The profile of genome-wide DNA methylation, transcriptome, and proteome in streptomycin-resistant Mycobacterium tuberculosis.

Streptomycin-resistant (SM-resistant) Mycobacterium tuberculosis (M. tuberculosis) is a major concern in tuberculosis (TB) treatment. However, the mechanisms underlying streptomycin resistance remain unclear. This study primarily aimed to perform preliminary screening of genes associated with streptomycin resistance through conjoint analysis of multiple genomics. Genome-wide methylation, transcriptome, and proteome analyses were used to elucidate the associations between specific genes and streptomycin resistance in M. tuberculosis H37Rv. Methylation analysis revealed that 188 genes were differentially methylated between the SM-resistant and normal groups, with 89 and 99 genes being hypermethylated and hypomethylated, respectively. Furthermore, functional analysis revealed that these 188 differentially methylated genes were enriched in 74 pathways, with most of them being enriched in metabolic pathways. Transcriptome analysis revealed that 516 genes were differentially expressed between the drug-resistant and normal groups, with 263 and 253 genes being significantly upregulated and downregulated, respectively. KEGG analysis indicated that these 516 genes were enriched in 79 pathways, with most of them being enriched in histidine metabolism. The methylation level was negatively related to mRNA abundance. Proteome analysis revealed 56 differentially expressed proteins, including 14 upregulated and 42 downregulated proteins. Moreover, three hub genes (coaE, fadE5, and mprA) were obtained using synthetic analysis. The findings of this study suggest that an integrated DNA methylation, transcriptome, and proteome analysis can provide important resources for epigenetic studies in SM-resistant M. tuberculosis H37Rv.

Comparison of the MeltPro TB assay and whole-genome sequencing assay for rapid molecular diagnosis of drug resistant tuberculosis in guangdong province.

BACKGROUND: Rifampicin (RIF) and multidrug-resistant tuberculosis (TB) are major public health threats. As conventional phenotypic drug susceptibility testing requires two-eight weeks, molecular diagnostic assays are widely used to determine drug resistance. METHODS: Clinical Mycobacterium tuberculosis isolates with consistent drug susceptibility results, tested using microbroth dilution and proportion methods in Löwenstein-Jensen medium from patients with TB in Guangdong province were utilized to evaluate MeltPro TB and whole-genome sequencing (WGS) assays in detecting resistance to RIF, isoniazid (INH), ethambutol (EMB), fluoroquinolones (FQ), and streptomycin (SM). Solid phenotypic drug susceptibility testing was used as the gold standard to evaluate the detection capacity of MeltPro TB on clinical sputum samples of patients with TB. RESULTS: Similar to WGS, MeltPro TB successfully detected RIF, INH, and SM resistance with sensitivities of 86.3, 84.8, and 86.6 %, respectively. However, the resistant isolate detection rates were only 58.1 and 69.6 % for EMB and FQ-resistant strains. For clinical specimens, MeltPro TB still showed good detectable rates of RIF and INH resistance, with sensitivities of 82.4 % and 95.2 %, respectively. Detectable rates of FQ and EMB resistance were low: 77.8 % and 35.3 %, respectively. CONCLUSIONS: MeltPro TB can detect known DNA mutations associated with drug resistance in Mycobacterium tuberculosis strains with comparable efficacy to WGS. For FQ and EMB resistance testing, MeltPro TB requires optimization and is unsuitable for general use. MeltPro TB can be used for diagnosis of RIF and multidrug-resistant tuberculosis to rapidly initiate appropriate anti-TB drug therapy.

Enhancing Faraday and Kerr rotations based on the toroidal dipole mode in an all-dielectric magneto-optical metasurface.

The magneto-optical Faraday and Kerr effects are widely used in modern optical devices. In this Letter, we propose an all-dielectric metasurface composed of perforated magneto-optical thin films, which can support the highly confined toroidal dipole resonance and provide full overlap between the localized electromagnetic field and the thin film, and consequently enhance the magneto-optical effects to an unprecedented degree. The numerical results based on the finite element method show that the Faraday and Kerr rotations can reach -13.59° and 8.19° in the vicinity of toroidal dipole resonance, which are 21.2 and 32.8 times stronger than those in the equivalent thickness of thin films. In addition, we design an environment refractive index sensor based on the resonantly enhanced Faraday and Kerr rotations, with sensitivities of 62.96 nm/RIU and 73.16 nm/RIU, and the corresponding maximum figures of merit 132.22°/RIU and 429.45°/RIU, respectively. This work provides a new, to the best of our knowledge, strategy for enhancing the magneto-optical effects at nanoscale, and paves the way for the research and development of magneto-optical metadevices such as sensors, memories, and circuits.

