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Abstract Phosphorylation of substrates by cyclin-dependent kinases (CDKs) is the driving force of cell cycle progression. Several CDK-activating cyclins are involved, yet how they contribute to substrate specificity is still poorly understood. Here, we discover that a positively charged pocket in cyclin B1, which is exclusively conserved within B-type cyclins and binds phosphorylated serine- or threonine-residues, is essential for correct execution of mitosis. HeLa cells expressing pocket mutant cyclin B1 are strongly delayed in anaphase onset due to multiple defects in mitotic spindle function and timely activation of the E3 ligase APC/C. Pocket integrity is essential for APC/C phosphorylation particularly at non-consensus CDK1 sites and full in vitro ubiquitylation activity. Our results support a model in which cyclin B1’s pocket facilitates sequential substrate phosphorylations involving initial priming events that assist subsequent pocket-dependent phosphorylations even at non-consensus CDK1 motifs.

Aberrant liquid-to-solid phase transitions of biomolecular condensates have been linked to various neurodegenerative diseases. However, the underlying molecular interactions that drive aging remain enigmatic. Here, we develop quantitative time-resolved crosslinking mass spectrometry to monitor protein interactions and dynamics inside condensates formed by the protein fused in sarcoma (FUS). We identify misfolding of the RNA recognition motif of FUS as a key driver of condensate aging. We demonstrate that the small heat shock protein HspB8 partitions into FUS condensates via its intrinsically disordered domain and prevents condensate hardening via condensate-specific interactions that are mediated by its α-crystallin domain (αCD). These αCD-mediated interactions are altered in a disease-associated mutant of HspB8, which abrogates the ability of HspB8 to prevent condensate hardening. We propose that stabilizing aggregation-prone folded RNA-binding domains inside condensates by molecular chaperones may be a general mechanism to prevent aberrant phase transitions.

Introduction: Physiotherapy, a non-invasive method of conservative treatment, that includes manual therapy, exercises and physical procedures, is used in the treatment of temporomandibular dysfunction and polyarticular laxity. It has been proven that physiotherapy focused on temporomandibular dysfunction is an essential element of treatment that leads to a reduction in pain, an improvement in temporomandibular joint (TMJ) function, and an improvement in quality of life. Physiotherapy treatment aims to alleviate the symptoms and try to restore the normal function of the masticatory system, for which various techniques can be used. The aim of the study was to determine the type, intensity, and frequency of TMJ dysfunction pain before and after physiotherapy. Methods: The study was conducted in a private practice in Munich, Germany. All subjects who met the criteria for inclusion in the study completed a standardized questionnaire on TMJ dysfunction (TMJ disorder [TMJ/TMD] Questionnaire). The questionnaire was used to obtain data on the type and location of pain, duration of pain, and other symptoms related to the TMJ. The anamnestic data of the respondents who came to the doctor’s office with the symptoms mentioned were examined, and the orthodontic or dental findings were analyzed. Results: The analysis showed that before treatment some of the joints were affected in 28 subjects, while after treatment the joints were affected in 19 subjects. After treatment a significant decrease in affected TMJ was observed (χ2 = 9.516, p = 0.008). Ear pain occurred in 17 (54.84%) subjects before treatment, and in 5 (16.13%) after treatment. A significant reduction was observed at the p = 0.003 level. Pain around the eyes occurred in 9 (29.03%) of the respondents before treatment, and after treatment in 2 (6.45%) of the respondents. Facial pain was reported in 16.13% of the anamnestic data before the treatment, and it was not reported in any of the respondents after treatment. There was also a significant reduction in the occurrence of tinnitus (p = 0.004). The occurrence of headaches also decreased after treatment (p < 0.001). Ear pressure was present before treatment in 45.16% of cases, and after treatment in 22.58% of cases, so there was no significant decrease. Conclusion: Physiotherapy for people with TMJ dysfunction contributed significantly in reducing pain and alleviating other TMD symptoms.

