Metabolic homeostasis and growth in abiotic cells.

Metabolism constitutes the core chemistry of life. How it began on the early Earth and whether it had a cellular origin are still uncertain. A leading hypothesis for life's origins postulates that metabolism arose from geochemical CO2-fixing pathways, driven by inorganic catalysts and energy sources, long before enzymes or genes existed. The acetyl-CoA pathway and the reductive tricarboxylic acid cycle are considered ancient reaction networks that hold relics of early carbon-fixing pathways. Although transition metals can promote many steps of these pathways, whether they form a functional metabolic network in abiotic cells has not been demonstrated. Here, we formulate a nonenzymatic carbon-fixing network from these pathways and determine its functional feasibility in abiotic cells by imposing fundamental physicochemical constraints. Using first principles, we show that abiotic cells can sustain a steady carbon-fixing cycle that performs a systemic function over a relatively narrow range of conditions. Furthermore, we find that in all feasible steady states, the operation of the cycle elevates the osmotic pressure, leading to volume expansion. These results suggest that achieving homeostatic metabolic states under prebiotic conditions was possible, but challenging, and volume growth was a fundamental property of early metabolism.

Inferred regulons are consistent with regulator binding sequences in E. coli.

The transcriptional regulatory network (TRN) of E. coli consists of thousands of interactions between regulators and DNA sequences. Regulons are typically determined either from resource-intensive experimental measurement of functional binding sites, or inferred from analysis of high-throughput gene expression datasets. Recently, independent component analysis (ICA) of RNA-seq compendia has shown to be a powerful method for inferring bacterial regulons. However, it remains unclear to what extent regulons predicted by ICA structure have a biochemical basis in promoter sequences. Here, we address this question by developing machine learning models that predict inferred regulon structures in E. coli based on promoter sequence features. Models were constructed successfully (cross-validation AUROC > = 0.8) for 85% (40/47) of ICA-inferred E. coli regulons. We found that: 1) The presence of a high scoring regulator motif in the promoter region was sufficient to specify regulatory activity in 40% (19/47) of the regulons, 2) Additional features, such as DNA shape and extended motifs that can account for regulator multimeric binding, helped to specify regulon structure for the remaining 60% of regulons (28/47); 3) investigating regulons where initial machine learning models failed revealed new regulator-specific sequence features that improved model accuracy. Finally, we found that strong regulatory binding sequences underlie both the genes shared between ICA-inferred and experimental regulons as well as genes in the E. coli core pan-regulon of Fur. This work demonstrates that the structure of ICA-inferred regulons largely can be understood through the strength of regulator binding sites in promoter regions, reinforcing the utility of top-down inference for regulon discovery.

Ideal conductor/dielectric model (ICDM): A generalized technique to correct for finite-size effects in molecular simulations of hindered ion transport.

Molecular simulations serve as indispensable tools for investigating the kinetics and elucidating the mechanism of hindered ion transport across nanoporous membranes. In particular, recent advancements in advanced sampling techniques have made it possible to access translocation timescales spanning several orders of magnitude. In our prior study [Shoemaker et al., J. Chem. Theory Comput. 18, 7142 (2022)], we identified significant finite size artifacts in simulations of pressure-driven hindered ion transport through nanoporous graphitic membranes. We introduced the ideal conductor model, which effectively corrects for such artifacts by assuming the feed to be an ideal conductor. In the present work, we introduce the ideal conductor dielectric model (Icdm), a generalization of our earlier model, which accounts for the dielectric properties of both the membrane and the filtrate. Using the Icdm model substantially enhances the agreement among corrected free energy profiles obtained from systems of varying sizes, with notable improvements observed in regions proximate to the pore exit. Moreover, the model has the capability to consider secondary ion passage events, including the transport of a co-ion subsequent to the traversal of a counter-ion, a feature that is absent in our original model. We also investigate the sensitivity of the new model to various implementation details. The Icdm model offers a universally applicable framework for addressing finite size artifacts in molecular simulations of ion transport. It stands as a significant advancement in our quest to use molecular simulations to comprehensively understand and manipulate ion transport processes through nanoporous membranes.

Dental service utilization and the COVID-19 pandemic, a micro-data analysis.