Vector maps and spatial autocorrelation of carbon emissions at land patch level based on multi-source data.

An accurate carbon emissions map is of great significance for urban planning to reduce carbon emissions, mitigate the heat island effect, and avoid the impact of high temperatures on human health. However, little research has focused on carbon emissions maps at the land patch level, which makes poor integration with small and medium-sized urban planning based on land patches. In this study, a vectorization method for spatial allocation of carbon emissions at the land patch level was proposed. The vector maps and spatial autocorrelation of carbon emissions in Zhangdian City, China were explored using multi-source data. In addition, the differences between different streets were analyzed, and the carbon emissions ratio of the land patch was compared. The results show that the vector carbon emissions map can help identify the key carbon reduction land patches and the impact factors of carbon emissions. The vector maps of Zhangdian City show that in 2021, the total carbon emissions and carbon absorptions were 4.76 × 109kg and 4.28 × 106kg respectively. Among them, industrial land accounted for 70.16% of carbon emissions, mainly concentrated in three industrial towns. Forest land carbon absorption accounted for 98.56%, mainly concentrated in the peripheral streets away from urban areas. The Moran's I of land patch level carbon emissions was 0.138, showing a significant positive spatial correlation. The proportion of land patches is an important factor in determining carbon emissions, and the adjustment of industrial structure is the most critical factor in reducing carbon emissions. The results achieved can better help governments develop different carbon reduction strategies, mitigate the heat island effect, and support low-carbon and health-oriented urban planning.

Manipulating the optical beam width in topological pseudospin-dependent waveguides using all-dielectric photonic crystals.

We propose a width-tunable topological pseudospin-dependent waveguide (TPDW) which can manipulate the optical beam width using a heterostructure of all-dielectric photonic crystals (PhCs). The heterostructure can be realized by introducing a PhC featuring double Dirac cones into the other two PhCs with different topological indices. The topological pseudospin-dependent waveguide states (TPDWSs) achieved from the TPDW exhibit unidirectional transport and immunity against defects. As a potential application of our work, using these characteristics of TPDWSs, we further design a topological pseudospin-dependent beam expander which can expand a narrow beam into a wider one at the communication wavelength of 1.55 µm and is robust against three kinds of defects. The proposed TPDW with widely adjustable width can better dock with other devices to achieve stable and efficient transmission of light. Meanwhile, all-dielectric PhCs have negligible losses at optical wavelengths, which provides the prospect of broad application in photonic integrated devices.

mbtD and celA1 association with ethambutol resistance in Mycobacterium tuberculosis: A multiomics analysis.

Ethambutol (EMB) is a first-line antituberculosis drug currently being used clinically to treat tuberculosis. Mutations in the embCAB operon are responsible for EMB resistance. However, the discrepancies between genotypic and phenotypic EMB resistance have attracted much attention. We induced EMB resistance in Mycobacterium tuberculosis in vitro and used an integrated genome-methylome-transcriptome-proteome approach to study the microevolutionary mechanism of EMB resistance. We identified 509 aberrantly methylated genes (313 hypermethylated genes and 196 hypomethylated genes). Moreover, some hypermethylated and hypomethylated genes were identified using RNA-seq profiling. Correlation analysis revealed that the differential methylation of genes was negatively correlated with transcription levels in EMB-resistant strains. Additionally, two hypermethylated candidate genes (mbtD and celA1) were screened by iTRAQ-based quantitative proteomics analysis, verified by qPCR, and corresponded with DNA methylation differences. This is the first report that identifies EMB resistance-related genes in laboratory-induced mono-EMB-resistant M. tuberculosis using multi-omics profiling. Understanding the epigenetic features associated with EMB resistance may provide new insights into the underlying molecular mechanisms.

Clostridium, Bacteroides and Prevotella associates with increased fecal metabolites Trans-4-Hydroxy-L-proline and Genistein in active pulmonary tuberculosis patients during anti-tuberculosis chemotherapy with isoniazid-rifampin-pyrazinamide-ethambutol (HRZE).

