The two autophagy-related proteins 8a and 8b play distinct physiological roles in Drosophila.

Atg8 family proteins play crucial roles in autophagy to maintain cellular homeostasis. However, the physiological roles of Atg8 family proteins have not been systematically determined. In this study, we generated Atg8a and Atg8b (homologs of Atg8 in Drosophila melanogaster) knockout flies. We found that the loss of Atg8a affected autophagy and resulted in partial lethality, abnormal wings, decreased lifespan, and decreased climbing ability in flies. Furthermore, the loss of Atg8a resulted in reduced muscle integrity and the progressive degeneration of the neuron system. We also found that the phosphorylation at Ser88 of Atg8a is important for autophagy and neuronal integrity. The loss of Atg8b did not affect autophagy but induced male sterility in flies. Here, we take full advantage of the fly system to elucidate the physiological function of Atg8a and Atg8b in Drosophila.

Baricitinib protects ICIs-related myocarditis by targeting JAK1/STAT3 to regulate Macrophage polarization.

PURPOSE: The emergence of immune checkpoint inhibitors (ICIs) has revolutionized cancer treatment, but these drugs can also cause severe immune-related adverse effects (irAEs), including myocarditis. Researchers have become interested in exploring ways to mitigate this side effect, and one promising avenue is the use of baricitinib, a Janus kinase inhibitor known to have anti-inflammatory properties. This study aimed to examine the potential mechanism by which baricitinib in ICIs-related myocarditis. METHODS: To establish an ICIs-related myocarditis model, BALB/c mice were administered murine cardiac troponin I (cTnI) peptide and anti-mouse programmed death 1 (PD-1) antibodies. Subsequently, baricitinib was administered to the mice via intragastric administration. Echocardiography, HE staining, and Masson staining were performed to evaluate myocardial functions, inflammation, and fibrosis. Immunofluorescence was used to detect macrophages in the cardiac tissue of the mice.In vitro experiments utilized raw264.7 cells to induce macrophage polarization using anti-PD-1 antibodies. Different concentrations of baricitinib were applied to assess cell viability, and the release of pro-inflammatory cytokines was measured. The activation of the JAK1/STAT3 signaling pathway was evaluated through western blot analysis. RESULTS: Baricitinib demonstrated its ability to improve cardiac function and reduce cardiac inflammation, as well as fibrosis induced by ICIs. Mechanistically, baricitinib treatment promoted the polarization of macrophages towards the M2 phenotype. In vitro and in vivo experiments showed that anti-PD-1 promoted the release of inflammatory factors. However, treatment with baricitinib significantly inhibited the phosphorylation of JAK1 and STAT3. Additionally, the use of RO8191 reversed the effects of baricitinib, further confirming our findings. CONCLUSION: Baricitinib demonstrated its potential as a protective agent against ICIs-related myocarditis by modulating macrophage polarization. These findings provide a solid theoretical foundation for the development of future treatments for ICIs-related myocarditis.

Longitudinal antimicrobial susceptibility trends of canine Staphylococcus pseudintermedius.

Antimicrobial resistance within Staphylococcus pseudintermedius poses a significant risk for the treatment of canine pyoderma and as a reservoir for resistance and potential zoonoses, but few studies examine long-term temporal trends of resistance. This study assesses the antimicrobial resistance prevalence and minimum inhibitory concentration (MIC) trends in S. pseudintermedius (n=1804) isolated from canine skin samples at the Cornell University Animal Health Diagnostic Center (AHDC) between 2007 and 2020. Not susceptible (NS) prevalence, Cochran-Armitage tests, logrank tests, MIC50 and MIC90 quantiles, and survival analysis models were used to evaluate resistance prevalence and temporal trends to 23 antimicrobials. We use splines as predictors in accelerated failure time (AFT) models to model non-linear temporal trends in MICs. Multidrug resistance was common among isolates (47%), and isolates had moderate to high NS prevalence to the beta-lactams, chloramphenicol, the fluoroquinolones, gentamicin, the macrolides/lincosamides, the tetracyclines, and trimethoprim-sulfamethoxazole. However, low levels of NS to amikacin, rifampin, and vancomycin were observed. Around one third of isolates (38%) were found to be methicillin resistant S. pseudintermedius (MRSP), and these isolates had a higher prevalence of NS to all tested antimicrobials than methicillin susceptible isolates. Amongst the MRSP isolates, one phenotypically vancomycin resistant isolate (MIC >16 µg/mL) was identified, but genomic sequence data was unavailable. AFT models showed increasing MICs across time to the beta-lactams, chloramphenicol, the fluoroquinolones, gentamicin, and the macrolides/lincosamides, and decreasing temporal resistance (decreasing MICs) to doxycycline was observed amongst isolates. Notably, ATF modeling showed changes in MIC distributions that were not identified using Cochran-Armitage tests on prevalence, MIC quantiles, and logrank tests. Increasing resistance amongst these S. pseudintermedius isolates highlights the need for rational, empirical prescribing practices and increased antimicrobial resistance (AMR) surveillance to maintain the efficacy of current therapeutic agents. AFT models with non-linear predictors may be a useful, breakpoint-independent, surveillance tool alongside other modeling methods and antibiograms.