BACKGROUND: Global crises and disease pandemics, such as COVID-19, negatively affect dental care utilization by several factors, such as infection anxiety, disrupted supply chains, economic contraction, and household income reduction. Exploring the pattern of this effect can help policy makers to be prepared for future crises. The present study aimed to investigate the financial impact of COVID-19 disruptions on dental service utilization. METHODS: Data on the number of dental services offered in Dental School Clinics of Tehran University of Medical Sciences was collected over a period of two years, before and after the initial COVID-19 outbreak in Iran. School of Dentistry operates two clinics; one with competitive service fees and one with subsidies. Regression analyses were performed to determine the effect of the pandemic on the number of dental services divided by dental treatment groups and these clinics. The analyses were adjusted for seasonal patterns and the capacity of the clinics. RESULTS: There was a significant drop in dental services offered in both clinics across all dental groups in the post-COVID period (on average, 77 (39.44%) fewer services per day). The majority of the procedure loss happened in the Private clinic. Adjusting for seasonal patterns and the service capacity, regression results documented 54% and 12% service loss in Private and Subsidized clinics following the pandemic, respectively. Difference-in-difference analysis documented that the Subsidized clinic performed 40% more treatments than the Private clinic in the post-COVID period. CONCLUSIONS: Pandemic -reduction in dental care utilization could have long-term ramifications for the oral health of the population, and policymakers need to provide supportive packages to the affected segments of the economy to reverse this trend.

Identifying Men Who Can Remain on Active Surveillance Despite Biopsy Reclassification to Grade Group 2 Prostate Cancer.

PURPOSE: Men on active surveillance with Grade Group 1 prostate cancer who reclassify to Grade Group 2 on surveillance biopsy often leave active surveillance. We aimed to identify subgroups of men who can safely remain on active surveillance despite preoperative reclassification to Grade Group 2. MATERIALS AND METHODS: We studied 249 active surveillance patients with surveillance biopsies classified as Grade Group 1 or Grade Group 2 who underwent radical prostatectomy. Perineural invasion, cancer volume, linear length and maximum percentage of Gleason pattern 4, and prostate-specific antigen density were evaluated. Radical prostatectomy adverse pathology was defined by any of: pN1; ≥pT3; ≥Grade Group 2 with ≥20% Gleason pattern 4; intraductal carcinoma; large cribriform glands. RESULTS: A multivariable logistic regression model incorporating prostate-specific antigen density and perineural invasion stratified radical prostatectomy adverse pathology risk among Grade Group 1 and Grade Group 2 active surveillance patients. 57% (39/68) of Grade Group 1 men reclassified to Grade Group 2 while on active surveillance had favorable radical prostatectomy pathology. Those without biopsy perineural invasion and with low prostate-specific antigen density were more likely to have favorable radical prostatectomy pathology. CONCLUSIONS: Most Grade Group 1 men who enter active surveillance and subsequently reclassify to Grade Group 2 have favorable findings at radical prostatectomy and can remain on active surveillance. Among patients reclassified to Grade Group 2, those with low prostate-specific antigen density and without perineural invasion had the lowest risk of radical prostatectomy adverse pathology, comparable to (or below) that of Grade Group 1 patients who were not reclassified to Grade Group 2 preoperatively. Prostate-specific antigen density and perineural invasion stratify risk in active surveillance patients reclassified to Grade Group 2 and, if concordant with other clinicopathological and radiographic findings, can enable more patients to remain on active surveillance. Reclassification to Grade Group 2 alone should not disqualify men from remaining on active surveillance.

Influence of different modes of exercise training on inflammatory markers in older adults with and without chronic diseases: A systematic review and meta-analysis.