In this study, we focus on modeling the local spread of COVID-19 infections. As the pandemic continues and new variants or future pandemics can emerge, modelling the early stages of infection spread becomes crucial, especially as limited medical data might be available initially. Therefore, our aim is to gain a better understanding of the diffusion dynamics on smaller scales using partial differential equation (PDE) models. Previous works have already presented various methods to model the spatial spread of diseases, but, due to a lack of data on regional or even local scale, few actually applied their models on real disease courses in order to describe the behaviour of the disease or estimate parameters. We use medical data from both the Robert-Koch-Institute (RKI) and the Birkenfeld district government for parameter estimation within a single German district, Birkenfeld in Rhineland-Palatinate, during the second wave of the pandemic in autumn 2020 and winter 2020–21. This district can be seen as a typical middle-European region, characterized by its (mainly) rural nature and daily commuter movements towards metropolitan areas. A basic reaction-diffusion model used for spatial COVID spread, which includes compartments for susceptibles, exposed, infected, recovered, and the total population, is used to describe the spatio-temporal spread of infections. The transmission rate, recovery rate, initial infected values, detection rate, and diffusivity rate are considered as parameters to be estimated using the reported daily data and least square fit. This work also features an emphasis on numerical methods which will be used to describe the diffusion on arbitrary two-dimensional domains. Two numerical optimization techniques for parameter fitting are used: the Metropolis algorithm and the adjoint method. Two different methods, the Crank-Nicholson method and a finite element method, which are used according to the requirements of the respective optimization method are used to solve the PDE system. This way, the two methods are compared and validated and provide similar results with good approximation of the infected in both the district and the respective sub-districts.

We present a case of a persistent macular hole which was initially treated by pars plana vitrectomy with the inverted ILM flap technique. In a second procedure, the internal limiting membrane (ILM) flap was mobilized from its perifoveal adherence to the retina and peeled back to its adherence at the foveal ring. The eye was filled with 25% C2F6 gas. Three weeks after the second procedure, closure of the hole was observed. Best corrected visual acuity increased from 20/400 to 20/50. We assume that contractile elements within the ILM may cause perifoveal adhesion and centrifugal effects preventing macular hole closure. If macular hole closure does not occur after surgery with an inverted ILM flap, it is worth to peel back the existing flap again towards the foveal edge in order to induce hole closure and preserve the benefits of flap surgery. If the flap is only attached to the foveal ring, centripetal contraction could lead to annular closure of the macular hole. If the flap is lost, alternative surgical methods for refractory MHs should be considered.

Ophthalmic drug delivery for treating various eye diseases still remains a challenge in ophthalmology. One perspective way of overcoming this problem is to use nanoscale biodegradable drug carriers that are able to safely deliver pharmaceuticals directly to the locus of disease and maintain a therapeutic concentration of drug for a long time. The goal of the present study was the preparation of drug- (dexamethasone-, DEX-) loaded pseudo-protein nanoparticles (NPs) and investigation of drug encapsulation efficiency and drug release kinetics. DEX-loaded pseudo-protein NPs (DEX-NPs) were successfully prepared by the nanoprecipitation method. DEX-NPs were characterized by size (average diameter, AD), size distribution (polydispersity index, PDI), and surface charge (zeta-potential, ZP) using the dynamic light scattering technique. DEX encapsulation characteristics were determined using the UV-spectrophotometric method, and kinetics of DEX release from DEX-NPs was studied according to the dialysis method in PBS at 37°C. The obtained results showed that size of DEX-NPs varies within 143.6–164.1 nm depending on DEX content during the preparation. DEX incorporation characteristics were determined—encapsulation efficiency (EE) and actual drug loading (DL) were high enough and reached 55.1 and 10.2%, respectively. The kinetics of DEX release from DEX-NPs showed a typical biphasic release pattern—an initial rapid (burst) release and further much more continuous slow release. Based on the obtained data, we can conclude that the elaborated DEX-NPs have potential for the application in ophthalmology as ocular drug delivery nanocarriers.

