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Background The aim of the study was to evaluate the development of treatment of primary high-risk prostate cancer in regards to biochemical no evidence of disease (bNED), acute and late gastrointestinal (GI) and genitourinary (GU) side effects. Patients and methods Primary high-risk prostate cancer patients treated between 1994 and 2016 were included. Applied doses ranged from 60 to 80 Gy, with a dose of 1.8 or 2 Gy per fraction. Techniques were either 3D conformal or intensity modulated radiotherapy and volumetric intensity modulated arc therapy. Results 142 patients were treated with doses up to 70 Gy (median dose 66 Gy; 66 Gy group), 282 with doses between 70 and 76 Gy (median dose 74 Gy; 74 Gy group), and 141 with doses >76 Gy (median dose 78 Gy; 78 Gy group). The median follow-up was 48 months. The bNED rates were 50% after 5 years and 44% after 9 years in the 66 Gy group; 65% and 54%, respectively, in the 74 Gy group; and 83% and 66%, respectively, in the 78 Gy group (p = 0.03 vs. 74 Gy and p < 0.0001 vs. 66 Gy). We found a higher rate of acute GI side effects in the 78 Gy group compared to the other groups, but not in maximum acute GU side effects and late maximum GI and GU effects. Conclusions High-risk prostate cancer patients treated with doses of 78 Gy had significantly better bNED rates. Compared to the historical 66 Gy group, 50% more patients achieved bNED after a follow-up of 9 years.

PDF file - 85K, Supplementary Table S1: Characteristics of patients with MDS Supplementary Table S2: Characteristics of patients with AML Supplementary Table S3: Characteristics of patients with CML, CMML, MPS, AUL, and ALL Supplementary Table S4: Characteristics of patients with ICUS, normal/reactive bone marrow and long-term complete remission.

PDF file - 103K, Supplementary Figure S3: Expression of CD52 on CD34+/CD38− cells in different WHO subtypes of MDS and AML.

PDF file - 83K, Supplementary Table S6: Expression of CD52, CD123 and CLL-1 on CD34+/CD38−/CD90+/CD45RA− cells in patients with MDS and on CD34+/CD38−/CD90−/CD45RA− cells in patients with AML.

Purpose: The CD52-targeted antibody alemtuzumab induces major clinical responses in a group of patients with myelodysplastic syndromes (MDS). The mechanism underlying this drug effect remains unknown.Experimental Design: We asked whether neoplastic stem cells (NSC) in patients with MDS (n = 29) or acute myelogenous leukemia (AML; n = 62) express CD52.Results: As assessed by flow cytometry, CD52 was found to be expressed on NSC-enriched CD34+/CD38− cells in 8/11 patients with MDS and isolated del(5q). In most other patients with MDS, CD52 was weakly expressed or not detectable on NSC. In AML, CD34+/CD38− cells displayed CD52 in 23/62 patients, including four with complex karyotype and del(5q) and one with del(5q) and t(1;17;X). In quantitative PCR (qPCR) analyses, purified NSC obtained from del(5q) patients expressed CD52 mRNA. We were also able to show that CD52 mRNA levels correlate with EVI1 expression and that NRAS induces the expression of CD52 in AML cells. The CD52-targeting drug alemtuzumab, was found to induce complement-dependent lysis of CD34+/CD38−/CD52+ NSC, but did not induce lysis in CD52− NSC. Alemtuzumab also suppressed engraftment of CD52+ NSC in NSG mice. Finally, CD52 expression on NSC was found to correlate with a poor survival in patients with MDS and AML.Conclusions: The cell surface target Campath-1 (CD52) is expressed on NSC in a group of patients with MDS and AML. CD52 is a novel prognostic NSC marker and a potential NSC target in a subset of patients with MDS and AML, which may have clinical implications and may explain clinical effects produced by alemtuzumab in these patients. Clin Cancer Res; 20(13); 3589–602. ©2014 AACR.

PDF file - 143K, Supplementary Figure S2A: Effects of alemtuzumab on growth of HL60 cells transfected with empty vector or NRAS Q61K Supplementary Figure S2B: Effects of alemtuzumab in combination with IgG on growth of MDS and AML stem cells Supplementary Figure S2C: Correlation between CD52 surface expression and the cytotoxic effect of alemtuzumab.

PDF file - 162K, Supplementary Figure S1A: Expression of CD52 on CD34+/CD38+ cells in MDS and AML Supplementary Figure S1B: Expression of CD52 on CD34+/CD38+ cells in MDS, AML and control samples.

PDF file - 61K, Supplementary Table S9: Detection of del(5q) in sorted cells of MDS patients by FISH.

