PCR and peptide based PCMV detection in pig - development and application of a combined testing procedure differentiating newly from latent infected pigs.

Porcine cytomegalovirus (PCMV) is widely distributed in pigs and difficult to detect due to latency. PCMV infection of source pigs was associated with early graft failure after cardiac and renal xenotransplantation into nonhuman primates. Importantly, PCMV infection of the first genetically modified pig heart into a human may have contributed to the reduced survival of the patient. Sensitive and reliable assays for detection of latent PCMV infection are thus indispensable. Here, we report the development of five peptide-induced rabbit antisera specific for PCMV glycoprotein B (gB) and their validation for detection of PCMV in infected pig fallopian tube (PFT) cells by immunofluorescence and electron microscopy (EM). The anti-gB antibodies were also used for detection by Western blot analysis of PCMV purified from the supernatant of infected PFT cells. Sera of infected versus non-infected pigs have been compared. In parallel, PCMV viral load in blood samples of the animals was quantified by a novel highly sensitive nested-PCR and qPCR assay. A combination of four partly overlapping peptides from the gB C-terminus was used to establish a diagnostic ELISA for PCMV gB specific pig antibodies which is able to differentiate infected from non-infected animals and to quantify maternal antibodies in neonates. The combination of a highly sensitive nested PCR for direct virus detection with a sensitive peptide-based ELISA detecting anti-PCMV gB-antibodies, supplemented by Western blot analysis and/or immunohistochemistry for virus detection will reliably differentiate pigs with active infection, latently infected pigs, and non-infected pigs. It may significantly improve the virologic safety of xenotransplantation.

Toward integrated synchronously pumped optical parametric oscillators in silicon nitride.

We present a tunable, hybrid waveguide-fiber optical parametric oscillator (OPO) synchronously pumped by an ultra-fast fiber laser exploiting four-wave mixing (FWM) generated in silicon nitride waveguides. Parametric oscillation results in a 35 dB enhancement of the idler spectral power density in comparison to spontaneous FWM, with the ability of wide wavelength tuning over 86 nm in the O-band. Measurements of the oscillation threshold and the efficiency of the feedback loop reveal how an integration of the OPO on a single silicon nitride chip can be accomplished at standard repetition rates of pump lasers in the order of 100 MHz.

Optical parametric amplification in silicon nitride waveguides for coherent Raman imaging.

We present tunable waveguide-based optical parametric amplification by four-wave mixing (FWM) in silicon nitride waveguides, with the potential to be set up as an all-integrated device, for narrowband coherent anti-Stokes Raman scattering (CARS) imaging. Signal and idler pulses are generated via FWM with only 3 nJ pump pulse energy and stimulated by using only 4 mW of a continuous-wave seed source, resulting in a 35 dB enhancement of the idler spectral power density in comparison to spontaneous FWM. By using waveguides with different widths and tuning the wavelength of the signal wave seed, idler wavelengths covering the spectral region from 1.1 µm up to 1.6 µm can be generated. The versatility of the chip-based FWM light source is demonstrated by acquiring CARS images.

Forming megakaryocytes from murine-induced pluripotent stem cells by the inducible overexpression of supporting factors.