Abstract To investigate the effect of P2 receptor on microglia and its inhibitor PPADS on choroidal neovascularization. Forty CX3CR1GFP/+ mice were randomly divided into 8 groups. In addition to the normal group, the rest of groups were receiving laser treatment. The retina and choroid from the second, third, fourth and fifth group of mice were taken in the 1, 4, 7, 14 days after laser treatment. The mice in the sixth and seventh group received intravitreal injection of 2 µl PPADS or PBS respectively immediately after laser treatment. The mice in the eighth group received topical application of PPADS once per day of three days. The mice in sixth, seventh and eighth group received AF and FFA examination on the fourth day after laser treatment. Immunofluorescence histochemical staining and real-time quantitative PCR were used to evaluate P2 expression and its effect on choroidal neovascularization. After laser treatment, activated microglia can express P2 receptors (P2X4, P2X7, P2Y2 and P2Y12). The expression of P2 increased on the first day after laser damage, peaked on the fourth day (tP2X4 = 6.05, tP2X7 = 2.95, tP2Y2 = 3.67, tP2Y12 = 5.98, all P

Abstract This paper stresses its base contribution on a new SIR-type model including direct and fomite transmission as well as the effect of distinct household structures. The model derivation is modulated by several mechanistic processes inherent from typical airborne diseases. The notion of minimum contact radius is included in the direct transmission, facilitating the arguments on physical distancing. As fomite transmission heavily relates to former-trace of sneezes, the vector field of the system naturally contains an integral kernel with time delay indicating the contribution of undetected and non-quarantined asymptomatic cases in accumulating the historical contamination of surfaces. We then increase the complexity by including the different transmission routines within and between households. For airborne diseases, within-household interactions play a significant role in the propagation of the disease rendering countrywide effect. Two steps were taken to include the effect of household structure. The first step subdivides the entire compartments (susceptible, exposed, asymptomatic, symptomatic, recovered, death) into the household level and different infection rates for the direct transmission within and between households were distinguished. Under predefined conditions and assumptions, the governing system on household level can be raised to the community level. The second step then raises the governing system to the country level, where the final state variables estimate the total individuals from all compartments in the country. Two key attributes related to the household structure (number of local households and number of household members) effectively classify countries to be of low or high risk in terms of effective disease propagation. The basic reproductive number is calculated and its biological meaning is invoked properly. The numerical methods for solving the DIDE-system and the parameter estimation problem were mentioned. Our optimal model solutions are in quite good agreement with datasets of COVID-19 active cases and related deaths from Germany and Sri Lanka in early infection, allowing us to hypothesize several unobservable situations in the two countries. Focusing on extending minimum contact radius and reducing the intensity of individual activities, we were able to synthesize the key parameters telling what to practice.

Apoptosis, a key mechanism of programmed cell death, is triggered by caspase-3 protein and lowering its levels with gene therapy may rescue cell death after central nervous system damage. We developed a novel, non-viral gene therapy to block caspase-3 gene expression using small interfering RNA (siRNA) delivered by polybutylcyanoacrylate nanoparticles (CaspNPs). In vitro CaspNPs significantly blocked caspase-3 protein expression in C6 cells, and when injected intraocularly in vivo, CaspNPs lowered retinal capsase-3 immunofluorescence by 57.9% in rats with optic nerve crush. Longitudinal, repeated retinal ganglion cell counts using confocal neuroimaging showed that post-traumatic cell loss after intraocular CaspNPs injection was only 36.1% versus 63.4% in lesioned controls. Because non-viral gene therapy with siRNA-nanoparticles can selectively silence caspace-3 gene expression and block apoptosis in post-mitotic neurons, siRNA delivery with nanoparticles may be promising for neuroprotection or restoration of central visual system damage and other neurological disorders. The animal study procedures were approved by the German National Act on the use of experimental animals (Ethic Committee Referat Verbraucherschutz, Veterinärangelegenheiten; Landesverwaltungsamt Sachsen-Anhalt, Halle, Germany, # IMP/G/01-1150/12 and # IMP/G/01-1469/17).

Abstract The WW-and-C2-domain-containing (WWC) protein family is involved in the regulation of cell differentiation, cell proliferation, and organ growth control. As upstream components of the Hippo signaling pathway, WWC proteins activate the Large tumor suppressor (LATS) kinase that in turn phosphorylates Yes-associated protein (YAP) and its paralog Transcriptional coactivator-with-PDZ-binding motif (TAZ) preventing their nuclear import and transcriptional activity. Inhibition of WWC expression leads to downregulation of the Hippo pathway, increased expression of YAP/TAZ target genes and enhanced organ growth. In mice, a ubiquitous Wwc1 knockout (KO) induces a mild neurological phenotype with no impact on embryogenesis or organ growth. In contrast, we could show here that ubiquitous deletion of Wwc2 in mice leads to early embryonic lethality. Wwc2 KO embryos display growth retardation, a disturbed placenta development, impaired vascularization, and finally embryonic death. A whole-transcriptome analysis of embryos lacking Wwc2 revealed a massive deregulation of gene expression with impact on cell fate determination, cell metabolism, and angiogenesis. Consequently, a perinatal, endothelial-specific Wwc2 KO in mice led to disturbed vessel formation and vascular hypersprouting in the retina. In summary, our data elucidate a novel role for Wwc2 as a key regulator in early embryonic development and sprouting angiogenesis in mice.