Purpose: In chronic myelogenous leukemia (CML), leukemic stem cells (LSC) represent a critical target of therapy. However, little is known about markers and targets expressed by LSCs. The aim of this project was to identify novel relevant markers of CML LSCs.Experimental Design: CML LSCs were examined by flow cytometry, qPCR, and various bioassays. In addition, we examined the multipotent CD25+ CML cell line KU812.Results: In contrast to normal hematopoietic stem cells, CD34+/CD38− CML LSCs expressed the IL-2 receptor alpha chain, IL-2RA (CD25). STAT5 was found to induce expression of CD25 in Lin−/Sca-1+/Kit+ stem cells in C57Bl/6 mice. Correspondingly, shRNA-induced STAT5 depletion resulted in decreased CD25 expression in KU812 cells. Moreover, the BCR/ABL1 inhibitors nilotinib and ponatinib were found to decrease STAT5 activity and CD25 expression in KU812 cells and primary CML LSCs. A CD25-targeting shRNA was found to augment proliferation of KU812 cells in vitro and their engraftment in vivo in NOD/SCID-IL-2Rγ−/− mice. In drug-screening experiments, the PI3K/mTOR blocker BEZ235 promoted the expression of STAT5 and CD25 in CML cells. Finally, we found that BEZ235 produces synergistic antineoplastic effects on CML cells when applied in combination with nilotinib or ponatinib.Conclusions: CD25 is a novel STAT5-dependent marker of CML LSCs and may be useful for LSC detection and LSC isolation in clinical practice and basic science. Moreover, CD25 serves as a growth regulator of CML LSCs, which may have biologic and clinical implications and may pave the way for the development of new more effective LSC-eradicating treatment strategies in CML. Clin Cancer Res; 22(8); 2051–61. ©2015 AACR.

Tables: S1 Specification of monoclonal antibodies (mAb) used in this study; S2 Patients´ characteristics - chronic myeloid leukemia (CML); S3 Patients´ characteristics - ormal/reactive BM; S4 Detection of BCR/ABL1 in sorted CML cells by FISH; S5 Major genes up-regulated by gene array analysis on KU812 cells transduced with a CD25 shRNA (KU812 CD25 shRNA, clone #2) compared to KU812 cells transduced with a random control shRNA (KU812 RDM shRNA); Figures: S1 CML LSCs co-express CD25 together with other stem cell markers; S2 Expression of BCR/ABL1 in CD34+/CD38−/CD25+ stem cells obtained from a patient with CML as determined by fluorescence in situ hybridization (FISH); S3 Clonogenic growth of CML LSCs and normal stem cells obtained from two patients with Ph+ CML; S4 Western blotting confirmed the knock-down of STAT5 in KU812 cells; S5 Drug-induced upregulation of CD25 in CML cell lines; S6 Effects of imatinib on CD25 surface expression in Ba/F3 cells expressing diverse BCR/ABL1 mutants; S7 Effects of interleukin-2 (IL-2) on growth of untreated and BEZ235-treated CML cells; S8 Effect of transduced CD25 on growth of CML cells lines; S9 Pathway analysis of KU812 cells transduced with a CD25 shRNA; S10 Effects of shRNA-induced knock-down of CD25 on cell cycle progression and apoptosis in KU812 cells; S11 Effects of shRNA-induced knock-down of CD25 on the responsiveness of KU812 cells to imatinib, nilotinib, ponatinib and BEZ235.

PDF file - 48K, Supplementary Table S5: The oligonucleotide primer sequences for quantitative PCR (qPCR).

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 [Formula: see text] , influences the assembly kinetics. In fact, their [Formula: see text] 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 [Formula: see text]. 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 [Formula: see text] value. These data strongly indicate that differences in their individual amino acid sequences significantly impact the assembly rates. Nevertheless, using a single [Formula: see text] 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.

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 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 splitted 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(2)_h = 0.05 with a heralding efficiency of 𝜂_ℎ = 4.5 % at low pump powers.

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 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 810nm (signal) and 1550nm (idler) are generated via parametric down-conversion pumped at 532nm in the $\mathrm{LiNbO_3}$ waveguide. The pairs are splitted in the polymer board and routed to separate output ports. The module has a size of $(2 \times 1)\, \mathrm{cm^2}$ 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 $\eta_h=4.5\, \mathrm{\%}$ at low pump powers., Comment: 13 pages, 11 figures