BACKGROUND: Platelets are small anucleate cells that circulate in the blood in a resting state but can be activated by external cues. In case of need, platelets from blood donors can be transfused. As an alternative source, platelets can be produced from induced pluripotent stem cells (iPSCs); however, recovered numbers are low. OBJECTIVES: To optimize megakaryocyte (MK) and platelet output from murine iPSCs, we investigated overexpression of the transcription factors GATA-binding factor 1 (GATA1); nuclear factor, erythroid 2; and pre-B-cell leukemia transcription factor 1 (Pbx1) and a hyperactive variant of the small guanosine triphosphatase RhoA (RhoAhc). METHODS: To avoid off-target effects, we generated iPSCs carrying the reverse tetracycline-responsive transactivator M2 (rtTA-M2) in the Rosa26 locus and expressed the factors from Tet-inducible gammaretroviral vectors. Differentiation of iPSCs was initiated by embryoid body (EB) formation. After EB dissociation, early hematopoietic progenitors were enriched and cocultivated on OP9 feeder cells with thrombopoietin and stem cell factor to induce megakaryocyte (MK) differentiation. RESULTS: Overexpression of GATA1 and Pbx1 increased MK output 2- to 2.5-fold and allowed prolonged collection of MK. Cytologic and ultrastructural analyses identified typical MK with enlarged cells, multilobulated nuclei, granule structures, and an internal membrane system. However, GATA1 and Pbx1 expression did not improve MK maturation or platelet release, although in vitro-generated platelets were functional in spreading on fibrinogen or collagen-related peptide. CONCLUSION: We demonstrate that the use of rtTA-M2 transgenic iPSCs transduced with Tet-inducible retroviral vectors allowed for gene expression at later time points during differentiation. With this strategy we could identify factors that increased in vitro MK production.

Evaluation of the in vitro Function of Platelet Concentrates from Pooled Buffy Coats or Apheresis.

BACKGROUND: Platelet concentrates play an important role in transfusion medicine. Their short lifespan and lack of robustness require efforts to ensure adequate product quality. In this study, we compared the in vitro quality of the main concentrate types, pooled platelet concentrate (PPC) from whole blood donations, and platelet concentrate from single-donor apheresis (APC). METHODS: Twenty PPCs and 20 APCs prepared in plasma were analyzed on days 2, 4, and 7 of storage. Variables related to metabolism, degranulation, platelet aggregation, P-selectin expression, and annexin V binding were analyzed. Morphology was assessed by transmission electron microscopy of ultrathin sections. A microfluidic device was applied to test the effects of shear stress on platelet function. RESULTS: The metabolic parameters indicated stable storage conditions throughout the 7-day period. The resting discoid form was the prevailing morphology on days 2 and 4 in the PPCs and APCs. Chemokine release and receptor shedding of soluble P-selectin and soluble CD40L equally increased in PPCs and APCs. Aggregation responses to ADP and collagen were heterogeneous, with marked losses in collagen responsiveness on day 4 in individual concentrates. Baseline expression of P-selectin in PPCs and APCs was low, and inducibility of P-selectin was well preserved until day 4. Under shear stress, equal adhesiveness and stability were found with platelets from PPCs and APCs. CONCLUSIONS: Platelets from PPCs and APCs showed similar in vitro function and stability parameters. However, platelet concentrates presented a high variability and individual concentrates an impaired functional capability. Identifying the factors contributing to this would help increase product reliability.

The N-Terminus Makes the Difference: Impact of Genotype-Specific Disparities in the N-Terminal Part of The Hepatitis B Virus Large Surface Protein on Morphogenesis of Viral and Subviral Particles.

The N-terminus of the hepatitis B virus (HBV) large surface protein (LHB) differs with respect to genotypes. Compared to the amino terminus of genotype (Gt)D, in GtA, GtB and GtC, an additional identical 11 amino acids (aa) are found, while GtE and GtG share another similar 10 aa. Variants of GtB and GtC affecting this N-terminal part are associated with hepatoma formation. Deletion of these amino-terminal 11 aa in GtA reduces the amount of LHBs and changes subcellular accumulation (GtA-like pattern) to a dispersed distribution (GtD-like pattern). Vice versa, the fusion of the GtA-derived N-terminal 11 aa to GtD causes a GtA-like phenotype. However, insertion of the corresponding GtE-derived 10 aa to GtD has no effect. Deletion of these 11aa decreases filament size while neither the number of released viral genomes nor virion size and infectivity are affected. A negative regulatory element (aa 2-8) and a dominant positive regulatory element (aa 9-11) affecting the amount of LHBs were identified. The fusion of this motif to eGFP revealed that the effect on protein amount and subcellular distribution is not restricted to LHBs. These data identify a novel region in the N-terminus of LHBs affecting the amount and subcellular distribution of LHBs and identify release-promoting and -inhibiting aa residues within this motive.

