Bacteriophages evolve enhanced persistence to a mucosal surface.

The majority of viruses within the gut are obligate bacterial viruses known as bacteriophages (phages). Their bacteriotropism underscores the study of phage ecology in the gut, where they modulate and coevolve with gut bacterial communities. Traditionally, these ecological and evolutionary questions were investigated empirically via in vitro experimental evolution and, more recently, in vivo models were adopted to account for physiologically relevant conditions of the gut. Here, we probed beyond conventional phage-bacteria coevolution to investigate potential tripartite evolutionary interactions between phages, their bacterial hosts, and the mammalian gut mucosa. To capture the role of the mammalian gut, we recapitulated a life-like gut mucosal layer using in vitro lab-on-a-chip devices (to wit, the gut-on-a-chip) and showed that the mucosal environment supports stable phage-bacteria coexistence. Next, we experimentally coevolved lytic phage populations within the gut-on-a-chip devices alongside their bacterial hosts. We found that while phages adapt to the mucosal environment via de novo mutations, genetic recombination was the key evolutionary force in driving mutational fitness. A single mutation in the phage capsid protein Hoc-known to facilitate phage adherence to mucus-caused altered phage binding to fucosylated mucin glycans. We demonstrated that the altered glycan-binding phenotype provided the evolved mutant phage a competitive fitness advantage over its ancestral wild-type phage in the gut-on-a-chip mucosal environment. Collectively, our findings revealed that phages-in addition to their evolutionary relationship with bacteria-are able to evolve in response to a mammalian-derived mucosal environment.

Phage-antibiotic combination is a superior treatment against Acinetobacter baumannii in a preclinical study.

BACKGROUND: Clinical phage therapy is often delivered alongside antibiotics. However, the phenomenon of phage-antibiotic synergy has been mostly studied in vitro. Here, we assessed the in vivo bactericidal effect of a phage-antibiotic combination on Acinetobacter baumannii AB900 using phage øFG02, which binds to capsular polysaccharides and leads to antimicrobial resensitisation in vitro. METHODS: We performed a two-stage preclinical study using a murine model of severe A. baumannii AB900 bacteraemia. In the first stage, with an endpoint of 11 h, mice (n = 4 per group) were treated with either PBS, ceftazidime, phage øFG02, or the combination of phage and ceftazidime. The second stage involved only the latter two groups (n = 5 per group), with a prolonged endpoint of 16 h. The primary outcome was the average bacterial burden from four body sites (blood, liver, kidney, and spleen). Bacterial colonies from phage-treated mice were retrieved and screened for phage-resistance. FINDINGS: In the first stage, the bacterial burden (CFU/g of tissue) of the combination group (median: 4.55 × 105; interquartile range [IQR]: 2.79 × 105-2.81 × 106) was significantly lower than the PBS (median: 2.42 × 109; IQR: 1.97 × 109-3.48 × 109) and ceftazidime groups (median: 3.86 × 108; IQR: 2.15 × 108-6.35 × 108), but not the phage-only group (median: 1.28 × 107; IQR: 4.71 × 106-7.13 × 107). In the second stage, the combination treatment (median: 1.72 × 106; IQR: 5.11 × 105-4.00 × 106) outperformed the phage-only treatment (median: 7.46 × 107; IQR: 1.43 × 107-1.57 × 108). Phage-resistance emerged in 96% of animals receiving phages, and all the tested isolates (n = 11) had loss-of-function mutations in genes involved in capsule biosynthesis and increased sensitivity to ceftazidime. INTERPRETATION: øFG02 reliably drives the in vivo evolution of A. baumannii AB900 towards a capsule-deficient, phage-resistant phenotype that is resensitised to ceftazidime. This mechanism highlights the clinical potential of using phage therapy to target A. baumannii and restore antibiotic activity. FUNDING: National Health and Medical Research Council (Australia).

New Insights Into Sperm Ultrastructure Through Enhanced Scanning Electron Microscopy.

The analysis of spermatozoa morphology is fundamental to understand male fertility and the etiology of infertility. Traditionally scanning electron microscopy (SEM) has been used to define surface topology. Recently, however, it has become a critical tool for three-dimensional analysis of internal cellular ultrastructure. Modern SEM provides nanometer-scale resolution, but the meaningfulness of such information is proportional to the quality of the sample preservation. In this study, we demonstrate that sperm quickly and robustly adhere to gold-coated surfaces. Leveraging this property, we developed three step-by-step protocols fulfilling different needs for sperm imaging: chemically fixed monolayers for SEM examination of the external morphology, and two high-pressure freezing-based protocols for fast SEM examination of full cell internal morphology and focused ion-beam SEM tomography. These analyses allow previously unappreciated insights into mouse sperm ultrastructure, including the identification of novel structures within the fibrous sheath and domain-specific interactions between the plasma membrane and exosome-like structures.