Abstract Since the end of 2019 an outbreak of a new strain of coronavirus, called SARS-CoV-2, is reported from China and later other parts of the world. Since January 21, World Health Organization (WHO) reports daily data on confirmed cases and deaths from both China and other countries ( www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports ). The Johns Hopkins University ( github.com/CSSEGISandData/COVID-19/blob/master/csse_COVID_19_data/csse_COVID_19_time_series/time_series_COVID19_confirmed_global.csv ) collects those data from various sources worldwide on a daily basis. For Germany, the Robert-Koch-Institute (RKI) also issues daily reports on the current number of infections and infection related fatal cases ( www.rki.de/DE/Content/InfAZ/N/Neuartiges_Coronavirus/Situationsberichte/Gesamt.html ). However, due to delays in the data collection, the data from RKI always lags behind those reported by Johns Hopkins. In this work we present an extended SEIRD-model to describe the disease dynamics in Germany. The parameter values are identified by matching the model output to the officially reported cases. An additional parameter to capture the influence of unidentified cases is also included in the model.

Since the end of 2019 an outbreak of a new strain of coronavirus, called SARS–CoV–2, is reported from China and later also from other parts of the world. Since 21 January 2020, World Health Organization (WHO) reports daily data on confirmed cases and deaths from both China and other countries [1]. The Johns Hopkins University [2] collects those data from various sources worldwide on a daily basis. For Germany, the Robert–Koch–Institute (RKI) also issues daily reports on the current number of infections and infection related fatal cases and also provides estimates of several disease-related parameters [3]. In this work we present an extended SEIRD–model to describe these disease dynamics in Germany. The model takes into account the susceptible, exposed, infected, recovered and deceased fractions of the population. Epidemiological parameters like the transmission rate, lethality or the detection rate of infected individuals are estimated by fitting the model output to available data. For the parameter estimation itself we compare two methods: an adjoint based approach and a Monte–Carlo based Metropolis algorithm.

Abstract Background This study aimed to investigate the feasibility of optical coherence tomography angiography (OCT-A) for quantitative analysis of flow density to assess changes in retinal perfusion in an experimental model of haemorrhagic shock. Methods Haemorrhagic shock was induced in five healthy, anaesthetized sheep by stepwise blood withdrawal of 3 × 10 ml∙kg− 1 body weight. OCT-A imaging of retinal perfusion was performed using an OCT device. Incident dark-field illumination microscopy videos were obtained for the evaluation of conjunctival microcirculation. Haemodynamic variables and flow density data in the OCT angiogram were analysed before and during progressive haemorrhage resulting in haemorrhagic shock as well as after fluid resuscitation with 10 ml∙kg− 1 body weight of balanced hydroxyethyl starch solution (6% HES 130/0.4). Videos of the conjunctival microcirculation were recorded at baseline, in haemorrhagic shock, and after resuscitation. Data are presented as median with interquartile range. Comparisons between time points were made using Friedman’s test and the degree of correlation between two variables was expressed as Spearman’s rank correlation coefficient. Results Mean arterial pressure and cardiac index (CI) decreased and lactate concentration increased after induction of shock, and haemodynamics recovered after resuscitation. The flow density in the superficial retinal OCT angiogram decreased significantly after shock induction (baseline 44.7% (40.3; 50.5) vs haemorrhagic shock 34.5% (32.8; 40.4); P = 0.027) and recovered after fluid resuscitation (46.9% (41.7; 50.7) vs haemorrhagic shock; P = 0.027). The proportion of perfused vessels of the conjunctival microcirculation showed similar changes. The flow density measured using OCT-A correlated with the conjunctival microcirculation (perfused vessel density: Spearman’s rank correlation coefficient ρ = 0.750, P = 0.001) and haemodynamic parameters (CI: ρ = 0.693, P