Reactive mesothelial hyperplasia mimicking mesothelioma in an African green monkey (Chlorocebus aethiops).

A spontaneous reactive mesothelial hyperplasia occurred in a female, 15.7-year-old African green monkey (grivet; Chlorocebus aethiops). At necropsy, massive effusions were found in the abdomen, the thorax, and the pericardium. Additionally, multiple small, beige-gray nodules were detected on the serosal surfaces of the abdominal organs. Histopathologically, the mesothelial cells resembled the epithelioid subtype of a mesothelioma, but no infiltrative or invasive growth could be demonstrated. The mesothelial cells on the thoracis, liver, and intestinal serosa were accompanied by chronic serositis. Mesothelial cells expressed cytokeratin, vimentin, calretinin, desmin, Wilms Tumor 1 (WT-1) protein, and epithelial membrane antigen (EMA). Cells were negative for carcinoembryonic antigen (CEA), cluster of differentiation 15 (CD15), and podoplanin. Ultrastructurally, cells revealed a moderate amount of microvilli of medium length, perinuclear tonofilament bundles, and long desmosomes. In fluorescence in situ hybridization (FISH) for the detection of characteristic gene loss (p16; CDKN2A), NF2, and MTAP, no deletions were detected. No asbestos fibers and no presence of Simian virus 40 antigen (SV40) could be demonstrated.

Hybrid integrated InP-Si3N4 diode laser with a 40-Hz intrinsic linewidth.

We demonstrate a hybrid integrated and widely tunable diode laser with an intrinsic linewidth as narrow as 40 Hz, achieved with a single roundtrip through a low-loss feedback circuit that extends the cavity length to 0.5 meter on a chip. Employing solely dielectrics for single-roundtrip, single-mode resolved feedback filtering enables linewidth narrowing with increasing laser power, without limitations through nonlinear loss. We achieve single-frequency oscillation with up to 23 mW fiber coupled output power, 70-nm wide spectral coverage in the 1.55 µm wavelength range with 3 mW output and obtain more than 60 dB side mode suppression. Such properties and options for further linewidth narrowing render the approach of high interest for direct integration in photonic circuits serving microwave photonics, coherent communications, sensing and metrology with highest resolution.

Spontaneous four-wave mixing in silicon nitride waveguides for broadband coherent anti-Stokes Raman scattering spectroscopy.

We present a light source for coherent anti-Stokes Raman scattering (CARS) based on broadband spontaneous four-wave mixing, with the potential to be further integrated. By using 7 mm long silicon nitride waveguides, which offer tight mode confinement and a high nonlinear refractive index coefficient, broadband signal and idler pulses were generated with 4 nJ of input pulse energy. In comparison to fiber-based experiments, the input energy and the waveguide length were reduced by two orders of magnitude, respectively. The idler and residual pump pulses were used for CARS measurements, enabling chemically selective and label-free spectroscopy over the entire fingerprint region, with an ultrafast fiber-based pump source at 1033 nm wavelength. The presented simple light source paves the path towards cost-effective, integrated lab-on-a-chip CARS applications.

HPV caught in the tetraspanin web?

Tetraspanins are master organizers of the cell membrane. Recent evidence suggests that tetraspanins themselves may become crowded by virus particles and that these crowds/aggregates co-internalize with the viral particles. Using microscopy, we studied human papillomavirus (HPV) type 16-dependent aggregates on the cell surface of tetraspanin overexpressing keratinocytes. We find that aggregates are (1) rich in at least two different tetraspanins, (2) three-dimensional architectures extending up to several micrometers into the cell, and (3) decorated intracellularly by filamentous actin. Moreover, in cells not overexpressing tetraspanins, we note that obscurin-like protein 1 (OBSL1), which is thought to be a cytoskeletal adaptor, associates with filamentous actin. We speculate that HPV contact with the cell membrane could trigger the formation of a large tetraspanin web. This web may couple the virus contact site to the intracellular endocytic actin machinery, possibly involving the cytoskeletal adaptor protein OBSL1. Functionally, such a tetraspanin web could serve as a virus entry platform, which is co-internalized with the virus particle.