Purpose: To investigated the changes of gut microbiome and fecal metabolome during anti-tuberculosis chemotherapy with isoniazid (H)-rifampin (R)-pyrazinamide (Z)-ethambutol (E). Patients and methods: (1) In this study, we recruited 168 stool specimens from 49 healthy volunteers without M. tuberculosis (Mtb), 30 healthy volunteers with latently infected by Mtb, 41 patients with active tuberculosis (ATB), 28 patients with 2-month HRZE treatment and 20 patients with 2-month HRZE followed by 4-month HR treatment. (2) We used 16S rRNA sequencing and an untargeted Liquid Chromatograph Mass Spectrometer-based metabolomics to investigate the changes of gut microbiome and the alteration of fecal metabolome, respectively, during anti-TB chemotherapy. Results: Mtb infection can reduce the diversity of intestinal flora of ATB patients and change their taxonomic composition, while the diversity of intestinal flora of ATB patients were restored during anti-TB chemotherapy. Especially, family Veillonellacea and Bateroidaceae and their genera Veillonella and Bacteroides significantly increased in the gut microbiota during anti-TB chemotherapy. Additionally, Mtb infection dynamically regulates fecal metabolism in ATB patients during anti-TB chemotherapy. Interestingly, the altered abundance of fecal metabolites correlated with the altered gut microbiota, especially the change of gut Clostridium, Bacteroides and Prevotella was closely related to the change of fecal metabolites such as Trans-4-Hydroxy-L-proline and Genistein caused by Mtb infection or anti-TB chemotherapy. Conclusion: Anti-TB chemotherapy with HRZE can disrupt both gut microbiotas and metabolome in ATB patients. Some specific genera and metabolites are depleted or enriched during anti-TB chemotherapy. Therefore, revealing potential relevance between gut microbiota and anti-TB chemotherapy will provide potential biomarkers for evaluating the therapeutic efficacy in ATB patients. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-022-01003-2.

Structural and mechanistic insights into the cleavage of clustered O-glycan patches-containing glycoproteins by mucinases of the human gut.

Mucinases of human gut bacteria cleave peptide bonds in mucins strictly depending on the presence of neighboring O-glycans. The Akkermansia muciniphila AM0627 mucinase cleaves specifically in between contiguous (bis) O-glycans of defined truncated structures, suggesting that this enzyme may recognize clustered O-glycan patches. Here, we report the structure and molecular mechanism of AM0627 in complex with a glycopeptide containing a bis-T (Galß1-3GalNAcα1-O-Ser/Thr) O-glycan, revealing that AM0627 recognizes both the sugar moieties and the peptide sequence. AM0627 exhibits preference for bis-T over bis-Tn (GalNAcα1-O-Ser/Thr) O-glycopeptide substrates, with the first GalNAc residue being essential for cleavage. AM0627 follows a mechanism relying on a nucleophilic water molecule and a catalytic base Glu residue. Structural comparison among mucinases identifies a conserved Tyr engaged in sugar-π interactions in both AM0627 and the Bacteroides thetaiotaomicron BT4244 mucinase as responsible for the common activity of these two mucinases with bis-T/Tn substrates. Our work illustrates how mucinases through tremendous flexibility adapt to the diversity in distribution and patterns of O-glycans on mucins.

Enhancement of Casimir Friction between Graphene-Covered Topological Insulator.

Casimir friction is theoretically studied between graphene-covered undoped bismuth selenide (Bi2Se3) in detail. In the graphene/Bi2Se3 composite structure, the coupling of the hyperbolic phonon polaritons supported by Bi2Se3 with the surface plasmons supported by graphene can lead to the hybrid surface plasmon-phonon polaritons (SPPPs). Compared with that between undoped Bi2Se3, Casimir friction can be enhanced by more than one order of magnitude due to the contribution of SPPPs. It is found that the chemical potential that can be used to modulate the optical characteristic of SPPPs plays an important role in Casimir friction. In addition, the Casimir friction between doped Bi2Se3 is also studied. The friction coefficient between doped Bi2Se3 can even be larger than that between graphene-covered undoped Bi2Se3 for suitable chemical potential due to the contribution of unusual electron surface states. The results obtained in this work are not only beneficial to the study of Casimir frictions but also extend the research ranges of topological insulators.

Enhancement of lateral Casimir force on a rotating particle near hyperbolic metamaterial.