Numerous cell functions are accompanied by phenotypic changes in viscoelastic properties, and measuring them can help elucidate higher level cellular functions in health and disease. We present a high-throughput, simple and low-cost microfluidic method for quantitatively measuring the elastic (storage) and viscous (loss) modulus of individual cells. Cells are suspended in a high-viscosity fluid and are pumped with high pressure through a 5.8 cm long and 200 µm wide microfluidic channel. The fluid shear stress induces large, ear ellipsoidal cell deformations. In addition, the flow profile in the channel causes the cells to rotate in a tank-treading manner. From the cell deformation and tank treading frequency, we extract the frequency-dependent viscoelastic cell properties based on a theoretical framework developed by R. Roscoe [1] that describes the deformation of a viscoelastic sphere in a viscous fluid under steady laminar flow. We confirm the accuracy of the method using atomic force microscopy-calibrated polyacrylamide beads and cells. Our measurements demonstrate that suspended cells exhibit power-law, soft glassy rheological behavior that is cell-cycle-dependent and mediated by the physical interplay between the actin filament and intermediate filament networks.Cells in the human body are viscoelastic: they have some of the properties of an elastic solid, like rubber, as well as properties of a viscous fluid, like oil. To carry out mechanical tasks – such as, migrating through tissues to heal a wound or to fight inflammation – cells need the right balance of viscosity and elasticity. Measuring these two properties can therefore help researchers to understand important cell tasks and how they are impacted by disease. However, quantifying these viscous and elastic properties is tricky, as both depend on the time-scale they are measured: when pressed slowly, cells appear soft and liquid, but they turn hard and thick when rapidly pressed. Here, Gerum et al. have developed a new system for measuring the viscosity and elasticity of individual cells that is fast, simple, and inexpensive. In this new method, cells are suspended in a specialized solution with a consistency similar to machine oil which is then pushed with high pressure through channels less than half a millimeter wide. The resulting flow of fluid shears the cells, causing them to elongate and rotate, which is captured using a fast camera that takes 500 images per second. Gerum et al. then used artificial intelligence to extract each cell’s shape and rotation speed from these images, and calculated their viscosity and elasticity based on existing theories of how viscoelastic objects behave in fluids. Gerum et al. also investigated how the elasticity and viscosity of cells changed with higher rotation frequencies, which corresponds to shorter time-scales. This revealed that while higher frequencies made the cells appear more viscous and elastic, the ratio between these two properties remained the same. This means that researchers can compare results obtained from different experimental techniques, even if the measurements were carried out at completely different frequencies or time-scales. The method developed by Gerum et al. provides a fast an inexpensive way for analyzing the viscosity and elasticity of cells. It could also be a useful tool for screening the effects of drugs, or as a diagnostic tool to detect diseases that affect the mechanical properties of cells.

Summary Background The aim was to assess the prognostic value of the newly proposed prognostic index (PI) in patients with p16-positive oropharyngeal squamous cell carcinoma. Methods Patients treated with primary surgery from 2012 to 2019 with available preoperative (0–2 days) values of C‑reactive protein and white blood cell counts needed for calculation of the PI, were included. Main outcome measures were overall survival (OS) and disease-free survival (DFS). The PI was dichotomized into low (PI = 0) and high (PI ≥ 1). Results In this study 36 patients were included. Average overall (OS) and disease-free survival (DFS) were 3.3 years (range 0.2–12.3 years) and 2.8 years (0.0–9.8 years), respectively. The overall mortality was 16.7% (n = 6) and a recurrent disease was observed in 30.6% of patients (n = 11). Low PI was associated with better overall survival (mean OS 10.1 ± 1.4 years, 95% confidence interval, CI 7.3–12.9 years vs. 1.9 ± 0.4, 95% CI 1.3–2.6 years, p p Conclusion The PI might be an easily obtainable outcome prognosticator in p16-positive oropharyngeal squamous cell carcinoma patients. Analyzing routinely obtained blood samples can contribute to identifying high-risk patients.

Neuronal activity is the key modulator of nearly every aspect of behavior, affecting cognition, learning, and memory as well as motion. Hence, disturbances of the transmission of synaptic signals are the main cause of many neurological disorders. Lesions to nervous tissues are associated with phenotypic changes mediated by astrocytes becoming reactive. Reactive astrocytes form the basis of astrogliosis and glial scar formation. Astrocyte reactivity is often targeted to inhibit axon dystrophy and thus promote neuronal regeneration. Here, we aim to understand the impact of gravitational loading induced by hypergravity to potentially modify key features of astrocyte reactivity. We exposed primary murine astrocytes as a model system closely resembling the in vivo reactivity phenotype on custom-built centrifuges for cultivation as well as for live-cell imaging under hypergravity conditions in a physiological range (2

SummaryBrain metastases (BM) are the most frequent intracranial tumors in adults. About 10–20% of the patients with cancer will develop them. Historically, most of the patients with brain metastases were treated with whole brain radiotherapy (WBRT). The intention was to control the metastases and to eliminate distant micrometastases. Randomized control trials showed no difference in survival in patients with single and oligometastases treated with WBRT compared with stereotactic radiosurgery (SRS). To avoid treatment-related toxicities with neurocognitive decline, indications for WBRT are changing. High precision therapy with SRS or postoperative stereotactic treatments have become increasingly important. Only in exceptional cases is WBRT still the treatment of choice.

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.