De novo GRIN variants in M3 helix associated with neurological disorders control channel gating of NMDA receptor.

N-methyl-D-aspartate receptors (NMDARs) are members of the glutamate receptor family and participate in excitatory postsynaptic transmission throughout the central nervous system. Genetic variants in GRIN genes encoding NMDAR subunits are associated with a spectrum of neurological disorders. The M3 transmembrane helices of the NMDAR couple directly to the agonist-binding domains and form a helical bundle crossing in the closed receptors that occludes the pore. The M3 functions as a transduction element whose conformational change couples ligand binding to opening of an ion conducting pore. In this study, we report the functional consequences of 48 de novo missense variants in GRIN1, GRIN2A, and GRIN2B that alter residues in the M3 transmembrane helix. These de novo variants were identified in children with neurological and neuropsychiatric disorders including epilepsy, developmental delay, intellectual disability, hypotonia and attention deficit hyperactivity disorder. All 48 variants in M3 for which comprehensive testing was completed produce a gain-of-function (28/48) compared to loss-of-function (9/48); 11 variants had an indeterminant phenotype. This supports the idea that a key structural feature of the M3 gate exists to stabilize the closed state so that agonist binding can drive channel opening. Given that most M3 variants enhance channel gating, we assessed the potency of FDA-approved NMDAR channel blockers on these variant receptors. These data provide new insight into the structure-function relationship of the NMDAR gate, and suggest that variants within the M3 transmembrane helix produce a gain-of-function.

Comparison of extraction processes, characterization and intestinal protection activity of Bletilla striata polysaccharides.

We optimized the extraction process of Bletilla striata polysaccharides using orthogonal design, Box-Behnken design (BBD), and genetic algorithm-back propagation (GA-BP), then compared and evaluated them to confirm that the combination of BBD and GA-BP neural networks was capable of increasing polysaccharide yields and antioxidant activity. The optimal extraction parameters were as follows: liquid-to-solid ratio of 15 mL/g, extraction power of 450 W, and extraction time of 34 min. Under these conditions, the polysaccharide yield and antioxidant activity were 8.29 ± 0.50 % and 26.20 ± 0.28 (mM FE/mg). Subsequently, the polysaccharide was purified to obtain purified Bletilla striata polysaccharides 1 (pBSP1) with a Mw of 255.172 kDa. Scanning electron microscope (SEM), ultraviolet-visible detector (UV), fourier transform infrared spectrometer (FTIR), high performance liquid chromatography (HPLC), X-ray diffraction (XRD), nuclear magnetic resonance (NMR) and periodate oxidation were used to analyze the structure of pBSP1. The results showed pBSP1 had a smooth surface and a rough interior, with a composition of α-D conformation glucose (18.23 %) and ß-D conformation mannose (53.77 %), and an amorphous crystal structure. According to the results of thermogravimetric and rheological tests, pBSP1 exhibits good thermal stability and viscoelastic behavior. Furthermore, pBSP1 protected lipopolysaccharide (LPS)-induced GES - 1 and Caco2 cells, the results showed pBSP1(400 µg/mL) lowered TEER synthesis in Caco2 cells as well as apoptosis and reactive oxygen species (ROS) production in both cells, indicating that pBSP1 may have an intestine protective effect.

Mn-Doped M2CdCl4 (M = CH3NH3+, C2H8N+, and C3H10N+) Layered Hybrid Perovskite and Its Flexible Film Based on Simple Mechanochemical Synthesis.