Enhancement of weak Casimir forces is extremely important for their practical detection and subsequent applications in variety of scientific and technological fields. We study the lateral Casimir forces acting on the rotating particles with small radius of 50 nm as well as that with large radius of 500 nm near the hyperbolic metamaterial made of silicon carbide (SiC) nanowires. It is found that the lateral Casimir force acting on the small particle of 50 nm near hyperbolic metamaterial with appropriate filling fraction can be enhanced nearly four times comparing with that acting on the same particle near SiC bulk in the previous study. Such enhancement is caused by the coupling between the resonance mode excited by nanoparticle and the hyperbolic mode supported by hyperbolic metamaterial. The results obtained in this study provide an efficient method to enhance the interaction of nanoscale objects.

Dirac cones with zero refractive indices in phoxonic crystals.

In this paper, simultaneous zero refractive indices (ZRIs) for both sound and light are realized on the basis of a 2D triangular lattice phoxonic crystal (PxC) with C6v symmetry. For the phononic mode, accidental phononic Dirac degeneracy at the center of Brillouin zone (BZ) occurs at a relatively high frequency which leads to the failure of the efficient medium theory; hence, it is no longer applicable to the realization of acoustic ZRI. We thus turn to a low-frequency phononic Dirac cone located at K point, the corner of the BZ, which shows in-phase pressure field oscillations in expanded unit cells. Using zone folding, we further reveal the cause for the characteristic of acoustic ZRI. For the photonic mode, a low-frequency photonic Dirac-like cone can be achieved by adjusting the geometric parameter due to the high contrast permittivity between scatterers and the matrix. When the phononic and photonic low-frequency Dirac dispersions coexist, the PxC can be mapped into a zero-index material for both sound and light at the same time. The new mechanism for simultaneously controlling sound and light helps to achieve acousto-optic synchronous cloaking and unidirectional transmission, which are numerically demonstrated.

Evaluation of an artificial intelligence (AI) system to detect tuberculosis on chest X-ray at a pilot active screening project in Guangdong, China in 2019.

BACKGROUND: Although computer-aided detection (CAD) software employed with Artificial Intelligence (AI) system has been developed aiming to assist tuberculosis (TB) triage, screening, and diagnosis, its clinical performance for tuberculosis screening remains unknown. OBJECTIVE: To evaluate performance of an CAD software for detecting TB on chest X-ray images at a pilot active TB screening project. METHODS: A CAD software scheme employed with AI was used to screen chest X-ray images of participants and produce probability scores of cases being positive for TB. CAD-generated TB detection scores were compared with on-site and senior radiologists via several performance evaluation indices including area under the ROC curves (AUC), specificity, sensitive, and positive predict value. Pycharm CE and SPSS statistics software packages were used for data analysis. RESULTS: Among 2,543 participants, eight TB patients were identified from this screening pilot program. The AI-based CAD system outperformed the onsite (AUC = 0.740) and senior radiologists (AUC = 0.805) either using thresholds of 30% (AUC = 0.978) and 50% (AUC = 0.859) when taking the final diagnosis as the ground truth. CONCLUSIONS: The AI-based CAD software successfully detects all TB patients as identified from this study at a threshold of 30%. It demonstrates feasibility and easy accessibility to carry out large scale TB screening using this CAD software equipped in medical vans with chest X-ray imaging machine.

Strong coupling between excitons and magnetic dipole quasi-bound states in the continuum in WS2-TiO2 hybrid metasurfaces.

Enhancing the light-matter interactions in two-dimensional materials via optical metasurfaces has attracted much attention due to its potential to enable breakthrough in advanced compact photonic and quantum information devices. Here, we theoretically investigate a strong coupling between excitons in monolayer WS2 and quasi-bound states in the continuum (quasi-BIC). In the hybrid structure composed of WS2 coupled with asymmetric titanium dioxide nanobars, a remarkable spectral splitting and typical anticrossing behavior of the Rabi splitting can be observed, and such strong coupling effect can be modulated by shaping the thickness and asymmetry parameter of the proposed metasurfaces, and the angle of incident light. It is found that the balance of line width of the quasi-BIC mode and local electric field enhancement should be considered since both of them affect the strong coupling, which is crucial to the design and optimization of metasurface devices. This work provides a promising way for controlling the light-matter interactions in strong coupling regime and opens the door for the future novel quantum, low-energy, distinctive nanodevices by advanced meta-optical engineering.

The N-terminal Helix-Turn-Helix Motif of Transcription Factors MarA and Rob Drives DNA Recognition.

