LBR and lamin A/C sequentially tether peripheral heterochromatin and inversely regulate differentiation.

Eukaryotic cells have a layer of heterochromatin at the nuclear periphery. To investigate mechanisms regulating chromatin distribution, we analyzed heterochromatin organization in different tissues and species, including mice with mutations in the lamin B receptor (Lbr) and lamin A (Lmna) genes that encode nuclear envelope (NE) proteins. We identified LBR- and lamin-A/C-dependent mechanisms tethering heterochromatin to the NE. The two tethers are sequentially used during cellular differentiation and development: first the LBR- and then the lamin-A/C-dependent tether. The absence of both LBR and lamin A/C leads to loss of peripheral heterochromatin and an inverted architecture with heterochromatin localizing to the nuclear interior. Myoblast transcriptome analyses indicated that selective disruption of the LBR- or lamin-A-dependent heterochromatin tethers have opposite effects on muscle gene expression, either increasing or decreasing, respectively. These results show how changes in NE composition contribute to regulating heterochromatin positioning, gene expression, and cellular differentiation during development.

Tailored second harmonic generation in Ti-diffused PPLN waveguides using micro-heaters.

Frequency conversion based on χ(2) nonlinear optical interactions can be made very efficient in waveguide structures. Fabrication imperfections remain very often a limiting factor. They can induce strong distortions in the spectral shape and lower the efficiency. To overcome these imperfections a post-trimming method based on a cascade of ten micro-heaters on top of the waveguide along the interaction length is demonstrated. As an example, the second harmonic generation in a Ti-indiffused waveguide in periodically poled LiNbO3 is investigated. A tailoring of the spectral characteristics of the phase-matching curves could be successfully demonstrated.

Expression and regulation of Siglec-6 (CD327) on human mast cells and basophils.

BACKGROUND: Mast cells (MC) and basophils are effector cells of allergic reactions and display a number of activation-linked cell surface antigens. Of these antigens, however, only a few are functionally relevant and specifically expressed in these cells. OBJECTIVE: We sought to identify MC- and basophil-specific surface molecules and to study their cellular distribution and regulation during cytokine-induced and IgE-dependent activation. METHODS: Multicolor flow cytometry was performed to recognize surface antigens and to determine changes in antigen expression upon activation. RESULTS: We identified Siglec-6 (CD327) as a differentially regulated surface antigen on human MC and basophils. In the bone marrow, Siglec-6 was expressed abundantly on MC in patients with mastocytosis and in reactive states, but it was not detected on other myeloid cells, with the exception of basophils and monocytes. In healthy individuals, allergic patients, and patients with chronic myeloid leukemia (CML), Siglec-6 was identified on CD203c+ blood basophils, a subset of CD19+ B lymphocytes, and few CD14+ monocytes, but not on other blood leukocytes. CML basophils expressed higher levels of Siglec-6 than normal basophils. IL-3 promoted Siglec-6 expression on normal and CML basophils, and stem cell factor increased the expression of Siglec-6 on tissue MC. Unexpectedly, IgE-dependent activation resulted in downregulation of Siglec-6 in IL-3-primed basophils, whereas in MC, IgE-dependent activation augmented stem cell factor-induced upregulation of Siglec-6. CONCLUSIONS: Siglec-6 is a dynamically regulated marker of MC and basophils. Activated MC and basophils exhibit unique Siglec-6 responses, including cytokine-dependent upregulation and unique, cell-specific, responses to IgE-receptor cross-linking.

Fiber-coupled plug-and-play heralded single photon source based on Ti:LiNbO3 and polymer technology.

A reliable, but cost-effective generation of single-photon states is key for practical quantum communication systems. For real-world deployment, waveguide sources offer optimum compatibility with fiber networks and can be embedded in hybrid integrated modules. Here, we present what we believe to be the first chip-size fully integrated fiber-coupled heralded single photon source (HSPS) module based on a hybrid integration of a nonlinear lithium niobate waveguide into a polymer board. Photon pairs at 810 nm (signal) and 1550 nm (idler) are generated via parametric down-conversion pumped at 532 nm in the LiNbO3 waveguide. The pairs are split in the polymer board and routed to separate output ports. The module has a size of (2 × 1) cm2 and is fully fiber-coupled with one pump input fiber and two output fibers. We measure a heralded second-order correlation function of g h(2)=0.05 with a heralding efficiency of η h=3.5% at low pump powers.