Layered hybrid perovskites show significant advantages in the field of optoelectronics. However, the low quantum efficiency and complex preparation methods limit their applications. In this work, we developed a series of perovskite powders with a two-dimensional (2D) layered structure of organic-inorganic hybrid metal halides M2CdCl4:x%Mn (M = CH3NH3+, C2H8N+, C3H10N+) via facile mechanochemical methods. The prepared manganese Mn-doped MA2CdCl4 produces orange emission at 605 nm under both 254 and 420 nm excitation, which originates from a dual excitation channel competition mechanism, and its excitation channel could be changed with the increase of Mn2+ ion concentration. Typically, MA2CdCl4:20%Mn powder exhibits high photoluminescence quantum yield (PLQY) close to 90% at 605 nm due to the organic amine ions enlarging the Mn-Mn interlayer distances. In addition, we prepared MA2CdCl4:x%Mn@PVA flexible films, which also exhibit good luminescence at 254 nm excitation and were unexpectedly found to have a better response to Cs+, which could be a candidate for anticounterfeiting applications.

Scalable Anatomically-Tunable Fully In-Ear Dry-Electrode Array for User-Generic Unobtrusive Electrophysiology.

Traditional scalp EEG instrumentation is bulky and arduous to set up, requiring wires that constrain the subject's movement, conductive wet gels that dry over time which limits long-term recording, and/or is socially stigmatized. Therefore, there is growing research in in-ear EEG to increase user wearability, ease of use, and concealability. However, the fabrication of in-ear EEG sensors utilizes complex equipment and materials to capture the intricate geometry of the ear and to fabricate custom earpieces and electrodes. This work aims to lower the barrier of entry by decreasing the fabrication complexity by using PCB components with versatile, user-generic designs. Measured results on the assembled earpiece demonstrate that it viably captures eye blinks, jaw clench, auditory steady-state response (ASSR), and alpha modulation. Additionally, electrochemical impedance spectroscopy (EIS) experiments show reliable electrode-skin contact with impedance comparable to conventional dry-electrode designs at substantially greater channel density.

A Machine Learning Approach to COVID-19 Detection via Graphene Field-Effect-Transistor (GFET).

In the wake of the COVID-19 pandemic, there has been a need for reliable diagnostic testing. However, state-of-the-art detection methods rely on laboratory tests and also vary in accuracy. We evaluate that the usage of a graphene field-effect-transistor (GFET) coupled with machine learning can be a promising alternate diagnostic testing method. We processed the current-voltage data gathered from the GFET sensors to assess information about the presence of COVID-19 in biosamples. We perform binary classification using the following machine learning algorithms: Linear Discriminant Analysis (LDA), Support Vector Machines (SVM) with the Radial Basis Function (RBF) kernel, and K-Nearest Neighbors (KNN) in conjunction with Principal Component Analysis (PCA). We find that LDA and SVM with RBF proved to be the most accurate in identifying positive and negative samples, with accuracies of 99% and 98.5%, respectively. Based on these results, there is promise to develop a bioelectronic diagnostic method for COVID-19 detection by combining GFET technology with machine learning.

Clinical and functional consequences of GRIA variants in patients with neurological diseases.

AMPA receptors are members of the glutamate receptor family and mediate a fast component of excitatory synaptic transmission at virtually all central synapses. Thus, their functional characteristics are a critical determinant of brain function. We evaluate intolerance of each GRIA gene to genetic variation using 3DMTR and report here the functional consequences of 52 missense variants in GRIA1-4 identified in patients with various neurological disorders. These variants produce changes in agonist EC50, response time course, desensitization, and/or receptor surface expression. We predict that these functional and localization changes will have important consequences for circuit function, and therefore likely contribute to the patients' clinical phenotype. We evaluated the sensitivity of variant receptors to AMPAR-selective modulators including FDA-approved drugs to explore potential targeted therapeutic options.

Development and validation of a nomogram model for prediction of stroke-associated pneumonia associated with intracerebral hemorrhage.