Objective Recent developments on synthetically generated CTs (sCT), hybrid MRI linacs and MR-only simulations underlined the clinical feasibility and acceptance of MR guided radiation therapy . However, considering clinical application of open and low field MR with a limited field of view can result in truncation of the patient's anatomy which further affects the MR to sCT conversion. In this study an acquisition protocol and subsequent MR image stitching is proposed to overcome the limited field of view restriction of open MR scanners, for MR-only photon and proton therapy . Material and Methods 12 prostate cancer patients scanned with an open 0.35T scanner were included. To obtain the full body contour an enhanced imaging protocol including two repeated scans after bilateral table movement was introduced. All required structures (patient contour, target and organ at risk) were delineated on a post-processed combined transversal image set (stitched MRI). The postprocessed MR was converted into a sCT by a pretrained neural network generator. Inversely planned photon and proton plans (VMAT and SFUD) were designed using the sCT and recalculated for rigidly and deformably registered CT images and compared based on D2%, D50%, V70 Gy for organs at risk and based on D2%, D50%, D98% for the CTV and PTV. The stitched MRI and the untruncated MRI were compared to the CT, and the maximum surface distance was calculated. The sCT was evaluated with respect to delineation accuracy by comparing on stitched MRI and sCT using the DICE coefficient for femoral bones and the whole body. Results Maximum surface distance analysis revealed uncertainties in lateral direction of 1–3 mm on average. DICE coefficient analysis confirms good performance of the sCT conversion, i.e. 92%, 93%, and 100% were obtained for femoral bone left and right and whole body. Dose comparison resulted in uncertainties below 1% between deformed CT and sCT and below 2% between rigidly registered CT and sCT in the CTV for photon and proton treatment plans. Discussion A newly developed acquisition protocol for open MR scanners and subsequent Sct generation revealed good acceptance for photon and proton therapy. Moreover, this protocol tackles the restriction of the limited FOVs and expands the capacities towards MR guided proton therapy with horizontal beam lines.

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: i) lateral assembly of tetramers into unit-length filaments (ULFs); ii) longitudinal annealing of ULFs; iii) 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.Significance statementThe proper formation of intermediate filament (IF) networks in the cytoplasm is important for numerous cell functions. Here, we present a stopped-flow method for measuring the in-vitro assembly kinetics of intermediate filaments with a temporal resolution of 3 ms using static light scattering at two different wavelengths. This allows us to compute the shape factor of the assembly products based on Rayleigh-Gans light scattering theory. From the shape factor, we can separately measure the lateral assembly of tetramers into unit-length filaments (ULFs), and the longitudinal annealing of ULFs and longer filaments. For the IF protein vimentin, we find that with increasing salt concentrations, both the lateral and longitudinal assembly rates increase, and unstable, hyper-aggregated assembly complexes emerge.

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 5‑year biochemical recurrence-free rates were 52% and 24%, respectively (p p = 0.001); and the 5‑year 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.

In an attempt to identify novel markers and immunological targets in leukemic stem cells (LSCs) in acute myeloid leukemia (AML) and chronic myeloid leukemia (CML), we screened bone marrow (BM) samples from patients with AML (n = 274) or CML (n = 97) and controls (n = 288) for expression of cell membrane antigens on CD34+/CD38− and CD34+/CD38+ cells by multicolor flow cytometry. In addition, we established messenger RNA expression profiles in purified sorted CD34+/CD38− and CD34+/CD38+ cells using gene array and quantitative polymerase chain reaction. Aberrantly expressed markers were identified in all cohorts. In CML, CD34+/CD38− LSCs exhibited an almost invariable aberration profile, defined as CD25+/CD26+/CD56+/CD93+/IL-1RAP+. By contrast, in patients with AML, CD34+/CD38− cells variably expressed “aberrant” membrane antigens, including CD25 (48%), CD96 (40%), CD371 (CLL-1; 68%), and IL-1RAP (65%). With the exception of a subgroup of FLT3 internal tandem duplication–mutated patients, AML LSCs did not exhibit CD26. All other surface markers and target antigens detected on AML and/or CML LSCs, including CD33, CD44, CD47, CD52, CD105, CD114, CD117, CD133, CD135, CD184, and roundabout-4, were also found on normal BM stem cells. However, several of these surface targets, including CD25, CD33, and CD123, were expressed at higher levels on CD34+/CD38− LSCs compared with normal BM stem cells. Moreover, antibody-mediated immunological targeting through CD33 or CD52 resulted in LSC depletion in vitro and a substantially reduced LSC engraftment in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Together, we have established surface marker and target expression profiles of AML LSCs and CML LSCs, which should facilitate LSC enrichment, diagnostic LSC phenotyping, and development of LSC-eradicating immunotherapies.