INTRODUCTION: Ageing can be accompanied by increased inflammation, which contributes to the development of sarcopenia. Exercise training could be effective for preventing sarcopenia and mitigate inflammation and thus a viable intervention in ageing. Therefore, we performed a systematic review and meta-analysis to investigate the effects of exercise training on markers of inflammation including interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and C-reactive protein (CRP) in older adults (≥65 years). Exercise-based interventions are most successful in preventing the decline in skeletal muscle mass and in preserving or ameliorating functional capacities with increasing age. METHOD: PubMed and Web of Science were searched through to December 2021 using "exercise", "inflammatory markers", "elderly", and "randomized controlled trial" to identify randomized trials evaluating the effects of exercise training versus control groups on IL-6, TNF-α, and CRP in older adults with mean ages ≥ 65 yrs. Standardized mean differences (SMD) and 95% confidence intervals (95% CIs) were determined using random effects models. RESULTS: Forty studies involving 49 trials and 1,898 older adults were included in the meta-analysis. Overall, exercise training reduced IL-6 [-0.17 (95% CI -0.32 to -0.02), p = 0.02], TNF-α [-0.30 (95% CI -0.46 to -0.13), p = 0.001], and CRP [-0.45 (95% CI -0.61 to -0.29), p = 0.001]. Subgroup analyses showed that IL-6 was reduced significantly by combined training, TNF-α by aerobic training, and CRP by aerobic, resistance, and combined training. In addition, exercise training reduced IL-6 and TNF-α in older adults with chronic diseases, and CRP in older adults with and without chronic diseases. CONCLUSION: The current results highlight that exercise training, regardless of exercise type, has small to moderate beneficial effects on markers of inflammation in older adults, particularly in those with chronic diseases.

Positively charged mineral surfaces promoted the accumulation of organic intermediates at the origin of metabolism.

Identifying plausible mechanisms for compartmentalization and accumulation of the organic intermediates of early metabolic cycles in primitive cells has been a major challenge in theories of life's origins. Here, we propose a mechanism, where positive membrane potentials elevate the concentration of the organic intermediates. Positive membrane potentials are generated by positively charged surfaces of protocell membranes due to accumulation of transition metals. We find that (i) positive membrane potentials comparable in magnitude to those of modern cells can increase the concentration of the organic intermediates by several orders of magnitude; (ii) generation of large membrane potentials destabilize ion distributions; (iii) violation of electroneutrality is necessary to induce nonzero membrane potentials; and (iv) violation of electroneutrality enhances osmotic pressure and diminishes reaction efficiency, resulting in an evolutionary driving force for the formation of lipid membranes, specialized ion channels, and active transport systems.

The impact of exercise training versus caloric restriction on inflammation markers: a systemic review and meta-analysis.

Obesity is associated with an increased risk of chronic, low-grade systematic inflammation for which exercise training (EX) and caloric restriction (CR) are potential treatments. We therefore performed a systematic meta-analysis to compare the effect of EX vs. CR and EX + CR vs. CR on inflammation markers in overweight and obese individuals. PubMed, Scopus, Web of Science and the Cochrane were searched up to April 2020 for EX vs. CR or EX + CR vs. CR interventions studies on inflammatory makers i.e. CRP, IL-6 and TNF-α in overweight and obese individuals. Standardized mean differences and 95% confidence intervals were calculated. Thirty two articles (reporting 38 trials) involving 2108 participants were included in the meta-analysis. Based on studies that directly compared EX and CR, there were no evidence for an effect of EX on IL-6 (p = 0.20) and TNF-α (p = 0.58), when compared with a CR. However, when compared to EX, CR has a statistically greater benefit on CRP (p = 0.01). In those studies, directly comparing EX + CR and CR, EX + CR caused a larger decrease in TNF-α (p = 0.002) and IL-6 (p = 0.02) and tended to decrease CRP (p = 0.06) when compared with CR. These results suggest that a combination of EX and CR may be more effective than CR alone at reducing inflammatory cytokines and CRP in overweight and obese individuals.

How to quantify and avoid finite size effects in computational studies of crystal nucleation: The case of homogeneous crystal nucleation.

Finite size artifacts arise in molecular simulations of nucleation when critical nuclei are too close to their periodic images. A rigorous determination of what constitutes too close is, however, a major challenge. Recently, we devised rigorous heuristics for detecting such artifacts based on our investigation of how system size impacts the rate of heterogeneous ice nucleation [S. Hussain and A. Haji-Akbari, J. Chem. Phys. 154, 014108 (2021)]. We identified the prevalence of critical nuclei spanning across the periodic boundary, and the thermodynamic and structural properties of the liquid occupying the inter-image region as indicators of finite size artifacts. Here, we further probe the performance of such heuristics by examining the dependence of homogeneous crystal nucleation rates in the Lennard-Jones (LJ) liquid on system size. The rates depend non-monotonically on system size and vary by almost six orders of magnitude for the range of system sizes considered here. We confirm that the prevalence of spanning critical nuclei is the primary indicator of finite size artifacts and almost fully explains the observed variations in rate. Proximity, or structuring of the inter-image liquid, however, is not as strong of an indicator due to the fragmented nature of crystalline nuclei. As a result, the dependence of rate on system size is subtle for the systems with a minuscule fraction of spanning critical nuclei. These observations indicate that our heuristics are universally applicable to different modes of nucleation (homogeneous and heterogeneous) in different systems even if they might be overly stringent for homogeneous nucleation, e.g., in the LJ system.

