Estimation of the ice melting point in molecular dynamics simulations based on the finite-size effects.

Predicting the ice melting point using molecular dynamics (MD) simulations is nontrivial due to uncertainty associated with the stochastic nature of the simulation and effect of finite domain sizes on the simulated ice-water phase transition. We developed a method based on the percolation theory to make use of the finite size effects to allow determination of a unique critical phase transition temperature as the melting point. The method involves construction of melting/freezing probability curves from multiple simulations with varying temperatures for different domain sizes. While the domain sizes affect the apparent melting/freezing probability and hence generate different curves with a wider probability distribution for a smaller size, the intersection of these curves is unique and locates the melting point. Based on MD simulations using the Tip4p/Ice water model, we tested and demonstrated the effectiveness of this method in locating the critical ice-water phase transition at a melting temperature of 268.78 K. Our analysis also showed that the apparent melting probability at this critical point is ∼0.69, not 0.5 assumed in the ad hoc method used previously. Our method, making no assumption about the system size, may provide a generic framework for analyzing phase transitions influenced by the finite size effects in MD simulations.

Functional evaluation of BRCA1/2 variants of unknown significance with homologous recombination assay and integrative in silico prediction model.

Numerous variants of unknown significance (VUSs) exist in hereditary breast and ovarian cancers. Although multiple methods have been developed to assess the significance of BRCA1/2 variants, functional discrepancies among these approaches remain. Therefore, a comprehensive functional evaluation system for these variants should be established. We performed conventional homologous recombination (HR) assays for 50 BRCA1 and 108 BRCA2 VUSs and complementarily predicted VUSs using a statistical logistic regression prediction model that integrated six in silico functional prediction tools. BRCA1/2 VUSs were classified according to the results of the integrative in vitro and in silico analyses. Using HR assays, we identified 10 BRCA1 and 4 BRCA2 VUSs as low-functional pathogenic variants. For in silico prediction, the statistical prediction model showed high accuracy for both BRCA1 and BRCA2 compared with each in silico prediction tool individually and predicted nine BRCA1 and seven BRCA2 variants to be pathogenic. Integrative functional evaluation in this study and the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines strongly suggested that seven BRCA1 variants (p.Glu272Gly, p.Lys1095Glu, p.Val1653Leu, p.Thr1681Pro, p.Phe1761Val, p.Thr1773Ile, and p.Gly1803Ser) and four BRCA2 variants (p.Trp31Gly, p.Ser2616Phe, p.Tyr2660Cys, and p.Leu2792Arg) were pathogenic. This study demonstrates that integrative evaluation using conventional HR assays and optimized in silico prediction comprehensively classified the significance of BRCA VUSs for future clinical applications.

luxS contributes to intramacrophage survival of Streptococcus agalactiae by positively affecting the expression of fruRKI operon.

The LuxS quorum sensing system is a widespread system employed by many bacteria for cell-to-cell communication. The luxS gene has been demonstrated to play a crucial role in intramacrophage survival of piscine Streptococcus agalactiae, but the underlying mechanism remains largely unknown. In this study, transcriptome analysis, followed by the luxS gene deletion and subsequent functional studies, confirmed that impaired bacterial survival inside macrophages due to the inactivation of luxS was associated with reduced transcription of the fruRKI operon, encoding the fructose-specific phosphotransferase system. Further, luxS was determined not to enhance the transcription of fruRKI operon by binding its promoter, but to upregulate the expression of this operon via affecting the binding ability of catabolite control protein A (CcpA) to the catabolite responsive element (cre) in the promoter of fruRKI. Collectively, our study identifies a novel and previously unappreciated role for luxS in bacterial intracellular survival, which may give a more thorough understanding of the immune evasion mechanism in S. agalactiae.

Passively-targeted mitochondrial tungsten-based nanodots for efficient acute kidney injury treatment.

Acute kidney injury (AKI) can lead to loss of kidney function and a substantial increase in mortality. The burst of reactive oxygen species (ROS) plays a key role in the pathological progression of AKI. Mitochondrial-targeted antioxidant therapy is very promising because mitochondria are the main source of ROS in AKI. Antioxidant nanodrugs with actively targeted mitochondria have achieved encouraging success in many oxidative stress-induced diseases. However, most strategies to actively target mitochondria make the size of nanodrugs too large to pass through the glomerular system to reach the renal tubules, the main damage site of AKI. Here, an ultra-small Tungsten-based nanodots (TWNDs) with strong ROS scavenging can be very effective for treatment of AKI. TWNDs can reach the tubular site after crossing the glomerular barrier, and enter the mitochondria of the renal tubule without resorting to complex active targeting strategies. To our knowledge, this is the first time that ultra-small negatively charged nanodots can be used to passively target mitochondrial therapy for AKI. Through in-depth study of the therapeutic mechanism, such passive mitochondria-targeted TWNDs are highly effective in protecting mitochondria by reducing mitochondrial ROS and increasing mitophagy. In addition, TWNDs can also reduce the infiltration of inflammatory cells. This work provides a new way to passively target mitochondria for AKI, and give inspiration for the treatment of many major diseases closely related to mitochondria, such as myocardial infarction and cerebral infarction.