BACKGROUND: We aimed to establish risk factors for stroke-associated pneumonia (SAP) following intracerebral hemorrhage (ICH) and develop an efficient and convenient model to predict SAP in patients with ICH. METHODS: Our study involved 1333 patients consecutively diagnosed with ICH and admitted to the Neurology Department of the First Affiliated Hospital of Wenzhou Medical University. The 1333 patients were randomly divided (3:1) into the derivation cohort (n = 1000) and validation Cohort (n = 333). Variables were screened from demographics, lifestyle-related factors, comorbidities, clinical symptoms, neuroimaging features, and laboratory tests. In the derivation cohort, we developed a prediction model with multivariable logistic regression analysis. In the validation cohort, we assessed the model performance and compared it to previously reported models. The area under the receiver operating characteristic curve (AUROC), GiViTI calibration belt, net reclassification index (NRI), integrated discrimination index (IDI) and decision curve analysis (DCA) were used to assess the prediction ability and the clinical decision-making ability. RESULTS: The incidence of SAP was 19.9% and 19.8% in the derivation (n = 1000) and validation (n = 333) cohorts, respectively. We developed a nomogram prediction model including age (Odds Ratio [OR] 1.037, 95% confidence interval [CI] 1.020-1.054), male sex (OR 1.824, 95% CI 1.206-2.757), multilobar involvement (OR 1.851, 95% CI 1.160-2.954), extension into ventricles (OR 2.164, 95% CI 1.456-3.215), dysphagia (OR 3.626, 95% CI 2.297-5.725), disturbance of consciousness (OR 2.113, 95% CI 1.327-3.362) and total muscle strength of the worse side (OR 0.93, 95% CI 0.876-0.987). Compared with previous models, our model was well calibrated and showed significantly higher AUROC, better reclassification ability (improved NRI and IDI) and a positive net benefit for predicted probability thresholds between 10% and 73% in DCA. CONCLUSIONS: We developed a simple, valid, and clinically useful model to predict SAP following ICH, with better predictive performance than previous models. It might be a promising tool to assess the individual risk of developing SAP for patients with ICH and optimize decision-making.

A 3D-Printed Dual Driving Forces Scaffold with Self-Promoted Cell Absorption for Spinal Cord Injury Repair.

Stem cells play critical roles in cell therapies and tissue engineering for nerve repair. However, achieving effective delivery of high cell density remains a challenge. Here, a novel cell delivery platform termed the hyper expansion scaffold (HES) is developed to enable high cell loading. HES facilitated self-promoted and efficient cell absorption via a dual driving force model. In vitro tests revealed that the HES rapidly expanded 80-fold in size upon absorbing 2.6 million human amniotic epithelial stem cells (hAESCs) within 2 min, representing over a 400% increase in loading capacity versus controls. This enhanced uptake benefited from macroscopic swelling forces as well as microscale capillary action. In spinal cord injury (SCI) rats, HES-hAESCs promoted functional recovery and axonal projection by reducing neuroinflammation and improving the neurotrophic microenvironment surrounding the lesions. In summary, the dual driving forces model provides a new rationale for engineering hydrogel scaffolds to facilitate self-promoted cell absorption. The HES platform demonstrates great potential as a powerful and efficient vehicle for delivering high densities of hAESCs to promote clinical treatment and repair of SCI.

Removal of broken screws on implant abutment by digital guide plate: A case report and literature review.

Implant restoration is currently the most mainstream method for repairing missing teeth. With the increasing number of plantings, various planting complications begin to be paid attention to. Among them, there are many reports of disability phenomena such as loose and broken abutment screws and broken top screws, which cause the implant to fail or fail to function. In recent years, with the development of computer-aided software and its application in the field of oral treatment, digital guide plates based on 3D printing of oral CBCT scanning data are widely used in oral implants. Therefore, we explore the application prospect of post-core crown restoration after removing broken screws from the implant abutment with a digital guide plate. We reported a case of upper right first molar implant abutment screws broken, which were removed by a digital guide plate and customized turning bur. The resin-matrix ceramics crown post core was prepared, and then the occlusal force was tested by the T-ScanIII system. It provides a reference for the application of digital guide plates in special cases such as broken screws of implant abutment.

Anti-osteoporosis mechanism of resistance exercise in ovariectomized rats based on transcriptome analysis: a pilot study.

