethylammoniocinnamate chloride. The optimum pH and temperature of the recombinant LfFE were around 6.5 and 37°C, respectively, as determined using either crude or purified recombinant LfFE. This study will be essential for the production of the LfFE in E. coli on a larger scale that could not be readily achieved by L. fermentum fermentation. IMPORTANCE: The production of feruloyl esterase (FE) from Lactobacillus fermentum NRRL B-1932 reported in this study will have immense potential commercial applications not only in biofuel production but also in pharmaceutical, polymer, oleo chemical, cosmetic additive, and detergent industries, as well as human health-related applications, including food flavoring, functional foods, probiotic agents, preventive medicine, and animal feed. Given the essential role FE plays in the production of hydroxycinnamic acids and ferulic acid, plus the generally regarded as safe status of lactobacilli, which therefore have less regulatory concerns, LfFE from the probiotic L. fermentum reported in this work can be directly used for increased production of high-value hydroxycinnamates and ferulic acid from natural or synthetic carbon sources.

Phylogenetic classification of Aureobasidium pullulans strains for production of feruloyl esterase.

OBJECTIVE: The objective was to phylogenetically classify diverse strains of Aureobasidium pullulans and determine their production of feruloyl esterase. RESULTS: Seventeen strains from the A. pullulans literature were phylogenetically classified. Phenotypic traits of color variation and endo-ß-1,4-xylanase overproduction were associated with phylogenetic clade 10 and particularly clade 8. Literature strains used for pullulan production all belonged to clade 7. These strains and 36 previously classified strains were tested for feruloyl esterase production, which was found to be associated with phylogenetic clades 4, 11, and particularly clade 8. Clade 8 strains NRRL 58552 and NRRL 62041 produced the highest levels of feruloyl esterase among strains tested. CONCLUSIONS: Production of both xylanase and feruloyl esterase are associated with A. pullulans strains in phylogenetic clade 8, which is thus a promising source of enzymes with potential biotechnological applications.

Fate of virginiamycin through the fuel ethanol production process.

Antibiotics are frequently used to prevent and treat bacterial contamination of commercial fuel ethanol fermentations, but there is concern that antibiotic residues may persist in the distillers grains coproducts. A study to evaluate the fate of virginiamycin during the ethanol production process was conducted in the pilot plant facilities at the National Corn to Ethanol Research Center, Edwardsville, IL. Three 15,000-liter fermentor runs were performed: one with no antibiotic (F1), one dosed with 2 parts per million (ppm) of a commercial virginiamycin product (F2), and one dosed at 20 ppm of virginiamycin product (F3). Fermentor samples, distillers dried grains with solubles (DDGS), and process intermediates (whole stillage, thin stillage, syrup, and wet cake) were collected from each run and analyzed for virginiamycin M and virginiamycin S using a liquid chromatography-mass spectrometry method. Virginiamycin M was detected in all process intermediates of the F3 run. On a dry-weight basis, virginiamycin M concentrations decreased approximately 97 %, from 41 µg/g in the fermentor to 1.4 µg/g in the DDGS. Using a disc plate bioassay, antibiotic activity was detected in DDGS from both the F2 and F3 runs, with values of 0.69 µg virginiamycin equivalent/g sample and 8.9 µg/g, respectively. No antibiotic activity (<0.6 µg/g) was detected in any of the F1 samples or in the fermentor and process intermediate samples from the F2 run. These results demonstrate that low concentrations of biologically active antibiotic may persist in distillers grains coproducts produced from fermentations treated with virginiamycin.

Irradiation of Yarrowia lipolytica NRRL YB-567 creating novel strains with enhanced ammonia and oil production on protein and carbohydrate substrates.