Demonstration of Hong-Ou-Mandel interference in an LNOI directional coupler.

Interference between single photons is key for many quantum optics experiments and applications in quantum technologies, such as quantum communication or computation. It is advantageous to operate the systems at telecommunication wavelengths and to integrate the setups for these applications in order to improve stability, compactness and scalability. A new promising material platform for integrated quantum optics is lithium niobate on insulator (LNOI). Here, we realise Hong-Ou-Mandel (HOM) interference between telecom photons from an engineered parametric down-conversion source in an LNOI directional coupler. The coupler has been designed and fabricated in house and provides close to perfect balanced beam splitting. We obtain a raw HOM visibility of (93.5 ± 0.7) %, limited mainly by the source performance and in good agreement with off-chip measurements. This lays the foundation for more sophisticated quantum experiments in LNOI.

Clinical outcome in metastatic prostate cancer after primary radiotherapy.

PURPOSE: To describe a local radio-oncological treatment for patients with prostate cancer that metastasized to either the lymph nodes or distant regions. METHODS AND MATERIALS: We included 133 patients with prostate cancer that displayed either distant metastases (DM) or lymph node metastases alone (NM) and were treated between 2004 and 2019. All patients underwent computed tomography and a bone scan or 18F- or prostate-specific membrane antigen-targeted positron emission tomography. Patients received local external beam radiation therapy to the prostate to achieve local control (60-81.4 Gy to the prostate, and 45-50.4 Gy to pelvic lymph nodes), with either the 3D conformal (4-field box) or volumetric modulated arc therapy technique. A urologist prescribed additional therapy. RESULTS: We included 51 patients with DM and 82 patients with NM. The mean follow-up was 42 months for all patients. The groups were similar in T stage, initial prostate-specific antigen, histology, androgen deprivation therapy, age, treatment techniques, and prescribed doses, but different in lymph node inclusion and follow-up times. In the NM and DM groups, the 5­year biochemical recurrence-free rates were 52% and 24%, respectively (p < 0.0001); the 5­year disease-specific survival rates were 92% and 61%, respectively (p = 0.001); and the 5­year OS rates were 77% and 48%, respectively (p = 0.01). The groups had similar acute and late gastrointestinal and genitourinary side effects, except that late genitourinary side effects occurred significantly more frequently in the NM group (p = 0.01). CONCLUSIONS: DM was associated with significantly worse outcomes than NM. The long-term survival of patients with metastatic prostate cancer was low.

Fully guided and phase locked Ti:PPLN waveguide squeezing for applications in quantum sensing.

This work reports a fully guided setup for single-mode squeezing on integrated titanium-indiffused periodically poled nonlinear resonators. A continuous-wave laser beam is delivered and the squeezed field is collected by single-mode fibers; up to -3.17(9) dB of useful squeezing is available in fibers. To showcase the usefulness of such a fiber-coupled device, we applied the generated squeezed light in a fiber-based phase sensing experiment, showing a quantum enhancement in the signal-to-noise ratio of 0.35 dB. Moreover, our investigation of the effect of photorefraction on the cavity resonance condition suggests that it causes system instabilities at high powers.