Type 1 Choledochal Cyst with Ectopic Pancreas and Septate Gallbladder.

Background Choledochal cysts (CCs), congenital cystic dilatation of the biliary tract, are more commonly identified in females and have been associated with a myriad of other developmental abnormalities. Case Report: We present a male infant who was diagnosed with type I CC prenatally. He subsequently underwent cyst and gallbladder resection with hepaticoduodenostomy reconstruction at the age of 6 months. Pathologic examination confirmed type I CC with co-existing septate gallbladder and ectopic pancreas (Heinrich type 1). Conclusions: Although the clinical significance is unclear, this second case of CC with septate gallbladder and ectopic pancreas highlights the embryologic association of these abnormalities.

Role of Nanoscale Interfacial Proximity in Contact Freezing in Water.

Contact freezing is a mode of atmospheric ice nucleation in which a collision between a dry ice nucleating particle (INP) and a water droplet results in considerably faster heterogeneous nucleation. The molecular mechanism of such an enhancement is, however, still a mystery. While earlier studies had attributed it to collision-induced transient perturbations, recent experiments point to the pivotal role of nanoscale proximity of the INP and the free interface. By simulating the heterogeneous nucleation of ice within INP-supported nanofilms of two model water-like tetrahedral liquids, we demonstrate that such nanoscale proximity is sufficient for inducing rate increases commensurate with those observed in contact freezing experiments, but only if the free interface has a tendency to enhance homogeneous nucleation. Water is suspected of possessing this latter property, known as surface freezing propensity. Our findings therefore establish a connection between the surface freezing propensity and kinetic enhancement during contact nucleation. We also observe that faster nucleation proceeds through a mechanism markedly distinct from classical heterogeneous nucleation, involving the formation of hourglass-shaped crystalline nuclei that conceive at either interface and that have a lower free energy of formation due to the nanoscale proximity of the interfaces and the modulation of the free interfacial structure by the INP. In addition to providing valuable insights into the physics of contact nucleation, our findings can assist in improving the accuracy of heterogeneous nucleation rate measurements in experiments and in advancing our understanding of ice nucleation on nonuniform surfaces such as organic, polymeric, and biological materials.

Androgenetic/Biparental Mosaic/Chimeric Conceptions With a Molar Component: A Diagnostic and Clinical Challenge.

Hydatidiform moles (HM) are gestational trophoblastic diseases which arise due to an imbalance in genetic material and which are morphologically characterized by enlarged and irregular chorionic villi and trophoblastic hyperplasia, among other features. The morphologic differential diagnosis for HM encompasses a number of entities including androgenetic/biparental mosaic/chimeric (ABMC) conceptions, an interesting duo of lesions with a nonmolar form (placental mesenchymal dysplasia) and a molar form (typically with a complete HM component). ABMC conceptions contain a mixture of 2 cell populations (1 androgenetic and 1 biparental) and arise as a result of mosaicism (mitotic error in a zygote) or chimerism (fusion of 2 zygotes). Because of their unique molecular underpinnings, these rare lesions show a number of findings including the presence of multiple villous populations, discordant p57 immunostaining, and mixed genotypes. ABMC conceptions are important to accurately diagnose as the molar form in particular carries a risk for persistent gestational trophoblastic diseases and thus requires appropriate treatment and follow-up. In this report, we provide detailed characterizations of 2 such cases of ABMC conceptions with a molar component. Both patients (ages 34 and 31) were in the first trimester of pregnancy and had ultrasound findings concerning for HM. Increased comprehension of the pathogenesis and morphology of ABMC conceptions, combined with ancillary techniques including p57 immunohistochemistry, fluorescence in situ hybridization, and molar genotyping, has allowed us to accurately and efficiently identify these lesions. However, a number of pitfalls exist which may lead to misdiagnosis.

How to quantify and avoid finite size effects in computational studies of crystal nucleation: The case of heterogeneous ice nucleation.