Increased interest in sustainable production of renewable diesel and other valuable bioproducts is redoubling efforts to improve economic feasibility of microbial-based oil production. Yarrowia lipolytica is capable of employing a wide variety of substrates to produce oil and valuable co-products. We irradiated Y. lipolytica NRRL YB-567 with UV-C to enhance ammonia (for fertilizer) and lipid (for biodiesel) production on low-cost protein and carbohydrate substrates. The resulting strains were screened for ammonia and oil production using color intensity of indicators on plate assays. Seven mutant strains were selected (based on ammonia assay) and further evaluated for growth rate, ammonia and oil production, soluble protein content, and morphology when grown on liver infusion medium (without sugars), and for growth on various substrates. Strains were identified among these mutants that had a faster doubling time, produced higher maximum ammonia levels (enzyme assay) and more oil (Sudan Black assay), and had higher maximum soluble protein levels (Bradford assay) than wild type. When grown on plates with substrates of interest, all mutant strains showed similar results aerobically to wild-type strain. The mutant strain with the highest oil production and the fastest doubling time was evaluated on coffee waste medium. On this medium, the strain produced 0.12 g/L ammonia and 0.20 g/L 2-phenylethanol, a valuable fragrance/flavoring, in addition to acylglycerols (oil) containing predominantly C16 and C18 residues. These mutant strains will be investigated further for potential application in commercial biodiesel production.

Antibacterial activity of a cell wall hydrolase from Lactobacillus paracasei NRRL B-50314 produced by recombinant Bacillus megaterium.

The cell-free supernatant (CFS) from Lactobacillus paracasei NRRL B-50314 culture has been previously reported as containing antibacterial activity against a wide variety of Gram-positive bacteria. The CFS protein gel slice corresponding to antibacterial activities was subjected to trypsin digestion and ion trap MASS (Gel/LC-MS/MS) analysis. BlastP search of the resulted IQAVISIAEQQIGKP sequence led to a hypothetical cell-wall associated hydrolase (designated as CWH here) from Lactobacillus paracasei ATCC 25302. Further analyses of CWH revealed that the IQAVISIAEQQIGKP belongs to a highly conserved region of the NlpC/P60 superfamily. The L. paracasei NRRL B-50314 CWH gene, cloned in pStrepHIS1525CWH477, was introduced into Bacillus megaterium MS 941. The production of CWH477 protein was induced by xylose. The CWH477 protein was purified by using NiNTA column, and elution fraction E2 showed highest antibacterial activity. This study and bioinformatics analyses suggested that the antibacterial activity of CWH could originate from its cell wall degrading enzymatic function.

Acoustofluidic scanning fluorescence nanoscopy with a large field of view.

Large-field nanoscale fluorescence imaging is invaluable for many applications, such as imaging subcellular structures, visualizing protein interactions, and high-resolution tissue imaging. Unfortunately, conventional fluorescence microscopy requires a trade-off between resolution and field of view due to the nature of the optics used to form the image. To overcome this barrier, we developed an acoustofluidic scanning fluorescence nanoscope that simultaneously achieves superior resolution, a large field of view, and strong fluorescent signals. The acoustofluidic scanning fluorescence nanoscope utilizes the superresolution capabilities of microspheres that are controlled by a programmable acoustofluidic device for rapid fluorescence enhancement and imaging. The acoustofluidic scanning fluorescence nanoscope resolves structures that cannot be resolved with conventional fluorescence microscopes with the same objective lens and enhances the fluorescent signal by a factor of ~5 without altering the field of view of the image. The improved resolution realized with enhanced fluorescent signals and the large field of view achieved via acoustofluidic scanning fluorescence nanoscopy provides a powerful tool for versatile nanoscale fluorescence imaging for researchers in the fields of medicine, biology, biophysics, and biomedical engineering.

Assessing the agreement of chronic lung disease of prematurity diagnosis between radiologists and clinical criteria.

BACKGROUND: Chronic lung disease of prematurity (CLD) is the most prevalent complication of preterm birth and indicates an increased likelihood of long-term pulmonary complications. The accurate diagnosis of this condition is critical for long-term health management. Numerous definitions define CLD with different clinical parameters and radiology findings, making diagnosis of the disease ambiguous and potentially inaccurate. METHODS: 95 patients were identified for this study, as determined by the diagnosis or confirmation of CLD in the impression of the radiologist's report on chest x-ray. Pulmonary function and complications were recorded at multiple benchmark timeframes within each patient's first few months of life and used for determining eligibility under each definition. RESULTS: Each clinical definition of CLD had a high sensitivity for patients identified to have CLD by radiologists, correctly fitting over 90% of patients. Most patients included required invasive mechanical ventilation or positive pressure ventilation at 36 weeks postmenstrual age, indicating patients with radiographically confirmed CLD tended to have more severe disease. Radiologists tended to diagnose CLD before 36 weeks postmenstrual age, a timepoint used by multiple standard clinical definitions, with cases called earlier fitting under a larger percentage of definitions than those called later. CONCLUSIONS: Radiologists tend to diagnose CLD in young patients with severe respiratory compromise, and can accurately diagnose the condition before developmental milestones for clinical definitions are met.