Phenotypic characterization of disease-initiating stem cells in JAK2- or CALR-mutated myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPN) are characterized by uncontrolled expansion of myeloid cells, disease-related mutations in certain driver-genes including JAK2, CALR, and MPL, and a substantial risk to progress to secondary acute myeloid leukemia (sAML). Although behaving as stem cell neoplasms, little is known about disease-initiating stem cells in MPN. We established the phenotype of putative CD34+ /CD38- stem cells and CD34+ /CD38+ progenitor cells in MPN. A total of 111 patients with MPN suffering from polycythemia vera, essential thrombocythemia, or primary myelofibrosis (PMF) were examined. In almost all patients tested, CD34+ /CD38- stem cells expressed CD33, CD44, CD47, CD52, CD97, CD99, CD105, CD117, CD123, CD133, CD184, CD243, and CD274 (PD-L1). In patients with PMF, MPN stem cells often expressed CD25 and sometimes also CD26 in an aberrant manner. MPN stem cells did not exhibit substantial amounts of CD90, CD273 (PD-L2), CD279 (PD-1), CD366 (TIM-3), CD371 (CLL-1), or IL-1RAP. The phenotype of CD34+ /CD38- stem cells did not change profoundly during progression to sAML. The disease-initiating capacity of putative MPN stem cells was confirmed in NSGS mice. Whereas CD34+ /CD38- MPN cells engrafted in NSGS mice, no substantial engraftment was produced by CD34+ /CD38+ or CD34- cells. The JAK2-targeting drug fedratinib and the BRD4 degrader dBET6 induced apoptosis and suppressed proliferation in MPN stem cells. Together, MPN stem cells display a unique phenotype, including cytokine receptors, immune checkpoint molecules, and other clinically relevant target antigens. Phenotypic characterization of neoplastic stem cells in MPN and sAML should facilitate their enrichment and the development of stem cell-eradicating (curative) therapies.

A survey on oral health-related standard of care for head and neck cancer patients in the EU.

OBJECTIVE: To map oral health-related standard of care in the context of head and neck cancer (HNC) treatment across the European Union (EU). MATERIALS AND METHODS: Six hundred and ninety centers across the European Union were contacted. The questionnaire contained questions focusing on the team/department structure, HNC treatment planning routines, and assessment and handling of dental treatment needs prior to cancer treatment. RESULTS: Eighty-seven centers across the EU responded. Department structure and number of HNC patients treated per year varied widely and dental professionals are included as part of the team in about 25% of the centers. Standard of care, in terms of dental assessment and preventive dentistry routines, such as recording an orthopantomogram, offering dental treatment, and providing a radiation protection splint and splint for fluoride application, differed significantly among the European regions. Independent of the region, these aspects are positively affected if dental professionals are part of the interdisciplinary treatment team and if dental treatment is offered within the center. CONCLUSION: Dental professionals are still only to a very limited extent included in interdisciplinary treatment planning teams of HNC patients. However, their inclusion and/or offering dental treatment within the same hospital/center appears to improve oral health-related standard of care. CLINICAL RELEVANCE: Inclusion of dental professionals in treatment planning teams of HNC patients appears to improve oral health-related standard of care within HNC treatment.

A general mathematical model for the in vitro assembly dynamics of intermediate filament proteins.

Intermediate filament (IF) proteins assemble into highly flexible filaments that organize into complex cytoplasmic networks: keratins in all types of epithelia, vimentin in endothelia, and desmin in muscle. Since IF elongation proceeds via end-to-end annealing of unit-length filaments and successively of progressively growing filaments, it is important to know how their remarkable flexibility, i.e., their persistence length lp, influences the assembly kinetics. In fact, their lp ranges between 0.3 µm (keratin K8/K18) and 1.0 µm (vimentin and desmin), and thus is orders of magnitude lower than that of microtubules and F-actin. Here, we present a unique mathematical model, which implements the semiflexible nature of the three IF types based on published semiflexible polymers theories and depends on a single free parameter k0. Calibrating this model to filament mean length dynamics of the three proteins, we demonstrate that the persistence length is indeed essential to accurately describe their assembly kinetics. Furthermore, we reveal that the difference in flexibility alone does not explain the significantly faster assembly rate of keratin filaments compared with that of vimentin. Likewise, desmin assembles approximately six times faster than vimentin, even though both their filaments exhibit the same lp value. These data strongly indicate that differences in their individual amino acid sequences significantly impact the assembly rates. Nevertheless, using a single k0 value for each of these three key representatives of the IF protein family, our advanced model does accurately describe the length distribution and mean length dynamics and provides effective filament assembly rates. It thus provides a tool for future investigations on the impact of posttranslational modifications or amino acid changes of IF proteins on assembly kinetics. This is an important issue, as the discovery of mutations in IF genes causing severe human disease, particularly for desmin and keratins, is steadily increasing.