Computational studies of crystal nucleation can be impacted by finite size effects, primarily due to unphysical interactions between crystalline nuclei and their periodic images. It is, however, not always feasible to systematically investigate the sensitivity of nucleation kinetics and mechanism to system size due to large computational costs of nucleation studies. Here, we use jumpy forward flux sampling to accurately compute the rates of heterogeneous ice nucleation in the vicinity of square-shaped model structureless ice nucleating particles (INPs) of different sizes and identify three distinct regimes for the dependence of rate on the INP dimension, L. For small INPs, the rate is a strong function of L due to the artificial spanning of critical nuclei across the periodic boundary. Intermediate-sized INPs, however, give rise to the emergence of non-spanning "proximal" nuclei that are close enough to their periodic images to fully structure the intermediary liquid. While such proximity can facilitate nucleation, its effect is offset by the higher density of the intermediary liquid, leading to artificially small nucleation rates overall. The critical nuclei formed at large INPs are neither spanning nor proximal. Yet, the rate is a weak function of L, with its logarithm scaling linearly with 1/L. The key heuristic emerging from these observations is that finite size effects will be minimal if critical nuclei are neither spanning nor proximal and if the intermediary liquid has a region that is structurally indistinguishable from the supercooled liquid under the same conditions.

Differentiated exophytic vulvar intraepithelial lesion: Clinicopathologic and molecular analysis documenting its relationship with verrucous carcinoma of the vulva.

Verruciform proliferations of the vulva unrelated to HPV infection are rare. The term differentiated exophytic vulvar intraepithelial lesion (DEVIL) was recently proposed for these lesions, which harbor recurrent PIK3CA mutations. It is still unclear whether DEVIL is related to verrucous carcinoma, a neoplasm characterized by persistence and local recurrence but nil risk of distant spread. Specimens identified using the words "verruciform" and "verrucous" were reviewed. Diagnosis of DEVIL required verruciform acanthosis, hyper and/or parakeratosis, hypogranulosis, cytoplasmic pallor, and bland nuclei. Verrucous carcinoma required, in addition, discontinuous, bulbous, puzzle-like nests in the stroma. A targeted next-generation sequencing using a custom 11-gene panel was performed. Eighteen specimens corresponding to ten patients with DEVIL and/or verrucous carcinoma were included. Median age at presentation was 66 years for DEVIL and 70 years for verrucous carcinoma. A similar spectrum of prevalent mutations was found in both lesions involving HRAS, PIK3CA, and BRAF. DEVIL preceded verrucous carcinoma and/or was diagnosed concurrently or in subsequent follow-up in five patients. In four of these, the same mutation was identified in DEVIL and synchronous or metachronous carcinoma. All cases showed wild-type 53 staining and lacked pathogenic TP53 mutations. DEVIL is a rare form of squamous proliferation characterized by prevalent PIK3CA and HRAS mutations. Its temporal relationship with verrucous carcinoma and their shared mutational profile in some patients suggest that DEVIL is a precursor of verrucous carcinoma. Moreover, given their morphologic and molecular overlap and the nil risk of verrucous carcinoma for distant spread, it is conceivable that DEVIL and verrucous carcinoma represent a spectrum of the same entity.

Single crystal texture by directed molecular self-assembly along dual axes.

Creating well-defined single-crystal textures in materials requires the biaxial alignment of all grains into desired orientations, which is challenging to achieve in soft materials. Here we report the formation of single crystals with rigorously controlled texture over macroscopic areas (>1 cm2) in a soft mesophase of a columnar discotic liquid crystal. We use two modes of directed self-assembly, physical confinement and magnetic fields, to achieve control of the orientations of the columnar axes and the hexagonal lattice along orthogonal directions. Field control of the lattice orientation emerges in a low-temperature phase of tilted discogens that breaks the field degeneracy around the columnar axis present in non-tilted states. Conversely, column orientation is controlled by physical confinement and the resulting imposition of homeotropic anchoring at bounding surfaces. These results extend our understanding of molecular organization in tilted systems and may enable the development of a range of new materials for distinct applications.

Studying rare events using forward-flux sampling: Recent breakthroughs and future outlook.