Abstract Background The aim of the study was to measure pH changes of the human vitreous caused by the intravitreal drugs bevacizumab, ranibizumab, aflibercept, and ziv-aflibercept. Methods Fresh human vitreous samples were obtained during core vitrectomy (23-gauge) from patients with epiretinal gliosis. Aliquots of bevacizumab, ranibizumab, aflibercept or ziv-aflibercept (2 µl) were added consecutively to 200 µl of vitreous samples or 0.9% NaCl saline. The pH was measured using a pH-sensitive microelectrode. Rituximab, in off-label use against intraocular lymphoma, was tested as an IgG1 antibody. Results The pH of the administered drugs was 5.91 for bevacizumab (95% CI 5.63–6.19), 5.32 for ranibizumab (95% CI 5.0–5.63), 6.05 for aflibercept (95% CI 5.78–6.31), ziv-aflibercept 6.1 (95% CI 6.05–6.15), and 6.29 for rituximab (95% CI 5.97–6.61). While the fresh and undiluted vitreous fluid showed pH values of 7.0–7.4, pH values increased if saline or rituximab were added. In contrast, the pH decreased slightly if aflibercept, bevacizumab, ranibizumab or ziv-aflibercept were supplemented. The observed pH decreases were not significant after ranibizumab was added. Significant changes were only notable with higher-than-normal amounts of bevacizumab (26–40 µl). The vitreous showed the most robust buffering capacity towards ranibizumab and rituximab. Conclusions The pH changes in vitreous samples elicited by the usual intravitreal anti-VEGF drugs differed clearly, but only by much higher concentrations than used in the clinical routine. Although the ingredient solution of ranibizumab showed the lowest pH, it caused only moderate changes of vitreal pH compared to bevacizumab, aflibercept or ziv-aflibercept.

Background/aimsTo analyse the distribution of macular ganglion cell layer thickness (GCLT) in patients with foveal hypoplasia (FH) with or without albinism to obtain new insights into visual pathway anomalies in albinos.MethodsPatients with FH who presented at our institution between 2013 and 2018 were retrospectively drawn for analysis. Mean GCLT was calculated after automated segmentation of spectral domain-optical coherence tomography (SD-OCT) scans. Patients with FH due to albinism (n = 13, termed 'albinism FH') or other kinds (n = 10, termed 'non-albinism FH') were compared with control subjects (n = 15). The areas: fovea (central), parafovea (nasal I, temporal I) and perifovea (nasal II, temporal II) along the horizontal meridian were of particular interest. Primary endpoints of this study were the ratios (GCLT-I- and GCLT-II-Quotient) between the GCLT measured in the temporal I or II and nasal I or II areas.ResultsThere was a significant difference between the GCLT-I-Quotient of healthy controls and albinism FH (pConclusionMacular GCLT-distribution showed a characteristic temporal to central shift in patients with FH due to albinism. Calculation of the GCLT-II-Quotient at a cut-off of

Abstract After the publication of this article [1], we were made aware that the osmolarity of aflibercept (Eylea) was incorrect and should have been 286 mOsm and not 1000 mOsm. The correct version of Table 1 is shown in this correction.

BACKGROUND:Vascular endothelial growth factor-A (VEGF-A) is intensively investigated in various medical fields. However, comparing VEGF-A measurements is difficult because sample acquisition and pre-analytic procedures differ between studies. We therefore investigated which variables act as confounders of VEGF-A measurements. METHODS:Following a standardized protocol, blood was taken at three clinical sites from six healthy participants (one male and one female participant at each center) twice one week apart. The following pre-analytical parameters were varied in order to analyze their impact on VEGF-A measurements: analyzing center, anticoagulant (EDTA vs. PECT / CTAD), cannula (butterfly vs. neonatal), type of centrifuge (swing-out vs. fixed-angle), time before and after centrifugation, filling level (completely filled vs. half-filled tubes) and analyzing method (ELISA vs. multiplex bead array). Additionally, intrapersonal variations over time and sex differences were explored. Statistical analysis was performed using a linear regression model. RESULTS:The following parameters were identified as statistically significant independent confounders of VEGF-A measurements: analyzing center, anticoagulant, centrifuge, analyzing method and sex of the proband. The following parameters were no significant confounders in our data set: intrapersonal variation over one week, cannula, time before and after centrifugation and filling level of collection tubes. CONCLUSION:VEGF-A measurement results can be affected significantly by the identified pre-analytical parameters. We recommend the use of CTAD anticoagulant, a standardized type of centrifuge and one central laboratory using the same analyzing method for all samples.