Ring resonator enhanced mode-hop-free wavelength tuning of an integrated extended-cavity laser.

Extending the cavity length of diode lasers with feedback from Bragg structures and ring resonators is highly effective for obtaining ultra-narrow laser linewidths. However, cavity length extension also decreases the free-spectral range of the cavity. This reduces the wavelength range of continuous laser tuning that can be achieved with a given phase shift of an intracavity phase tuning element. We present a method that increases the range of continuous tuning to that of a short equivalent laser cavity, while maintaining the ultra-narrow linewidth of a long cavity. Using a single-frequency hybrid integrated InP-Si3N4 diode laser with 120 nm coverage around 1540 nm, with a maximum output of 24 mW and lowest intrinsic linewidth of 2.2 kHz, we demonstrate a six-fold increased continuous and mode-hop-free tuning range of 0.22 nm (28 GHz) as compared to the free-spectral range of the laser cavity.

Characterization of porcine endogenous retrovirus particles released by the CRISPR/Cas9 inactivated cell line PK15 clone 15.

The infection of human transplant recipients by porcine endogenous retrovirus (PERV) is a safety issue for xenotransplantation (XTx). CRISPR/Cas9 technology has enabled the generation of pigs free of functional PERVs, and the susceptibility of these animals to reinfection by PERVs remains unclear. To assess virological safety, we characterized a cell line in which PERVs have been inactivated by CRISPR/Cas9 (PK15 clone 15) for its susceptibility to infectious PERV. First, basal expression of PERV pol, the porcine PERV-A receptor (POPAR), and reverse transcriptase (RT) activity of PERV were determined. PK15 clone 15 cells were inoculated with PERV and monitored post infection for virus expression and RT activity. Particles were visualized by electron microscopy. Our data show that PK15 clone 15 cells still produce viral proteins that assemble to produce impaired viral particles. These virions have an irregular morphology that diverges from that of mature wild type. The particles are no longer infectious when tested in a downstream infection assay using supernatants of PK15 clone 15 cells to infect susceptible swine testis-IOWA (ST-IOWA) cells. The expression of POPAR was quantified to exclude the possibility that lack of susceptibility to reinfection, for PERV-A, is caused by absence of viral host receptor(s). PK15 and PK15 clone 15 cells do, in fact, express POPAR equally. PERV RT inactivation mediated by CRISPR/Cas9 does not compromise virus assembly but affects virion structure and proviral integration. The constitutive virion production seems to maintain cellular resistance to superinfection and possibly indicates a protective side effect of this specific CRISPR/Cas9 mediated RT inactivation.

8×8 reconfigurable quantum photonic processor based on silicon nitride waveguides.

The development of large-scale optical quantum information processing circuits ground on the stability and reconfigurability enabled by integrated photonics. We demonstrate a reconfigurable 8×8 integrated linear optical network based on silicon nitride waveguides for quantum information processing. Our processor implements a novel optical architecture enabling any arbitrary linear transformation and constitutes the largest programmable circuit reported so far on this platform. We validate a variety of photonic quantum information processing primitives, in the form of Hong-Ou-Mandel interference, bosonic coalescence/anti-coalescence and high-dimensional single-photon quantum gates. We achieve fidelities that clearly demonstrate the promising future for large-scale photonic quantum information processing using low-loss silicon nitride.

Formation of semi-enveloped particles as a unique feature of a hepatitis B virus PreS1 deletion mutant.