Postmenopausal osteoporosis is the main cause of fractures in women. Resistance exercise has a positive effect on bone mineral density in postmenopausal osteoporosis patients, but its mechanism is unclear. The purpose of this study was to explore the mechanism of resistance exercise in improving ovariectomized osteoporotic rats based on the transcriptome sequencing technique. Eighteen female Sprague-Dawley rats were randomly divided into the sham-operated group, the non-exercise group, and the resistance exercise group. The rat model of postmenopausal osteoporosis was established by bilateral ovariectomy. Ten weeks after the operation, the resistance exercise group received 2 weeks of adaptive training, and 12 weeks of resistance exercise began in the 13th week. The rats were trained 5 days per week, in 4 sets of 3 repetitions per day. After the intervention, all rats were sacrificed, and the body weight, bone mineral density, trabecular bone microarchitecture, and bone biomechanics were examined. At the same time, RNA-seq and enrichment analysis of gene ontology and Kyoto Encyclopedia of Genes and Genomes were performed on the left tibias, followed by Elisa and RT-qPCR verification. It had been found that resistance exercise can effectively counteract the weight gain of ovariectomized osteoporotic rats, and has a good effect on bone mineral density and trabecular bone microarchitecture. Enrichment analysis showed that regulation of gene expression and osteoclast differentiation is the most closely related biological process and signaling pathway shared by RE/Ovx and NE/Ovx groups. Our results revealed that resistance exercise can play a role in inhibiting osteoclast activation and preventing the enhancement of osteoclast bone resorption function in ovariectomized osteoporotic rats by inhibiting Fos/Fosb-regulated TRAP activation and relieving Calcr inhibition, which has important application value in preventing bone loss caused by estrogen deficiency.

In-ear integrated sensor array for the continuous monitoring of brain activity and of lactate in sweat.

Owing to the proximity of the ear canal to the central nervous system, in-ear electrophysiological systems can be used to unobtrusively monitor brain states. Here, by taking advantage of the ear's exocrine sweat glands, we describe an in-ear integrated array of electrochemical and electrophysiological sensors placed on a flexible substrate surrounding a user-generic earphone for the simultaneous monitoring of lactate concentration and brain states via electroencephalography, electrooculography and electrodermal activity. In volunteers performing an acute bout of exercise, the device detected elevated lactate levels in sweat concurrently with the modulation of brain activity across all electroencephalography frequency bands. Simultaneous and continuous unobtrusive in-ear monitoring of metabolic biomarkers and brain electrophysiology may allow for the discovery of dynamic and synergetic interactions between brain and body biomarkers in real-world settings for long-term health monitoring or for the detection or monitoring of neurodegenerative diseases.

A Fast and Effective Spike Sorting Method Based on Multi-Frequency Composite Waveform Shapes.

Accurate spike sorting to the appropriate neuron is crucial for neural activity analysis. To improve spike sorting performance, it is essential to fully leverage each processing step, including filtering, spike detection, feature extraction, and clustering. However, compared to the latter two steps that were widely studied and optimized, the filtering process was largely neglected. In this study, we proposed a fast and effective spike sorting method (MultiFq) based on multi-frequency composite waveform shapes acquired through an optimized filtering process. When combined with the classical PCA-Km spiking sorting algorithm, our proposed MultiFq significantly improved its sorting performance and achieved as high performance as the complex Wave-clus did in both the simulated and in vivo datasets. But, the combined method was about 10 times faster than Wave-clus (0.16 s vs. 2.06 s in simulated datasets; 0.46 s vs. 2.03 s in in vivo datasets). Furthermore, we demonstrated the compatibility of our MultiFq by combining it with other sorting algorithms, which consistently resulted in significant improvement in sorting accuracy with the maximum improvement at 35.04%. The above results demonstrated that our proposed method could significantly improve the sorting performance with low computation cost and good compatibility by leveraging the multi-frequency composite waveform shapes.

Differential responses of disease-related GRIN variants located in pore-forming M2 domain of N-methyl-D-aspartate receptor to FDA-approved inhibitors.