Vimentin intermediate filaments are a significant component of the cytoskeleton in cells of mesenchymal origin. In vivo, filaments assemble and disassemble and thus participate in the dynamic processes of the cell. Post‐translational modifications (PTMs) such as protein phosphorylation regulate the multiphasic association of vimentin from soluble complexes to insoluble filaments and the reverse processes. The thiol side chain of the single vimentin cysteine at position 328 (Cys328) is a direct target of oxidative modifications inside cells. Here, we used atomic force microscopy, electron microscopy and a novel hydrogen–deuterium exchange mass spectrometry (HDex‐MS) procedure to investigate the structural consequences of S‐nitrosylation and S‐glutathionylation of Cys328 for in vitro oligomerisation of human vimentin. Neither modification affects the lateral association of tetramers to unit‐length filaments (ULF). However, S‐glutathionylation of Cys328 blocks the longitudinal assembly of ULF into extended filaments. S‐nitrosylation of Cys328 does not hinder but slows down the elongation. Likewise, S‐glutathionylation of preformed vimentin filaments causes their extensive fragmentation to smaller oligomeric species. Chemical reduction of the S‐glutathionylated Cys328 thiols induces reassembly of the small fragments into extended filaments. In conclusion, our in vitro results suggest S‐glutathionylation as a candidate PTM for an efficient molecular switch in the dynamic rearrangements of vimentin intermediate filaments, observed in vivo, in response to changes in cellular redox status. Finally, we demonstrate that HDex‐MS is a powerful method for probing the kinetics of vimentin filament formation and filament disassembly induced by PTMs., Vimentin intermediate filaments are a major component of the cytoskeleton in cells of mesenchymal origin. Oxidative modification of vimentin Cys328 is known to affect the assembly of the filament. Using atomic force microscopy, electron microscopy and a novel hydrogen–deuterium exchange mass spectrometry, the authors investigated the structural impact of S‐nitrosylation and S‐glutathionylation on human vimentin oligomerisation in vitro. They show that S‐glutathionylation does not affect the lateral association of tetramers to unit‐length filaments, but blocks the longitudinal assembly of vimentin into long filaments and causes extensive fragmentation of preformed vimentin filaments. Thus, this modification may modulate the dynamic rearrangements of vimentin intermediate filaments in vivo.

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.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.Multicolor flow cytometry was performed to recognize surface antigens and to determine changes in antigen expression upon activation.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 CD203cSiglec-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.

Interferometers provide a highly sensitive means to investigate and exploit the coherence properties of light in metrology applications. However, interferometers come in various forms and exploit different properties of the optical states within. In this paper, we introduce a classification scheme that characterizes any interferometer based on the number of involved nonlinear elements by studying their influence on single-photon and photon-pair states. Several examples of specific interferometers from these more general classes are discussed, and the theory describing the expected first-order and second-order coherence measurements for single-photon and single-photon-pair input states is summarized and compared. These theoretical predictions are then tested in an innovative experimental setup that is easily able to switch between implementing an interferometer consisting of only one or two nonlinear elements. The resulting singles and coincidence rates are measured in both configurations and the results are seen to fit well with the presented theory. The measured results of coherence are tied back to the presented classification scheme, revealing that our experimental design can be useful in gaining insight into the properties of the various interferometeric setups containing different degrees of nonlinearity., Comment: 12 pages, 5 figures

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. We investigate the effect of the R349P desmin mutation on the formation, differentiation, and contractile function of muscle micro‐tissues grown from satellite cells. Desmin‐mutated micro‐tissues show progressive degeneration over time, which is closely linked to extracellular matrix fibre breakage and associated with unevenly distributed tensile stress and locally increased contractile forces. Our results suggest 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. image

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.

Aim: The aim of this study was to assess the effects of including somatostatin receptor agonist (SSTR) PET imaging in meningioma radiotherapy planning by means of changes in inter-observer variability (IOV). Further, the possibility of using threshold-based delineation approaches for semiautomatic tumor volume definition was assessed. Patients and Methods: Sixteen patients with meningioma undergoing fractionated radiotherapy were delineated by five radiation oncologists. IOV was calculated by comparing each delineation to a consensus delineation, based on the simultaneous truth and performance level estimation (STAPLE) algorithm. The consensus delineation was used to adapt a threshold-based delineation, based on a maximization of the mean Dice coefficient. To test the threshold-based approach, seven patients with SSTR-positive meningioma were additionally evaluated as a validation group. Results: The average Dice coefficients for delineations based on MRI alone was 0.84 ± 0.12. For delineation based on MRI + PET, a significantly higher dice coefficient of 0.87 ± 0.08 was found (p < 0.001). The Hausdorff distance decreased from 10.96 ± 11.98 mm to 8.83 ± 12.21 mm (p < 0.001) when adding PET for the lesion delineation. The best threshold value for a threshold-based delineation was found to be 14.0% of the SUVmax, with an average Dice coefficient of 0.50 ± 0.19 compared to the consensus delineation. In the validation cohort, a Dice coefficient of 0.56 ± 0.29 and a Hausdorff coefficient of 27.15 ± 21.54 mm were found for the threshold-based approach. Conclusions: SSTR-PET added to standard imaging with CT and MRI reduces the IOV in radiotherapy planning for patients with meningioma. When using a threshold-based approach for PET-based delineation of meningioma, a relatively low threshold of 14.0% of the SUVmax was found to provide the best agreement with a consensus delineation.