BACKGROUND: Naturally occurring variants with deletions or mutations in the C-terminal PreS1 domain from hepatitis B virus (HBV) chronically infected patients have been shown to promote HBsAg retention, inhibit HBsAg secretion and change the extracellular appearance of PreS1-containing HBV particles (filaments and virions). AIMS: To study the impact of N-terminal deletion in preS1 domain on viral secretion and morphogenesis. METHODS: An HBV mutant with 15 amino acids (aa 25-39) deletion in N-terminal preS1 was isolated. Intracellular and extracellular HBsAg were quantified by Western blot. Subcellular HBsAg distribution was analysed by confocal laser scanning microscopy. The viral morphology was characterised by sucrose density gradient ultracentrifugation, Western blot, electron microscopy, HBV mixed ELISA and HBV particle gel essay. RESULTS: Expression of this mutant genome released higher amounts of HBsAg in the form of shorter filaments. A significant fraction of semi-enveloped virions was observed in the supernatant that has been unprecedented so far. Stepwise insertion of aa 25-31, aa 32-39 and aa 25-39 increased the length of filaments. The rescue of aa 25-31 and aa 25-39 drastically reduced the amounts of extracellular HBsAg and semi-enveloped virions, while such effects could not be observed after insertion of aa 32-39, arguing against a simple spacer function of this region. The deletion and rescued mutants do not differ in subcellular HBsAg distribution and colocalisation with ER, Golgi and multivesicular bodies markers arguing against differences in release pathways. CONCLUSION: N-terminal PreS1-domain (aa 25-31) determines HBsAg secretion and triggers proper assembly of PreS1-containing particles.

Linewidth narrowing via low-loss dielectric waveguide feedback circuits in hybrid integrated frequency comb lasers.

We present an integrated hybrid semiconductor-dielectric (InP-Si3N4) waveguide laser that generates frequency combs at a wavelength around 1.5 µm with a record-low intrinsic optical linewidth of 34 kHz. This is achieved by extending the cavity photon lifetime using a low-loss dielectric waveguide circuit. In our experimental demonstration, the on-chip, effective optical path length of the laser cavity is extended to 6 cm. The resulting linewidth narrowing shows the high potential of on-chip, highly coherent frequency combs with direct electrical pumping, based on hybrid and heterogeneous integrated circuits making use of low-loss dielectric waveguides.

Inactivation and Removal of Chikungunya Virus and Mayaro Virus from Plasma-derived Medicinal Products.

BACKGROUND: Chikungunya virus (CHIKV) and Mayaro virus (MAYV) are closely related members of the Semliki Forest complex within the genus alphavirus and are transmitted by arthropods, causing acute febrile illness in humans. CHIKV has spread to almost all continents, whereas autochthonous MAYV infections have been reported in South America and in the Caribbean. Nevertheless, there was concern about potential spread of MAYV to other regions similar to CHIKV in the past. The risk for transmission of emerging viruses by blood transfusion and the safety of plasma-derived medicinal products (PDMPs) are constant concerns. The manufacturing processes of PDMPs include procedures to inactivate/remove viruses. METHODS: In this study, we investigated the reduction of MAYV and CHIKV by heat inactivation in various matrices, solvent/detergent treatment and nanofiltration. RESULTS: Unexpectedly, MAYV was significantly more resistant to heat and solvent/detergent treatment compared to CHIKV. However, being similar in size, both MAYV and CHIKV were removed below the detection limit by 35 nm virus filters. CONCLUSIONS: The inactivation profiles of different alphavirus members vary considerably, even within the Semliki Forest Complex. However, robust dedicated viral inactivation/removal procedures commonly used in the plasma product industry are effective in inactivating or removing MAYV and CHIKV.

Surface acoustic waves for acousto-optic modulation in buried silicon nitride waveguides.