N-methyl-D-aspartate receptors (NMDAR), ionotropic glutamate receptors, mediate a slow component of excitatory synaptic transmission in the central nervous system and play a key role in normal brain function and development. Genetic variations in GRIN genes encoding NMDAR subunits that alter the receptor's functional characteristics are associated with a wide range of neurological and neuropsychiatric conditions. Pathological GRIN variants located in the M2 re-entrant loop lining the channel pore cause significant functional changes, the most consequential alteration being a reduction in voltage-dependent Mg2+ inhibition. Voltage-dependent Mg2+ block is a unique feature of NMDAR biology whereby channel activation requires both ligand binding and postsynaptic membrane depolarization. Thus, loss of NMDAR Mg2+ block will have a profound impact on synaptic function and plasticity. Here, we choose 11 missense variants within the GRIN1, GRIN2A, and GRIN2B genes that alter residues located in the M2 loop and significantly reduce Mg2+ inhibition. Each variant was evaluated for tolerance to genetic variation using the 3-dimensional structure and assessed for functional rescue pharmacology via electrophysiological recordings. Three FDA-approved NMDAR drugs-memantine, dextromethorphan, and ketamine-were chosen based on their ability to bind near the M2 re-entrant loop, potentially rectifying dysregulated NMDAR function by supplementing the reduced voltage-dependent Mg2+ block. These results provide insight of structural determinants of FDA-approved NMDAR drugs at their binding sites in the channel pore and may further define conditions necessary for the use of such agents as potential rescue pharmacology.

Genome-Wide Association Study Identifies a Plant-Height-Associated Gene OsPG3 in a Population of Commercial Rice Varieties.

Plant height is one of the most crucial components of plant structure. However, due to its complexity, the genetic architecture of rice plant height has not been fully elucidated. In this study, we performed a genome-wide association study (GWAS) to determine rice plant height using 178 commercial rice varieties and identified 37 loci associated with rice plant height (LAPH). Among these loci, in LAPH2, we identified a polygalacturonase gene, OsPG3, which was genetically and functionally associated with rice plant height. The rice plant exhibits a super dwarf phenotype when the knockout of the OsPG3 gene occurs via CRISPR-Cas9 gene-editing technology. RNA-Seq analysis indicated that OsPG3 modulates the expression of genes involved in phytohormone metabolism and cell-wall-biosynthesis pathways. Our findings suggest that OsPG3 plays a vital role in controlling rice plant height by regulating cell wall biosynthesis. Given that rice architecture is one of the most critical phenotypes in rice breeding, OsPG3 has potential in rice's molecular design breeding toward an ideal plant height.

One-step hydrothermal generation of oxygen-deficient N-doped blue TiO2-Ti3C2 for degradation of pollutants and antibacterial properties.

In this study, TiO2 was generated in situ on the surface of Ti3C2 by a hydrothermal process, and urea was added to form N-doped TiO2-Ti3C2. The surface morphology and functional group properties of the prepared materials were analyzed by SEM, TEM, XRD, XPS, etc. The results showed that anatase TiO2 formed on the surface of the Ti3C2 monolayer. Nitrogen-doped nanomaterials show good phenol degradation and good recyclability under visible light. At a urea content of 0.5 g, the photocatalytic degradation of phenol under visible light is best, reaching 88.9% in 3 h, with ·OH and ·O2- holes playing the leading role. However, at lower pH and higher ion concentration, the degradability of N-TiO2-Ti3C2 for phenol is reduced. Furthermore, the material prepared in this work is a two-dimensional layered material, and the adsorption of phenol best fits the Langmuir adsorption isotherm model and the pseudo-second-order kinetic equation. In terms of the antibacterial performance of the material, the N-doped TiO2-Ti3C2 nanomaterial made with 0.2 g of urea has an Escherichia coli scavenging efficiency of about 97.86%, which is an excellent antibacterial material. This study shows that the N-TiO2-Ti3C2 produced in this experiment can be used for environmental applications.

Addressing the Embeddability Problem in Transition Rate Estimation.

Markov State Models (MSM) and related techniques have gained significant traction as a tool for analyzing and guiding molecular dynamics (MD) simulations due to their ability to extract structural, thermodynamic, and kinetic information on proteins using computationally feasible MD simulations. The MSM analysis often relies on spectral decomposition of empirically generated transition matrices. This work discusses an alternative approach for extracting the thermodynamic and kinetic information from the so-called rate/generator matrix rather than the transition matrix. Although the rate matrix itself is built from the empirical transition matrix, it provides an alternative approach for estimating both thermodynamic and kinetic quantities, particularly in diffusive processes. A fundamental issue with this approach is known as the embeddability problem. The key contribution of this work is the introduction of a novel method to address the embeddability problem as well as the collection and utilization of existing algorithms previously used in the literature. The algorithms are tested on data from a one-dimensional toy model to show the workings of these methods and discuss the robustness of each method in dependence of lag time and trajectory length.