The realisation of light modulation based on single photon detection events is an important step towards feed-forward-modulation, which is required for many quantum optics protocols. This requires combining detection events with modulation in a device with a small physical footprint, for which integrated optics is ideal. Several platforms for integrated quantum optics have been developed to generate, modulate, and detect quantum features in the light over the last years. Titanium in-diffused waveguides in lithium niobate is a promising candidate for quantum communication applications [1] , [2] , however, single photon detection with superconducting photon detectors (SNSPDs) require cryogenic temperatures. Therefore, optimising the nonlinear optical properties of these waveguides under cryogenic operating conditions is an important task. Previously, we have demonstrated frequency conversion [3] , electro-optical modulation [4] and integrated single photon detection [5] at cryogenic temperatures.

Squeezed light forms the foundation of continuous-variable quantum optics. In most experimental realisations, bulk optics resonators utilising the second-order nonlinearity serve as the source of stable, high levels of squeezing. As one would expect, there is a drive towards producing squeezed light sources in integrated platforms, as it is expected that such a transition will ease the challenges in moving toward real-world applications. However, waveguides come with their own set of challenges; they generally exhibit increased losses, and some platforms suffer from photothermal or photorefractive effects.

In chronic myeloid leukemia (CML), resistance against second-generation tyrosine kinase inhibitors (TKI) remains a serious clinical challenge, especially in the context of multi-resistant BCR-ABL1 mutants, such as T315I. Treatment with ponatinib may suppress most of these mutants, including T315I, but is also associated with a high risk of clinically relevant side effects. We screened for alternative treatment options employing available tyrosine kinase inhibitors (TKI) in combination. Dasatinib and bosutinib are two second-generation TKI that bind to different, albeit partially overlapping, spectra of kinase targets in CML cells. This observation prompted us to explore anti-leukemic effects of the combination dasatinib + bosutinib in highly resistant primary CML cells, various CML cell lines (K562, K562R, KU812, KCL22) and Ba/F3 cells harboring various BCR-ABL1 mutant-forms. We found that bosutinib synergizes with dasatinib in inducing growth inhibition and apoptosis in all CML cell lines and in Ba/F3 cells exhibiting BCR-ABL1T315I. Clear synergistic effects were also observed in primary CML cells in all patients tested (n = 20), including drug-resistant cells carrying BCR-ABL1T315I. Moreover, the drug combination produced cooperative or even synergistic apoptosis-inducing effects on CD34+/CD38– CML stem cells. Finally, we found that the drug combination is a potent approach to block the activity of major additional CML targets, including LYN, KIT and PDGFRα. Together, bosutinib and dasatinib synergize in producing anti-leukemic effects in drug-resistant CML cells. Whether such cooperative TKI effects also occur in vivo in patients with drug-resistant CML, remains to be determined in forthcoming studies.

Vimentin intermediate filaments constitute a distinct filament system in mesenchymal cells that is instrumental for cellular mechanics and migration. In vitro, the rod-like monomers assemble in a multi-step, salt-dependent manner into micrometer long biopolymers. To disclose the underlying mechanisms further, we employed small angle X-ray scattering on two recombinant vimentin variants, whose assembly departs at strategic points from the normal assembly route: (i) vimentin with a tyrosine to leucine change at position 117; (ii) vimentin missing the non-α-helical carboxyl-terminal domain. Y117L vimentin assembles into unit-length filaments (ULFs) only, whereas ΔT vimentin assembles into filaments containing a higher number of tetramers per cross section than normal vimentin filaments. We show that the shape and inner structure of these mutant filaments is significantly altered. ULFs assembled from Y117L vimentin contain more, less tightly bundled vimentin tetramers, and ΔT vimentin filaments preserve the number density despite the higher number of tetramers per filament cross-section. peerReviewed

Lithium niobate is a promising platform for integrated quantum optics. In this platform we aim to efficiently manipulate and detect quantum states by combining superconducting single photon detectors and modulators. The cryogenic operation of a superconducting single photon detector dictates the optimisation of the electro-optic modulators under the same operating conditions. To that end, we characterise a phase modulator, directional coupler, and polarisation converter at both ambient and cryogenic temperatures. The operation voltage $V_{\pi/2}$ of these modulators increases due to the decrease of the electro-optic effect by 74% for the phase modulator, 84% for the directional coupler and 35% for the polarisation converter below 8.5$\,\mathrm{K}$. The phase modulator preserves its broadband nature and modulates light in the characterised wavelength range. The unbiased bar state of the directional coupler changed by a wavelength shift of 85$\,\mathrm{nm}$ while cooling the device down to 5$\,\mathrm{K}$. The polarisation converter uses periodic poling to phasematch the two orthogonal polarisations. The phasematched wavelength of the used poling changes by 112$\,\mathrm{nm}$ when cooling to 5$\,\mathrm{K}$, Comment: 18 pages, 5 figures

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.