Nonlinear systems are typically linearized to permit linear feedback control design, but, in some systems, the nonlinearities are so strong that their performance is called chaotic, and linear control designs can be rendered ineffective. One famous example is the van der Pol equation of oscillatory circuits. This study investigates the control design for the forced van der Pol equation using simulations of various control designs for iterated initial conditions. The results of the study highlight that even optimal linear, time-invariant (LTI) control is unable to control the nonlinear van der Pol equation, but idealized nonlinear feedforward control performs quite well after an initial transient effect of the initial conditions. Perhaps the greatest strength of ideal nonlinear control is shown to be the simplicity of analysis. Merely equate coefficients order-of-differentiation insures trajectory tracking in steady-state (following dissipation of transient effects of initial conditions), meanwhile the solution of the time-invariant linear-quadratic optimal control problem with infinite time horizon is needed to reveal constant control gains for a linear-quadratic regulator. Since analytical development is so easy for ideal nonlinear control, this article focuses on numerical demonstrations of trajectory tracking error.

PURPOSE: The aim of this study was to investigate prospectively whether MRI plaque imaging can identify patients with asymptomatic carotid artery stenosis who have an increased risk for future cerebral events. MRI plaque imaging allows categorization of carotid stenosis into different lesion types (I-VIII). Within these lesion types, lesion types IV-V and VI are regarded as rupture-prone plaques, whereas the other lesion types represent stable ones. METHODS: Eighty-three consecutive patients (45 male (54.2%); age 54-88 years (mean 73.2 years)) presenting with an asymptomatic carotid stenosis of 50-99% according to ECST-criteria were recruited. Patients were imaged with a 1.5-T scanner. T1-, T2-, time-of-flight-, and proton-density weighted studies were performed. The carotid plaques were classified as lesion type I-VIII. Clinical endpoints were ischemic stroke, TIA or amaurosis fugax. Survival analysis and log rank test were used to ascertain statistical significance. RESULTS: Six out of 83 patients (7.2%) were excluded: 4 patients had insufficient MR image quality; 1 patient was lost-to-follow-up; 1 patient died shortly after the baseline MRI plaque imaging. The following results were obtained by analyzing the remaining 77 patients. The mean time of follow-up was 41.1 months. During follow-up, n = 9 (11.7%) ipsilateral ischemic cerebrovascular events occurred. Only patients presenting with the high-risk lesion types IV-V and VI developed an ipsilateral cerebrovascular event versus none of the patients presenting with the stable lesion types III, VII, and VIII (n = 9 (11.7%) vs. n = 0 (0%) during follow-up). Event-free survival was higher among patients with the MRI-defined stable lesion types (III, VII, and VIII) than in patients with the high-risk lesion types (IV-V and VI) (log rank test P

Fractalkine (CX3CL1, FKN) is expressed in the inflamed vascular wall and absence of FKN reduces atherogenesis. Whether FKN is expressed throughout all stages of atherosclerotic disease and whether it directly contributes to monocyte recruitment to atherosclerotic lesions is not known. We collected human atherosclerotic plaque material and blood samples from patients with carotid artery disease undergoing endarterectomy. Plaques were analyzed by immunohistochemistry and qPCR. We found that FKN is expressed at all stages of atherosclerotic lesion formation, and that the number of FKN-expressing cells positively correlates with the number of CX3CR1-positive cells in human carotid artery plaques. In the circulation, soluble FKN levels are significantly elevated in the presence of high-grade (sub-occlusive) stenosis. To determine the role of the FKN-CX3CR1 axis for monocyte adhesion in vivo we then performed intravital videofluorescence microscopy of the carotid artery in ApoE(-/-) mice. Notably, FKN-CX3CR1 interactions are critical for recruitment of circulating monocytes to the injured atherosclerotic vascular wall. Thus, this chemokine dyad could represent an attractive target for anti-atherosclerotic strategies.