Advances in Microsphere-based Super-resolution Imaging.

Techniques to resolve images beyond the diffraction limit of light with a large field of view (FOV) are necessary to foster progress in various fields such as cell and molecular biology, biophysics, and nanotechnology, where nanoscale resolution is crucial for understanding the intricate details of large-scale molecular interactions. Although several means of achieving super-resolutions exist, they are often hindered by factors such as high costs, significant complexity, lengthy processing times, and the classical tradeoff between image resolution and FOV. Microsphere-based super-resolution imaging has emerged as a promising approach to address these limitations. In this review, we delve into the theoretical underpinnings of microsphere-based imaging and the associated photonic nanojet. This is followed by a comprehensive exploration of various microsphere-based imaging techniques, encompassing static imaging, mechanical scanning, optical scanning, and acoustofluidic scanning methodologies. This review concludes with a forward-looking perspective on the potential applications and future scientific directions of this innovative technology.

The impact of package selection and versioning on single-cell RNA-seq analysis.

Standard single-cell RNA-sequencing analysis (scRNA-seq) workflows consist of converting raw read data into cell-gene count matrices through sequence alignment, followed by analyses including filtering, highly variable gene selection, dimensionality reduction, clustering, and differential expression analysis. Seurat and Scanpy are the most widely-used packages implementing such workflows, and are generally thought to implement individual steps similarly. We investigate in detail the algorithms and methods underlying Seurat and Scanpy and find that there are, in fact, considerable differences in the outputs of Seurat and Scanpy. The extent of differences between the programs is approximately equivalent to the variability that would be introduced in benchmarking scRNA-seq datasets by sequencing less than 5% of the reads or analyzing less than 20% of the cell population. Additionally, distinct versions of Seurat and Scanpy can produce very different results, especially during parts of differential expression analysis. Our analysis highlights the need for users of scRNA-seq to carefully assess the tools on which they rely, and the importance of developers of scientific software to prioritize transparency, consistency, and reproducibility for their tools.

High-yield and rapid isolation of extracellular vesicles by flocculation via orbital acoustic trapping: FLOAT.

Extracellular vesicles (EVs) have been identified as promising biomarkers for the noninvasive diagnosis of various diseases. However, challenges in separating EVs from soluble proteins have resulted in variable EV recovery rates and low purities. Here, we report a high-yield ( > 90%) and rapid ( < 10 min) EV isolation method called FLocculation via Orbital Acoustic Trapping (FLOAT). The FLOAT approach utilizes an acoustofluidic droplet centrifuge to rotate and controllably heat liquid droplets. By adding a thermoresponsive polymer flocculant, nanoparticles as small as 20 nm can be rapidly and selectively concentrated at the center of the droplet. We demonstrate the ability of FLOAT to separate urinary EVs from the highly abundant Tamm-Horsfall protein, addressing a significant obstacle in the development of EV-based liquid biopsies. Due to its high-yield nature, FLOAT reduces biofluid starting volume requirements by a factor of 100 (from 20 mL to 200 µL), demonstrating its promising potential in point-of-care diagnostics.

Acoustothermal transfection for cell therapy.

Transfected stem cells and T cells are promising in personalized cell therapy and immunotherapy against various diseases. However, existing transfection techniques face a fundamental trade-off between transfection efficiency and cell viability; achieving both simultaneously remains a substantial challenge. This study presents an acoustothermal transfection method that leverages acoustic and thermal effects on cells to enhance the permeability of both the cell membrane and nuclear envelope to achieve safe, efficient, and high-throughput transfection of primary T cells and stem cells. With this method, two types of plasmids were simultaneously delivered into the nuclei of mesenchymal stem cells (MSCs) with efficiencies of 89.6 ± 1.2%. CXCR4-transfected MSCs could efficiently target cerebral ischemia sites in vivo and reduce the infarct volume in mice. Our acoustothermal transfection method addresses a key bottleneck in balancing the transfection efficiency and cell viability, which can become a powerful tool in the future for cellular and gene therapies.