DNA-binding proteins play an important role in gene regulation and cellular function. The transcription factors MarA and Rob are two homologous members of the AraC/XylS family that regulate multidrug resistance. They share a common DNA-binding domain, and Rob possesses an additional C-terminal domain that permits binding of low-molecular weight effectors. Both proteins possess two helix-turn-helix (HTH) motifs capable of binding DNA; however, while MarA interacts with its promoter through both HTH-motifs, prior studies indicate that Rob binding to DNA via a single HTH-motif is sufficient for tight binding. In the present work, we perform microsecond time scale all-atom simulations of the binding of both transcription factors to different DNA sequences to understand the determinants of DNA recognition and binding. Our simulations characterize sequence-dependent changes in dynamical behavior upon DNA binding, showcasing the role of Arg40 of the N-terminal HTH-motif in allowing for specific tight binding. Finally, our simulations demonstrate that an acidic C-terminal loop of Rob can control the DNA binding mode, facilitating interconversion between the distinct DNA binding modes observed in MarA and Rob. In doing so, we provide detailed molecular insight into DNA binding and recognition by these proteins, which in turn is an important step toward the efficient design of antivirulence agents that target these proteins.

A subcellular map of the human kinome.

The human kinome comprises 538 kinases playing essential functions by catalyzing protein phosphorylation. Annotation of subcellular distribution of the kinome greatly facilitates investigation of normal and disease mechanisms. Here, we present Kinome Atlas (KA), an image-based map of the kinome annotated to 10 cellular compartments. 456 epitope-tagged kinases, representing 85% of the human kinome, were expressed in HeLa cells and imaged by immunofluorescent microscopy under a similar condition. KA revealed kinase family-enriched subcellular localizations and discovered a collection of new kinase localizations at mitochondria, plasma membrane, extracellular space, and other structures. Furthermore, KA demonstrated the role of liquid-liquid phase separation in formation of kinase condensates. Identification of MOK as a mitochondrial kinase revealed its function in cristae dynamics, respiration, and oxidative stress response. Although limited by possible mislocalization due to overexpression or epitope tagging, this subcellular map of the kinome can be used to refine regulatory mechanisms involving protein phosphorylation.

Mechanical modulation of spontaneous emission of nearby nanostructured black phosphorus.

In this study, we investigate the spontaneous emission of a quantum emitter nearby black phosphorus (BP) sheet. The spontaneous emission can be modulated mechanically by rotating the BP sheet when the quantum emitter is placed parallel to the sheet. The spontaneous emission is dependent on the electron doping and rotation angle of BP with respect to the x-axis. The Purcell factor decreases with the increase in rotation angle under smaller electron doping. The Purcell factor increases with the increase in rotation angle under larger electron doping. The spontaneous emission of quantum emitter nearby two types of BP ribbon arrays tailored along armchair (type I) and zigzag (type II) directions is studied in detail. The spontaneous emission of quantum emitter parallel to type I is enhanced compared with that parallel to BP sheet. The spontaneous emission decreases remarkably for the quantum emitter parallel to type II compared with that parallel to BP sheet. The spontaneous emission can be flexibly modulated by rotating BP ribbon arrays mechanically in two types. The results obtained in this study provide a new method to actively modulate the spontaneous emission.

Validation of handheld fundus camera with mydriasis for retinal imaging of diabetic retinopathy screening in China: a prospective comparison study.

OBJECTIVES: To investigate the clinical validity of using a handheld fundus camera to detect diabetic retinopathy (DR) in China. DESIGN AND SETTINGS: Prospective comparison study of the handheld fundus camera with a standard validated instrument in detection of DR in hospital and a community screening clinic in Guangdong Province, China. PARTICIPANTS: Participants aged 18 years and over with diabetes who were able to provide informed consent and agreed to attend the dilated eye examination with handheld tests and a standard desktop camera. PRIMARY AND SECONDARY OUTCOME MEASURES: Primary outcome was the proportion of those with referable DR (R2 and above) identified by the handheld fundus camera (the index test) compared with the standard camera. Secondary outcome was the comparison of proportion of gradable images obtained from each test. RESULTS: In this study, we examined 304 people (608 eyes) with each of the two cameras under mydriasis. The handheld camera detected 119 eyes (19.5%) with some level of DR, 81 (13.3%) of them were referable, while the standard camera detected 132 eyes (21.7%) with some level of DR and 83 (13.7%) were referable. It seems that the standard camera found more eyes with referable DR, although McNemar's test detected no significant difference between the two cameras.Of the 608 eyes with images obtained by desktop camera, 598 (98.4%) images were of sufficient quality for grading, 12 (1.9%) images were not gradable. By the handheld camera, 590 (97.0%) were gradable and 20 (3.2%) images were not gradable.The two cameras reached high agreement on diagnosis of retinopathy and maculopathy at all the levels of retinopathy. CONCLUSION: Although it could not take the place of standard desktop camera on clinic fundus examination, the handheld fundus camera showed promising role on preliminary DR screening at primary level in China. To ensure quality images, mydriasis is required.