Dual-wavelength stopped-flow analysis of the lateral and longitudinal assembly kinetics of vimentin.

Vimentin is a highly charged intermediate filament protein that inherently forms extended dimeric coiled coils, which serve as the basic building blocks of intermediate filaments. Under low ionic strength conditions, vimentin filaments dissociate into uniform tetrameric complexes of two anti-parallel-oriented, half-staggered coiled-coil dimers. By addition of salt, vimentin tetramers spontaneously reassemble into filaments in a time-dependent process: 1) lateral assembly of tetramers into unit-length filaments, 2) longitudinal annealing of unit-length filaments, and 3) longitudinal assembly of filaments coupled with subsequent radial compaction. To independently determine the lateral and longitudinal assembly kinetics, we measure with a stopped-flow instrument the static light scattering signal at two different wavelengths (405 and 594 nm) with a temporal resolution of 3 ms and analyze the signals based on Rayleigh-Gans theory. This theory considers that the intensity of the scattered light depends not only on the molecular weight of the scattering object but also on its shape. This shape dependence is more pronounced at shorter wavelengths, allowing us to decompose the scattered light signal into its components arising from lateral and longitudinal filament assembly. We demonstrate that both the lateral and longitudinal filament assembly kinetics increase with salt concentration.

The desmin mutation R349P increases contractility and fragility of stem cell-generated muscle micro-tissues.

AIMS: Desminopathies comprise hereditary myopathies and cardiomyopathies caused by mutations in the intermediate filament protein desmin that lead to severe and often lethal degeneration of striated muscle tissue. Animal and single cell studies hinted that this degeneration process is associated with massive ultrastructural defects correlating with increased susceptibility of the muscle to acute mechanical stress. The underlying mechanism of mechanical susceptibility, and how muscle degeneration develops over time, however, has remained elusive. METHODS: Here, we investigated the effect of a desmin mutation on the formation, differentiation, and contractile function of in vitro-engineered three-dimensional micro-tissues grown from muscle stem cells (satellite cells) isolated from heterozygous R349P desmin knock-in mice. RESULTS: Micro-tissues grown from desmin-mutated cells exhibited spontaneous unsynchronised contractions, higher contractile forces in response to electrical stimulation, and faster force recovery compared with tissues grown from wild-type cells. Within 1 week of culture, the majority of R349P desmin-mutated tissues disintegrated, whereas wild-type tissues remained intact over at least three weeks. Moreover, under tetanic stimulation lasting less than 5 s, desmin-mutated tissues partially or completely ruptured, whereas wild-type tissues did not display signs of damage. CONCLUSIONS: Our results demonstrate that the progressive degeneration of desmin-mutated micro-tissues is closely linked to extracellular matrix fibre breakage associated with increased contractile forces and unevenly distributed tensile stress. This suggests that the age-related degeneration of skeletal and cardiac muscle in patients suffering from desminopathies may be similarly exacerbated by mechanical damage from high-intensity muscle contractions. We conclude that micro-tissues may provide a valuable tool for studying the organization of myocytes and the pathogenic mechanisms of myopathies.

Redistribution, homing and organ-invasion of neoplastic stem cells in myeloid neoplasms.

The development of a myeloid neoplasm is a step-wise process that originates from leukemic stem cells (LSC) and includes pre-leukemic stages, overt leukemia and a drug-resistant terminal phase. Organ-invasion may occur in any stage, but is usually associated with advanced disease and a poor prognosis. Sometimes, extra-medullary organ invasion shows a metastasis-like or even sarcoma-like destructive growth of neoplastic cells in local tissue sites. Examples are myeloid sarcoma, mast cell sarcoma and localized blast phase of chronic myeloid leukemia. So far, little is known about mechanisms underlying re-distribution and extramedullary dissemination of LSC in myeloid neoplasms. In this article, we discuss mechanisms through which LSC can mobilize out of the bone marrow niche, can transmigrate from the blood stream into extramedullary organs, can invade local tissue sites and can potentially create or support the formation of local stem cell niches. In addition, we discuss strategies to interfere with LSC expansion and organ invasion by targeted drug therapies.