An integrated source of squeezed states is required for many quantum optics applications. We present a 1cm long Ti:LiNbO3 waveguide resonator producing up to 4.9dB of single-mode squeezing and efforts towards incorporating an electro-optic modulator into the device.

The cytoskeleton is a major determinant of cell mechanics, a property that is altered during many pathological situations. To understand these alterations, 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 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 behavior compared to that of the pure networks. In stark contrast, the composites displayed increasing strain stiffening behavior as a result of increased keratin content when larger deformations were imposed. This strain stiffening behavior is fundamentally different from behavior encountered with vimentin IF as a composite network partner for actin. Our results provide new insights into the mechanical interplay between actin and keratin in which keratin provides reinforcement to actin. This interplay may contribute to the overall integrity of cells, providing an explanation for the stability of stressed epithelial tissues due to their high keratin contents. Additionally, this helps us to understand the physiological necessity to exchange IF systems during epithelial-mesenchymal transition (EMT) in order to suppress strain stiffening of the network, making cells more elastic and, thus, facilitating their migration through dense tissues.

Objective: To evaluate the impact of radiotherapy (RT) on dysphagia and long-term swallowing outcome in patients with stage III and IV head and neck squamous cell carcinomas (HNSCCs). Material and Methods: Between 2005 and 2008, 189 patients with HNSCCs underwent primary or adjuvant RT in a curative setting. Long-term swallowing outcome was evaluated in 50 patients. Among them, 26 were further eligible for prospective analysis of long-term swallowing and dysphagia outcome. Medical charts were retrospectively reviewed regarding pre- and post-treatment dysphagia (3 months after last irradiation setting) as well as persisting long-term dysphagia (2019–2021). Results: Pre-treatment dysphagia was observed in 24 (48%) of 50 patients, particularly in oropharyngeal or hypopharyngeal stage III–IV tumors (OR 9.3; p = 0.003). Conversely, 46 patients (92%) complained about post-treatment dysphagic symptoms, which were more commonly seen in patients with positive neck nodes (OR 10.5; p = 0.037). The post-treatment dysphagia rate dropped from 92% to 24% (p < 0.001) during surveillance, which was significantly linked to xerostomia (OR 5.77; p = 0.019), dysgeusia (OR 9.9; p = 0.036) and free flap reconstruction (OR 6.1; p = 0.022). Conclusion: Pretreatment dysphagia is common in advanced stage HNSCCs and almost all patients complain about dysphagia at the end of RT. Importantly, applied RT protocols did not affect long-term dysphagia, which improves significantly in the majority of patients over time. Meeting Information: Preliminary results have been presented at the 65th Annual Meeting of the Austrian Society of Otorhinolaryngology, 22–26 September 2021, Austria.

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.

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.

We demonstrate an electro-optic polarisation converter for 1550nm at cryo genic temperatures in titanium in-diffused lithium niobate waveguides. The switching voltage increases, the modulation depth remains unchanged, and we show operation up to 25 MHz.

We present advances in the use of gigahertz LiNbO3 modulators enabling us programmable delays, which we will use to implement a multiplexed single-photon source, based on birefringent phase-matching in KTP waveguides.

Summary In 2008 the Ludwig Boltzmann Cluster Oncology (LBC ONC) was established on the basis of two previous Ludwig Boltzmann Institutes working in the field of hematology and cancer research. The general aim of the LBC ONC is to improve treatment of hematopoietic neoplasms by eradicating cancer-initiating and disease-propagating cells, also known as leukemic stem cells (LSC) in the context of leukemia. In a first phase, the LBC ONC characterized the phenotype and molecular aberration profiles of LSC in various malignancies. The LSC phenotypes were established in acute and chronic myeloid leukemia, in acute lymphoblastic leukemia and in chronic lymphocytic leukemia. In addition, the concept of preleukemic (premalignant) neoplastic stem cells (pre-L-NSC) was coined by the LBC ONC and was tested in myelodysplastic syndromes and myeloproliferative neoplasms. Phenotypic characterization of LSC provided a solid basis for their purification and for the characterization of specific target expression profiles. In a second phase, molecular markers and targets were validated. This second phase is ongoing and should result in the development of new diagnostics parameters and novel, more effective, LSC-eradicating, treatment strategies; however, many issues still remain to be solved, such as sub-clonal evolution, LSC niche interactions, immunologic control of LSC, and LSC resistance. In the forthcoming years, the LBC ONC will concentrate on developing LSC-eradicating strategies, with special focus on LSC resistance, precision medicine and translation of LSC-eradicating concepts into clinical application.