Design of phoxonic virtual waveguides for both electromagnetic and elastic waves based on the self-collimation effect: an application to enhance acousto-optic interaction.

The dual beam guides for transverse-electric and transverse-magnetic polarizations of electromagnetic (EM) wave and elastic wave in defect-free phoxonic crystals are reported. The realization for phoxonic virtual waveguides relies on dual flat equifrequency contours (EFCs) enabling self-collimation for EM and elastic waves. As a possible application of our work, the enhanced acousto-optic (AO) interaction in this kind of defect-free phoxonic waveguide, just as it does in defect-based waveguides, is further studied. Results show that obvious shifts of the transmission peaks of EM waves exist for both polarizations during one period of the elastic wave, and single-phonon exchange dominates the AO interaction. This kind of phoxonic virtual waveguide provides an effective platform to enhance AO interaction and exhibits some advantage over defect-based waveguides by properly manipulating the photonic and phononic dispersion surfaces.

Integrated Quantitative Proteomics and Metabolome Profiling Reveal MSMEG_6171 Overexpression Perturbing Lipid Metabolism of Mycobacterium smegmatis Leading to Increased Vancomycin Resistance.

In recent years, the treatment of tuberculosis is once again facing a severe situation because the existing antituberculosis drugs have become weaker and weaker with the emergence of drug-resistant Mycobacterium tuberculosis (Mtb). The studies of cell division and cell cycle-related factors in Mtb are particularly important for the development of new drugs with broad-spectrum effects. Mycobacterium smegmatis (Msm) has been used as a model organism to study the molecular, physiological, and drug-resistant mechanisms of Mtb. Bioinformatics analysis has predicted that MSMEG_6171 is a MinD-like protein of the septum site-determining protein family associated with cell division in Mycobacterium smegmatis. In our study, we use ultrastructural analysis, proteomics, metabolomics, and molecular biology techniques to comprehensively investigate the function of MSMEG_6171. Overexpression of MSMEG_6171 in Msm resulted in elongated cells, suggesting an important role of MSMEG_6171 in regulating cell wall morphology. The MSMEG_6171 overexpression could enhance the bacterial resistance to vancomycin, ethionamide, meropenem, and cefamandole. The MSMEG_6171 overexpression could alter the lipid metabolism of Msm to cause the changes on cellular biofilm property and function, which enhances bacterial resistance to antibiotics targeting cell wall synthesis. MSMEG_6171 could also induce the glyceride and phospholipid alteration in vivo to exhibit the pleiotropic phenotypes and various cellular responses. The results showed that amino acid R249 in MSMEG_6171 was a key site that can affect the level of bacterial drug resistance, suggesting that ATPase activity is required for function.

Enhanced sampling and free energy calculations for protein simulations.

Molecular dynamics simulation is a powerful computational technique to study biomolecular systems, which complements experiments by providing insights into the structural dynamics relevant to biological functions at atomic scale. It can also be used to calculate the free energy landscapes of the conformational transitions to better understand the functions of the biomolecules. However, the sampling of biomolecular configurations is limited by the free energy barriers that need to be overcome, leading to considerable gaps between the timescales reached by MD simulation and those governing biological processes. To address this issue, many enhanced sampling methodologies have been developed to increase the sampling efficiency of molecular dynamics simulations and free energy calculations. Usually, enhanced sampling algorithms can be classified into methods based on collective variables (CV-based) and approaches which do not require predefined CVs (CV-free). In this chapter, the theoretical basis of free energy estimation is briefly reviewed first, followed by the reviews of the most common CV-based and CV-free methods including the presentation of some examples and recent developments. Finally, the combination of different enhanced sampling methods is discussed.