Dual Functional States of R406W-Desmin Assembly Complexes Cause Cardiomyopathy With Severe Intercalated Disc Derangement in Humans and in Knock-In Mice.

BACKGROUND: Mutations in the human desmin gene cause myopathies and cardiomyopathies. This study aimed to elucidate molecular mechanisms initiated by the heterozygous R406W-desmin mutation in the development of a severe and early-onset cardiac phenotype. METHODS: We report an adolescent patient who underwent cardiac transplantation as a result of restrictive cardiomyopathy caused by a heterozygous R406W-desmin mutation. Sections of the explanted heart were analyzed with antibodies specific to 406W-desmin and to intercalated disc proteins. Effects of the R406W mutation on the molecular properties of desmin were addressed by cell transfection and in vitro assembly experiments. To prove the genuine deleterious effect of the mutation on heart tissue, we further generated and analyzed R405W-desmin knock-in mice harboring the orthologous form of the human R406W-desmin. RESULTS: Microscopic analysis of the explanted heart revealed desmin aggregates and the absence of desmin filaments at intercalated discs. Structural changes within intercalated discs were revealed by the abnormal organization of desmoplakin, plectin, N-cadherin, and connexin-43. Next-generation sequencing confirmed the DES variant c.1216C>T (p.R406W) as the sole disease-causing mutation. Cell transfection studies disclosed a dual behavior of R406W-desmin with both its integration into the endogenous intermediate filament system and segregation into protein aggregates. In vitro, R406W-desmin formed unusually thick filaments that organized into complex filament aggregates and fibrillar sheets. In contrast, assembly of equimolar mixtures of mutant and wild-type desmin generated chimeric filaments of seemingly normal morphology but with occasional prominent irregularities. Heterozygous and homozygous R405W-desmin knock-in mice develop both a myopathy and a cardiomyopathy. In particular, the main histopathologic results from the patient are recapitulated in the hearts from R405W-desmin knock-in mice of both genotypes. Moreover, whereas heterozygous knock-in mice have a normal life span, homozygous animals die at 3 months of age because of a smooth muscle-related gastrointestinal phenotype. CONCLUSIONS: We demonstrate that R406W-desmin provokes its severe cardiotoxic potential by a novel pathomechanism, where the concurrent dual functional states of mutant desmin assembly complexes underlie the uncoupling of desmin filaments from intercalated discs and their structural disorganization.

Effects of Vimentin Intermediate Filaments on the Structure and Dynamics of In Vitro Multicomponent Interpenetrating Cytoskeletal Networks.

We investigate the rheological properties of interpenetrating networks reconstituted from the main cytoskeletal components: filamentous actin, microtubules, and vimentin intermediate filaments. The elastic modulus is determined largely by actin, with little contribution from either microtubules or vimentin. However, vimentin dramatically impacts the relaxation, with even small amounts significantly increasing the relaxation time of the interpenetrating network. This highly unusual decoupling between dissipation and elasticity may reflect weak attractive interactions between vimentin and actin networks.

Ion type and valency differentially drive vimentin tetramers into intermediate filaments or higher order assemblies.

Vimentin intermediate filaments, together with actin filaments and microtubules, constitute the cytoskeleton in cells of mesenchymal origin. The mechanical properties of the filaments themselves are encoded in their molecular architecture and depend on their ionic environment. It is thus of great interest to disentangle the influence of both the ion type and their concentration on vimentin assembly. We combine small angle X-ray scattering and fluorescence microscopy and show that vimentin in the presence of the monovalent ions, K+ and Na+, assembles into "standard filaments" with a radius of about 6 nm and 32 monomers per cross-section. In contrast, di- and multivalent ions, independent of type and valency, lead to the formation of thicker filaments associating over time into higher order structures. Hence, our results may indeed be of relevance for living cells, as local ion concentrations in the cytoplasm during certain physiological activities may differ considerably from average intracellular concentrations.