Bacteriophage-resistant Acinetobacter baumannii are resensitized to antimicrobials.

We characterized two bacteriophages, ΦFG02 and ΦCO01, against clinical isolates of Acinetobacter baumannii and established that the bacterial capsule is the receptor for these phages. Phage-resistant mutants harboured loss-of-function mutations in genes responsible for capsule biosynthesis, resulting in capsule loss and disruption of phage adsorption. The phage-resistant strains were resensitized to human complement, beta-lactam antibiotics and alternative phages and exhibited diminished fitness in vivo. Using a mouse model of A. baumannii infection, we showed that phage therapy was effective.

The host exosome pathway underpins biogenesis of the human cytomegalovirus virion.

Human Cytomegalovirus (HCMV) infects over half the world's population, is a leading cause of congenital birth defects, and poses serious risks for immuno-compromised individuals. To expand the molecular knowledge governing virion maturation, we analysed HCMV virions using proteomics, and identified a significant proportion of host exosome constituents. To validate this acquisition, we characterized exosomes released from uninfected cells, and demonstrated that over 99% of the protein cargo was subsequently incorporated into HCMV virions during infection. This suggested a common membrane origin, and utilization of host exosome machinery for virion assembly and egress. Thus, we selected a panel of exosome proteins for knock down, and confirmed that loss of 7/9 caused significantly less HCMV production. Saliently, we report that VAMP3 is essential for viral trafficking and release of infectious progeny, in various HCMV strains and cell types. Therefore, we establish that the host exosome pathway is intrinsic for HCMV maturation, and reveal new host regulators involved in viral trafficking, virion envelopment, and release. Our findings underpin future investigation of host exosome proteins as important modulators of HCMV replication with antiviral potential.

Hybrid refractive-diffractive axicons for Bessel-beam multiplexing and resolution improvement.

Optical elements rely on refraction, diffraction, or reflection for light manipulation. Fusing diffractive and refractive functions in a single element provides an extra layer of control over the wave propagation, allowing complex beam shaping through self-aligned, monolithic and miniaturized optics. Using gray-scale lithography with high-current focused Xe ion-beams, we realized hybrid refractive-diffractive micro-axicons that feature diffractive gratings engraved on their conical surfaces. Furthermore, we fabricated these devices in lithium niobate, which is a challenging piezo/optoelectronic material for processing with an as-yet unexploited potential in optical applications. The curvilinear surfaces of fabricated micro-axicons with a 230-µm diameter were engraved with diffraction linear and circular gratings of various depths (<400 nm), and the optical performance of these components was characterized, showing excellent agreement with theoretical expectations. The fusing of diffractive elements with carrier refractive surfaces introduces additional or enhanced device functionalities, such as beam multiplexing and resolution improvement. The potential applications of such monolithic and miniaturized hybrid micro-optical components include beamshaping for fluorescence microscopy.

Ultrastructural Aspects of Photodynamic Inactivation of Highly Pathogenic Avian H5N8 Influenza Virus.

Ultrastructural studies revealing morphological differences between intact and photodynamically inactivated virions can point to inactivation mechanisms and molecular targets. Using influenza as a model system, we show that photodynamic virus inactivation is possible without total virion destruction. Indeed, irradiation with a relatively low concentration of the photosensitizer (octacationic octakis(cholinyl) zinc phthalocyanine) inactivated viral particles (the virus titer was determined in Madin Darby Canine Kidney (MDCK) cells) but did not destroy them. Transmission electron microscopy (TEM) revealed that virion membranes kept structural integrity but lost their surface glycoproteins. Such structures are known as "bald" virions, which were first described as a result of protease treatment. At a higher photosensitizer concentration, the lipid membranes were also destroyed. Therefore, photodynamic inactivation of influenza virus initially results from surface protein removal, followed by complete virion destruction. This study suggests that photodynamic treatment can be used to manufacture "bald" virions for experimental purposes. Photodynamic inactivation is based on the production of reactive oxygen species which attack and destroy biomolecules. Thus, the results of this study can potentially apply to other enveloped viruses and sources of singlet oxygen.

Effect of Mo on the catalytic activity of Ni-based self-organizing catalysts for processing of dichloroethane into segmented carbon nanomaterials.