Based on actual advancements of historical research on Smith and Luther, the thesis of protestantism concerning the roots of competitive capitalism is being confirmed, but modified by aspects of paradigmatic revolution to individualistic anthropology. The present paper analyses the Lutheran theology of justification as a special kind of contingency doctrine and shows historical developments from Luther to Thomasius, Smith and Kant. As to the history of paradigma effects of individualism, European origins of competition ethics of responsibility can be shown, which do not refer to market performance or imperformance, but to accountability in sense of risk identification and risk control. Unfair competitive actions, therefore, are to be legally interdicted as unethical competition behaviour when market risks are transferred to market partners or to third persons without good reasons of risk control. Some further conclusions can be made to ethical rules of banking and companies’ law as well as to the general law of commerce.

Systemic mastocytosis (SM) is a mast cell (MC) neoplasm with complex pathology and a variable clinical course. In aggressive SM (ASM) and MC leukemia (MCL), responses to conventional drugs are poor and the prognosis is dismal. R763 is a multi-kinase inhibitor that blocks the activity of Aurora-kinase-A/B, ABL1, AKT and FLT3. We examined the effects of R763 on proliferation and survival of neoplastic MC. R763 produced dose-dependent inhibition of proliferation in the human MC lines HMC-1.1 (IC50 5–50 nM), HMC-1.2 (IC50 1–10 nM), ROSAKIT WT (IC50 1–10 nM), ROSAKIT D816V (IC50 50–500 nM) and MCPV-1.1 (IC50 100–1000 nM). Moreover, R763 induced growth inhibition in primary neoplastic MC in patients with ASM and MCL. Growth-inhibitory effects of R763 were accompanied by signs of apoptosis and a G2/M cell cycle arrest. R763 also inhibited phosphorylation of KIT, BTK, AKT and STAT5 in neoplastic MC. The most sensitive target appeared to be STAT5. In fact, tyrosine phosphorylation of STAT5 was inhibited by R763 at 10 nM. At this low concentration, R763 produced synergistic growth-inhibitory effects on neoplastic MC when combined with midostaurin or dasatinib. Together, R763 is a novel promising multi-kinase inhibitor that blocks STAT5 activation and thereby overrides drug-resistance in neoplastic MC.

The keratin filament cytoskeleton is vital to the normal function of epithelial cells. It provides structural support and regulates different aspects of cell metabolism. Mutations in keratins 5 and 14 cause a skin fragility disorder, epidermolysis bullosa simplex (EBS). Patients with severe EBS have an increased cumulative risk for basal cell carcinoma. In this study, we tested how keratin 5 and 14 mutant EBS patient-derived keratinocytes behave in the face of two different types of stressors that are able to induce cell death: ionizing radiation and cytokines TNF-α and TRAIL. The data point out to a substantial difference between how normal and keratin mutant keratinocytes deal with such stresses. When case of DNA damage, the ATM/Chk2-pathway is one of the two main tracks that can prevent the progression of mitosis and so allow repair. This was altered in all investigated keratin mutants with a particular down-regulation of the activated form of checkpoint kinase 2 (pChk2). Keratin mutants also appear less sensitive than normal cells to treatment with TNF-α or TRAIL, and this may be linked to the up-regulation of two pro-survival proteins, Bcl-2 and FLIP. Such changes are likely to have a profound effect on mutant keratinocytes ability to survive and withstand stress, and in theory this may be also a contributing factor to cell transformation.

Mutations in the small heat shock protein chaperone CRYAB (αB-crystallin/HSPB5) and the intermediate filament protein desmin, phenocopy each other causing cardiomyopathies. Whilst the binding sites for desmin on CRYAB have been determined, desmin epitopes responsible for CRYAB binding and also the parameters that determine CRYAB binding to desmin filaments are unknown. Using a combination of co-sedimentation centrifugation, viscometric assays and electron microscopy of negatively stained filaments to analyse the in vitro assembly of desmin filaments, we show that the binding of CRYAB to desmin is subject to its assembly status, to the subunit organization within filaments formed and to the integrity of the C-terminal tail domain of desmin. Our in vitro studies using a rapid assembly protocol, C-terminally truncated desmin and two disease-causing mutants (I451M and R454W) suggest that CRYAB is a sensor for the surface topology of the desmin filament. Our data also suggest that CRYAB performs an assembly chaperone role because the assembling filaments have different CRYAB-binding properties during the maturation process. We suggest that the capability of CRYAB to distinguish between filaments with different surface topologies due either to mutation (R454W) or assembly protocol is important to understanding the pathomechanism(s) of desmin-CRYAB myopathies.