Aerosol jet printing of surface acoustic wave microfluidic devices.

The addition of surface acoustic wave (SAW) technologies to microfluidics has greatly advanced lab-on-a-chip applications due to their unique and powerful attributes, including high-precision manipulation, versatility, integrability, biocompatibility, contactless nature, and rapid actuation. However, the development of SAW microfluidic devices is limited by complex and time-consuming micro/nanofabrication techniques and access to cleanroom facilities for multistep photolithography and vacuum-based processing. To simplify the fabrication of SAW microfluidic devices with customizable dimensions and functions, we utilized the additive manufacturing technique of aerosol jet printing. We successfully fabricated customized SAW microfluidic devices of varying materials, including silver nanowires, graphene, and poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS). To characterize and compare the acoustic actuation performance of these aerosol jet printed SAW microfluidic devices with their cleanroom-fabricated counterparts, the wave displacements and resonant frequencies of the different fabricated devices were directly measured through scanning laser Doppler vibrometry. Finally, to exhibit the capability of the aerosol jet printed devices for lab-on-a-chip applications, we successfully conducted acoustic streaming and particle concentration experiments. Overall, we demonstrated a novel solution-based, direct-write, single-step, cleanroom-free additive manufacturing technique to rapidly develop SAW microfluidic devices that shows viability for applications in the fields of biology, chemistry, engineering, and medicine.

The production of glucans via glucansucrases from Lactobacillus satsumensis isolated from a fermented beverage starter culture.

Several starter cultures used in the production of fermented beverages were screened for lactic acid bacteria that produced water-insoluble polysaccharides from sucrose. The strain producing the greatest amount was identified as Lactobacillus satsumensis by its 16S RNA sequence and was deposited in the ARS culture collection as NRRL B-59839. This strain produced at least two α-D-glucans from sucrose. One was a water-soluble dextran, consisting of predominantly α-(1 → 6)-linked D-glucose units, and the other was a water-insoluble glucan containing both α-(1 → 6)-linked and α-(1 → 3)-linked D-glucose units. The culture fluid was found to contain glucansucrases responsible for the two glucans, and no significant level of fructansucrase was detected. Glucansucrase activity was not present in the culture fluid when the bacteria were grown on glucose, fructose, or raffinose as the carbon source. Although the water-soluble glucans produced by cell-free enzyme and by cell suspensions were essentially identical, the same was not true for the water-insoluble glucans. The water-insoluble glucan produced by cell-free culture fluid contained a higher proportion of α-(1 → 3)-linked D-glucose units than the water-insoluble glucan produced by cell suspensions.

Combinatorial evaluation of laccase-mediator system in the oxidation of veratryl alcohol.

Laccases play an important role in the biological break down of lignin and have great potential in the deconstruction of lignocellulosic feedstocks. We examined 16 laccases, both commercially prepared and crude extracts, for their ability to oxidize veratryl alcohol in the presence of various solvents and mediators. Screening revealed complete conversion of veratryl alcohol to veratraldehyde catalyzed by a crude preparation of the laccase from Trametes versicolor ATCC 11235 and the mediator TEMPO in 20 % (v/v) tert-butanol.

Lipase production by diverse phylogenetic clades of Aureobasidium pullulans.

Thirty-nine strains representing 12 diverse phylogenetic clades of Aureobasidium pullulans were surveyed for lipase production using a quantitative assay. Strains in clades 4 and 10 produced 0.2-0.3 U lipase/ml, while color variant strain NRRL Y-2311-1 in clade 8 produced 0.54 U lipase/ml. Strains in clade 9, which exhibit a dark olivaceous pigment, produced the highest levels of lipase, with strain NRRL 62034 yielding 0.57 U lipase/ml. By comparison, Candida cylindracea strain NRRL Y-17506 produced 0.05 U lipase/ml under identical conditions. A. pullulans strain NRRL 62034 reached maximal lipase levels in 5 days on lipase induction medium, while A. pullulans strain NRRL Y-2311-1 and strains in clades 4 and 10 were highest after 6 days. A. pullulans strain NRRL Y-2311-1 and strains in clade 9 produced two extracellular proteins in common, at >50 and <37 kDa.