CRISPR Contributes to Adhesion, Invasion, and Biofilm Formation in Streptococcus agalactiae by Repressing Capsular Polysaccharide Production.

The clustered regularly interspaced palindromic repeat (CRISPR)-associated (Cas) system functions classically as a prokaryotic defense system against invading mobile genetic elements, such as phages, plasmids, and viruses. Our previous study revealed that CRISPR deletion caused increased transcription of capsular polysaccharide (CPS) synthesis-related genes and severely attenuated virulence in the hypervirulent piscine Streptococcus agalactiae strain GD201008-001. Here, we found that CRISPR deficiency resulted in reduced adhesion, invasion, and biofilm formation abilities in this strain by upregulating the production of CPS. However, enhanced CPS production was not responsible for the attenuated phenotype of the ΔCRISPR mutant. RNA degradation assays indicated that inhibited transcription of the cps operon by CRISPR RNA (crRNA) was not due to the base pairing of the crRNA with the cps mRNA but to the repression of the promoter activity of cpsA, which is a putative transcriptional regulator of the capsule locus. IMPORTANCE Beyond protection from invading nucleic acids, CRISPR-Cas systems have been shown to have an important role in regulating bacterial endogenous genes. In this study, we demonstrate that crRNA inhibits the transcription of the cps operon by repressing the activity of promoter PcpsA, leading to increases in the abilities of adhesion, invasion, and biofilm formation in S. agalactiae. This study highlights the regulatory role of crRNA in bacterial physiology and provides a new explanation for the mechanism of crRNA-mediated endogenous gene regulation in S. agalactiae.

TMEM16F mediated phosphatidylserine exposure and microparticle release on erythrocyte contribute to hypercoagulable state in hyperuricemia.

The link between hyperuricemia (HUA) and the risk of venous thromboembolism (VTE) has been well established. However, the mechanisms of thrombus generation and the effect of HUA on procoagulant activity (PCA) of erythrocytes remain unclear no matter in uremia or hyperuricemia. Here, phosphatidylserine (PS) exposure, microparticles (MPs) release, cytosolic Ca2+, TMEM16F expression, reactive oxygen species (ROS) and lipid peroxidation of erythrocyte were detected by flow cytometer. PCA was assessed by coagulation time, purified coagulation complex and fibrin production assays. The fibrin formation was observed by scanning electron microscopy (SEM). We found that PS exposure, MPs generation, TMEM16F expression and consequent PCA of erythrocyte in HUA patients significantly increased compared to those in healthy volunteers. Furthermore, high UA induced PS exposure, and MPs release of erythrocyte in concentration and time-dependent manners in vitro, which enhanced the PCA of erythrocyte and was inhibited by lactadherin, a PS inhibitor. Additionally, using SEM, we also observed compact fibrin clots with highly-branched networks and thin fibers supported by red blood cells (RBCs) and RBC-derived MPs (RMPs). Importantly, we demonstrated UA enhanced the production of ROS and lipid peroxidation and reduced the generation of glutathione (GSH) of erythrocyte, which enhanced TMEM16F activity and followed PS externalization and RMPs formation. Collectively, these results suggest that Ca2+-dependent TMEM16F activation may be responsible for UA-induced PS exposure and MPs release of RBC, which thereby contribute to the prothrombotic risk in HUA.

Scrutinizing high-risk patients from ASC-US cytology via a deep learning model.