Rare events are processes that occur upon the emergence of unlikely fluctuations. Unlike what their name suggests, rare events are fairly ubiquitous in nature, as the occurrence of many structural transformations in biology and material sciences is predicated upon crossing large free energy barriers. Probing the kinetics and uncovering the molecular mechanisms of possible barrier crossings in a system is critical to predicting and controlling its structural and functional properties. Due to their activated nature, however, rare events are exceptionally difficult to study using conventional experimental and computational techniques. In recent decades, a wide variety of specialized computational techniques-known as advanced sampling techniques-have been developed to systematically capture improbable fluctuations relevant to rare events. In this perspective, we focus on a technique called forward flux sampling [Allen et al., J. Chem. Phys. 124, 024102 (2006)] and overview its recent methodological variants and extensions. We also provide a detailed overview of its application to study a wide variety of rare events and map out potential avenues for further explorations.

Computational investigation of surface freezing in a molecular model of water.

Water freezes in a wide variety of low-temperature environments, from meteors and atmospheric clouds to soil and biological cells. In nature, ice usually nucleates at or near interfaces, because homogenous nucleation in the bulk can only be observed at deep supercoolings. Although the effect of proximal surfaces on freezing has been extensively studied, major gaps in understanding remain regarding freezing near vapor-liquid interfaces, with earlier experimental studies being mostly inconclusive. The question of how a vapor-liquid interface affects freezing in its vicinity is therefore still a major open question in ice physics. Here, we address this question computationally by using the forward-flux sampling algorithm to compute the nucleation rate in a freestanding nanofilm of supercooled water. We use the TIP4P/ice force field, one of the best existing molecular models of water, and observe that the nucleation rate in the film increases by seven orders of magnitude with respect to bulk at the same temperature. By analyzing the nucleation pathway, we conclude that freezing in the film initiates not at the surface, but within an interior region where the formation of double-diamond cages (DDCs) is favored in comparison with the bulk. This, in turn, facilitates freezing by favoring the formation of nuclei rich in cubic ice, which, as demonstrated by us earlier, are more likely to grow and overcome the nucleation barrier. The films considered here are ultrathin because their interior regions are not truly bulk-like, due to their subtle structural differences with the bulk.

Effect of material flexibility on the thermodynamics and kinetics of hydrophobically induced evaporation of water.

The evaporation of water induced by confinement between hydrophobic surfaces has received much attention due to its suggested functional role in numerous biophysical phenomena and its importance as a general mechanism of hydrophobic self-assembly. Although much progress has been made in understanding the basic physics of hydrophobically induced evaporation, a comprehensive understanding of the substrate material features (e.g., geometry, chemistry, and mechanical properties) that promote or inhibit such transitions remains lacking. In particular, comparatively little research has explored the relationship between water's phase behavior in hydrophobic confinement and the mechanical properties of the confining material. Here, we report the results of extensive molecular simulations characterizing the rates, free energy barriers, and mechanism of water evaporation when confined between model hydrophobic materials with tunable flexibility. A single-order-of-magnitude reduction in the material's modulus results in up to a nine-orders-of-magnitude increase in the evaporation rate, with the corresponding characteristic time decreasing from tens of seconds to tens of nanoseconds. Such a modulus reduction results in a 24-orders-of-magnitude decrease in the reverse rate of condensation, with time scales increasing from nanoseconds to tens of millions of years. Free energy calculations provide the barriers to evaporation and confirm our previous theoretical predictions that making the material more flexible stabilizes the confined vapor with respect to liquid. The mechanism of evaporation involves surface bubbles growing/coalescing to form a subcritical gap-spanning tube, which then must grow to cross the barrier.

Thermodynamically driven assemblies and liquid-liquid phase separations in biology.

The sustenance of life depends on the high degree of organization that prevails through different levels of living organisms, from subcellular structures such as biomolecular complexes and organelles to tissues and organs. The physical origin of such organization is not fully understood, and even though it is clear that cells and organisms cannot maintain their integrity without consuming energy, there is growing evidence that individual assembly processes can be thermodynamically driven and occur spontaneously due to changes in thermodynamic variables such as intermolecular interactions and concentration. Understanding the phase separation in vivo requires a multidisciplinary approach, integrating the theory and physics of phase separation with experimental and computational techniques. This paper aims at providing a brief overview of the physics of phase separation and its biological implications, with a particular focus on the assembly of membraneless organelles. We discuss the underlying physical principles of phase separation from its thermodynamics to its kinetics. We also overview the wide range of methods utilized for experimental verification and characterization of phase separation of membraneless organelles, as well as the utility of molecular simulations rooted in thermodynamics and statistical physics in understanding the governing principles of thermodynamically driven biological self-assembly processes.