Keratins determine network stress responsiveness in reconstituted actin-keratin filament systems.

The cytoskeleton is a major determinant of cell mechanics, and alterations in the central mechanical aspects of cells are observed during many pathological situations. Therefore, it is essential to investigate the interplay between the main filament systems of the cytoskeleton in the form of composite networks. Here, we investigate the role of keratin intermediate filaments (IFs) in network strength by studying in vitro reconstituted actin and keratin 8/18 composite filament networks via bulk shear rheology. We co-polymerized these structural proteins in varying ratios and recorded how their relative content affects the overall mechanical response of the various composites. For relatively small deformations, we found that all composites exhibited an intermediate linear viscoelastic behaviour compared to that of the pure networks. In stark contrast, when larger deformations were imposed the composites displayed increasing strain stiffening behaviour with increasing keratin content. The extent of strain stiffening is much more pronounced than in corresponding experiments performed with vimentin IF as a composite network partner for actin. Our results provide new insights into the mechanical interplay between actin and keratin filaments in which keratin provides reinforcement to actin. This interplay may contribute to the overall integrity of cells. Hence, the high keratin 8/18 content of mechanically stressed simple epithelial cell layers, as found in the lung and the intestine, provides an explanation for their exceptional stability.

Effect of Divalent Cations on the Structure and Mechanics of Vimentin Intermediate Filaments.

Divalent cations behave as effective cross-linkers of intermediate filaments (IFs) such as vimentin IF (VIF). These interactions have been mostly attributed to their multivalency. However, ion-protein interactions often depend on the ion species, and these effects have not been widely studied in IFs. Here, we investigate the effects of two biologically important divalent cations, Zn2+ and Ca2+, on VIF network structure and mechanics in vitro. We find that the network structure is unperturbed at micromolar Zn2+ concentrations, but strong bundle formation is observed at a concentration of 100 µM. Microrheological measurements show that network stiffness increases with cation concentration. However, bundling of filaments softens the network. This trend also holds for VIF networks formed in the presence of Ca2+, but remarkably, a concentration of Ca2+ that is two orders higher is needed to achieve the same effect as with Zn2+, which suggests the importance of salt-protein interactions as described by the Hofmeister effect. Furthermore, we find evidence of competitive binding between the two divalent ion species. Hence, specific interactions between VIFs and divalent cations are likely to be an important mechanism by which cells can control their cytoplasmic mechanics.

Spatially single mode photon pair source at 800 nm in periodically poled Rubidium exchanged KTP waveguides.

Photon pair sources in the visible to NIR wavelength region play a key role in quantum optics. The wavelength range around 800 nm provides an opportunity for using low cost detectors, which makes it highly interesting for practical, large scale quantum applications. Here, we report on the realization of single mode Rubidium (Rb) exchanged waveguides in periodically poled (PP) Potassium Titanyl Phosphate (Rb:KTiOPO4 or Rb:KTP) for frequency-non-degenerate type II parametric down-conversion pumped at 400 nm and generating pairs of photons at around 800 nm. The source exhibits a nonlinear conversion efficiency of 2.0%/(Wcm2), estimated from SHG measurements. Characterisation of the generated two-photon state confirms nonclassical photon-number correlations, characterized by g(1,1). The high nonlinear conversion efficiency and low temperature sensitivity make this source a promising candidate for operations in both classical and quantum integrated network applications.

Cryogenic electro-optic polarisation conversion in titanium in-diffused lithium niobate waveguides.

Many technologies in quantum photonics require cryogenic conditions to operate. However, the underlying platform behind active components such as switches, modulators and phase shifters must be compatible with these operating conditions. To address this, we demonstrate an electro-optic polarisation converter for 1550 nm light at 0.8 K in titanium in-diffused lithium niobate waveguides. To do so, we exploit the electro-optic properties of lithium niobate to convert between orthogonal polarisation modes with a fiber-to-fiber transmission >43%. We achieve a modulation depth of 23.6±3.3 dB and a conversion voltage-length product of 28.8 V cm. This enables the combination of cryogenic photonics and active components on a single integration platform.