We theoretically investigate the use of Rayleigh surface acoustic waves (SAWs) for refractive index modulation in optical waveguides consisting of amorphous dielectrics. Considering low-loss Si3N4 waveguides with a standard core cross-section of 4.4×0.03 µm2 size, buried 8-µm deep in a SiO2 cladding, we compare surface acoustic wave generation in various different geometries via a piezo-active, lead zirconate titanate film placed on top of the surface and driven via an interdigitized transducer (IDT). Using numerical solutions of the acoustic and optical wave equations, we determine the strain distribution of the SAW under resonant excitation. From the overlap of the acoustic strain field with the optical mode field, we calculate and maximize the attainable amplitude of index modulation in the waveguide. For the example of a near-infrared wavelength of 840 nm, a maximum shift in relative effective refractive index of 0.7x10-3 was obtained for TE polarized light, using an IDT period of 30-35 µm, a film thickness of 2.5-3.5 µm, and an IDT voltage of 10 V. For these parameters, the resonant frequency is in the range of 70-85 MHz. The maximum shift increases to 1.2x10-3, with a corresponding resonant frequency of 87 MHz, when the height of the cladding above the core is reduced to 3 µm. The relative index change is about 300 times higher than in previous work based on non-resonant proximity piezo-actuation, and the modulation frequency is about 200 times higher. Exploiting the maximum relative index change of 1.2×10-3 in a low-loss, balanced Mach-Zehnder modulator should allow full-contrast modulation in devices as short as 120 µm (half-wave voltage length product = 0.24 Vcm).

Density matrix study of ground state depletion towards sub-diffraction-limited spontaneous Raman scattering spectroscopy.

The suppression of Raman scattering is of high interest for the achievement of sub-diffraction-limited resolution in Raman scattering spectroscopy and microscopy. We present density matrix calculations of the suppression of spontaneous Raman scattering via ground state depletion in a level system based on the molecule tris(bipyridine)ruthenium(ii). This particular molecule has been earlier used for an experimental demonstration of the suppression of spontaneous Raman scattering, allowing us to successfully verify the validity of our numerical calculations by a comparison to the experimental results. We investigate the required level of detail of the molecule model as well as the influence of certain molecule and pulse parameters on the Raman scattering suppression. It was found that pulses with a duration longer than the lifetime of the electronic states allow for a high suppression of the Raman scattering. Pulses shorter than the coherence lifetime between the ground state and electronic states lead to a similarly high suppression but also accomplish the suppression with more than one order of magnitude lower pulse energy fluence. Additionally, using a laser wavelength that is in resonance with one of the electronic transitions of the sample should allow suppressing the Raman scattering with four to six orders of magnitude lower pulse energy fluence.

Low-power broadband all-optical switching via intermodal cross-phase modulation in integrated optical waveguides.

We demonstrate the potential of all-optical switches in integrated waveguides based on intermodal cross-phase modulation between transverse modes. For this purpose, the differential phase between two transverse modes of a probe beam was altered by cross-phase modulation with a control beam propagating only in the fundamental mode. A switching behavior was accomplished by spatially filtering the resulting multimode interference of the probe modes, which changed depending on the control beam power. All-optical switching with a contrast of 82% at 1280 nm over a frequency range of 4.4 THz at 1.6 nJ was achieved, representing an improvement of the product of necessary power and waveguide length by a factor of nearly 2000 compared to similar experiments in graded-index fibers. Additionally, we show that the center wavelength of the switch can be tailored by changing the cross-sectional geometry of the waveguide or the involved probe modes.

siRNA-mediated inhibition of skNAC and Smyd1 expression disrupts myofibril organization: Immunofluorescence and electron microscopy study in C2C12 cells.

skNAC (skeletal and heart muscle-specific variant of nascent polypeptide-associated complex) and Smyd1 (SET and MYND domain-containing 1) form a protein dimer which is specific for striated muscle cells. Its function is largely unknown. On the one hand, skNAC-Smyd1 appears to control transcriptional processes in the nucleus, on the other hand, specifically at later stages of myogenic differentiation, both proteins translocate to the sarcoplasm and at least Smyd1 specifically associates with sarcomeric structures and might control myofibrillogenesis and/or sarcomere architecture. Here, using immunofluorescence and electron microscopy, we analyzed sarcomere formation and myofibril organization after siRNA-mediated knockdown of skNAC or Smyd1 expression in murine C2C12 skeletal muscle cells. We found that inhibition of skNAC or Smyd1 expression indeed prevents myofibrillogenesis and sarcomere formation, leading to a disorganized array of myofilaments predominantly within the region immediately beneath the plasma membrane.