A series of micro-disperse Ni-Mo alloys with the sponge-like structure was prepared by a simultaneous precipitation method followed by sintering of the sediment in H2 atmosphere at 800 °C. According to XRD data, the formation of single-phase solid solution Ni1-xMox took place for the samples with Mo content of 0.6-8.3 wt.%. Synthesized samples were studied in a process of the catalytic CVD of C2H4Cl2 at 550-700 °C. In situ kinetic studies of carbon deposition process were carried out in a flow gravimetric setup equipped with McBain balances. An interaction of Ni-Mo alloys with C2H4Cl2 is accompanied by their rapid disintegration with formation of disperse active particles catalyzing the growth of carbon nanomaterials (CNM). The strong boosting effect of Mo on the catalytic performance of Ni was revealed. The maximum yield of CNM product (8.3 wt.% Mo, 600 °C, 120 min) was as high as 45 g/gM. The study on effect of the reaction temperature on the CNM yield allowed one to define an optimal temperature regime. The impact of Mo concentration upon the morphology, structural features and textural properties of the produced carbon fibers was investigated by means of SEM, TEM, Raman spectroscopy and low-temperature nitrogen adsorption. The role of chemisorbed chlorine species in a pulse-to-pulse regime of the segmented carbon filaments formation was discussed.

Characterization of avian paramyxovirus type 6 isolated from a Eurasian teal in the intersection of migratory flyways in Russia.

The complete genome sequence was determined for avian paramyxovirus (APMV-6) serotype 6 strain teal/Chany/455/2009, isolated from a teal (Anas crecca) in Siberia. Siberia is crossed by four major migration flyways and represents the major breeding area for many wild bird species in the Palearctic. Strain teal/Chany/455/2009 is genetically closely related to Kazakh and Chinese strains and belongs to the genetic group of duck/Hong Kong/18/199/77-like APMV-6 viruses. We show that the virus has low pathogenic potential according to genetic markers and animal model experiments.

The selection and optimization of the detection system for self-contained multiplexed dot-immunoassay.

This study performed a comparative estimation of the detection systems with the use of conjugates based on peroxidase, alkaline phosphatase, colloidal gold, and the amplification system "Super-CARD" for multiplex dot-immunoassay of antibodies. The results of the study show that the sensitivity of the detection system with colloidal gold was approximately 8 times higher than that of the system with amplification "Super-CARD", 30 times higher than that of the system with conjugate of alkaline phosphatase, and 250 times higher than the sensitivity of the system with the peroxidase conjugate. Gold immunosols limit the direct detection of human IgG of 10 pg with dynamic range of optical signal change from 5 ng to 10 pg. This limit corresponds to a range of concentrations of IgG from 2.5 µg/mL to 5 ng/mL and covers the range of specific IgG concentrations, to which a typical natural immune response leads. The most typical reasons for aggregate formation during obtainment of colloidal gold and the binding of colloidal gold with biocomponents were explored. The method of minimization of particle clusters formation was suggested as well as the method for increasing stability of diluted preparations of probe by means of sedimentation of aggregates from the ready-made product.

Force-induced globule-coil transition in laminin binding protein and its role for viral-cell membrane fusion.

The specific interactions of the pairs laminin binding protein (LBP)-purified tick-borne encephalitis viral surface protein E and certain recombinant fragments of this protein, as well as West Nile viral surface protein E and certain recombinant fragments of that protein, are studied by combined methods of single-molecule dynamic force spectroscopy (SMDFS), enzyme immunoassay and optical surface waves-based biosensor measurements. The experiments were performed at neutral pH (7.4) and acid pH (5.3) conditions. The data obtained confirm the role of LBP as a cell receptor for two typical viral species of the Flavivirus genus. A comparison of these data with similar data obtained for another cell receptor of this family, namely human αVß3 integrin, reveals that both these receptors are very important. Studying the specific interaction between the cell receptors in question and specially prepared monoclonal antibodies against them, we could show that both interaction sites involved in the process of virus-cell interaction remain intact at pH 5.3. At the same time, for these acid conditions characteristic for an endosome during flavivirus-cell membrane fusion, SMDFS data reveal the existence of a force-induced (effective already for forces as small as 30-70 pN) sharp globule-coil transition for LBP and LBP-fragments of protein E complexes. We argue that this conformational transformation, being an analog of abrupt first-order phase transition and having similarity with the famous Rayleigh hydrodynamic instability, might be indispensable for the flavivirus-cell membrane fusion process.