BACKGROUND: Atypical squamous cells of undetermined significance (ASC-US) is the most frequent but ambiguous abnormal Papanicolaou (Pap) interpretation and is generally triaged by high-risk human papillomavirus (hrHPV) testing before colposcopy. This study aimed to evaluate the performance of an artificial intelligence (AI)-based triage system to predict ASC-US cytology for cervical intraepithelial neoplasia 2+ lesions (CIN2+). METHODS: More than 60,000 images were used to train this proposed deep learning-based ASC-US triage system, where both cell-level and slide-level information were extracted. In total, 1967 consecutive ASC-US Paps from 2017 to 2019 were included in this study. Histological follow-ups were retrieved to compare the triage performance between the AI system and hrHPV in 622 patients with simultaneous hrHPV testing. RESULTS: In the triage of women with ASC-US cytology for CIN2+, our system attained equivalent sensitivity (92.9%; 95% confidence interval [CI], 75.0%-98.8%) and higher specificity (49.7%; 95% CI, 45.6%-53.8%) than hrHPV testing (sensitivity: 89.3%; 95% CI, 70.6%-97.2%; specificity: 34.3%; 95% CI, 30.6%-38.3%) without requiring additional patient examination or testing. Additionally, the independence of this system from hrHPV testing (κ = 0.138) indicated that these 2 different methods could be used to triage ASC-US as an alternative way. CONCLUSION: This de novo deep learning-based system can triage ASC-US cytology for CIN2+ with a performance superior to hrHPV testing and without incurring additional expenses.

Hyperuricemia enhances procoagulant activity of vascular endothelial cells through TMEM16F regulated phosphatidylserine exposure and microparticle release.

The link between serum uric acid (SUA) and the risk of venous thromboembolism (VTE) is well established. Recent data suggested a causative role of UA in endothelial cells (ECs) dysfunction. However, the molecular mechanism of high UA on thrombogenesis is unknown. We investigate whether high UA induce phosphatidylserine (PS) externalization and microparticle (MP) shedding in cultured EC, and contribute to UA-induced hypercoagulable state. In the present study, we demonstrate that UA induces PS exposure and EMP release of EC in a concentration- and time-dependent manner, which enhances the procoagulant activity (PCA) of EC and inhibited over 90% by lactadherin in vitro. Furthermore, hyperuricemic rat model was used to evaluate the development of thrombi following by flow stasis in the inferior vena cava (IVC). Hyperuricemia group is more likely to form large and hard thrombi compared with control. Importantly, we found that TMEM16F expression is significantly upregulated in UA-treated EC, which is crucial for UA-induced PS exposure and MP formation. Additionally, UA increases the generation of reactive oxygen species (ROS), lipid peroxidation, and cytosolic Ca2+ concentration in EC, which might contribute to increased TMEM16F expression. Using confocal microscopy, we also observed disruption of the actin cytoskeleton, suggesting that depolymerization of actin filaments might be required for TMEM16F activation and followed by PS exposure and membrane blebbing in UA-treated EC. Our results demonstrate a thrombotic role of EC in hyperuricemia through TMEM16F-mediated PS exposure and MPs release.

Atypical squamous cells of undetermined significance cervical cytology in the Chinese population: Age-stratified reporting rates, high-risk HPV testing, and immediate histologic correlation results.

BACKGROUND: The US American Society of Colposcopy and Cervical Pathology guidelines for cervical cancer screening have been largely adopted worldwide. Pooled high-risk human papillomavirus (hrHPV) testing has been routinely used to risk-stratify women who have atypical squamous cells of undetermined significance (ASC-US) cytology. However, it has been reported that there are distinguished differences in the distribution of hrHPV genotypes between the Chinese and American populations. METHODS: The objective of this study was to analyze the age-stratified reporting rates, hrHPV-positive rates, and genotyping by different cytology preparation methods and hrHPV testing assays, along with the immediate histopathologic correlation of ASC-US cytology, in the Chinese population. RESULTS: The ASC-US reporting rate of 1,597,136 Papanicolaou (Pap) tests was 4.2%, and the overall hrHPV-positive rate was 48.7% in the ASC-US cases. In total, 25,338 women with ASC-US Pap tests had immediate histologic follow-up, and the detection rate for cervical intraepithelial neoplasia 2 and higher lesions (CIN2+) was 7.1%, including 0.6% carcinomas. Among the women who underwent hrHPV testing, CIN2+ lesions were identified in 657 of 6154 (10.7%) who had hrHPV-positive results and in only 1.5% those who had hrHPV-negative results. Further genotyping analysis revealed that HPV types 16 and/or 18 were commonly identified genotypes among the Chinese women who had ASC-US cytology. CONCLUSIONS: This large-scale study demonstrated that the hrHPV-positive rate, the CIN2+ detection rate, and the distribution of hrHPV genotypes in Chinese women with ASC-US cytology were essentially consistent with those from the American population, further supporting that the current and newly released 2019 American Society of Colposcopy and Cervical Pathology guidelines should be applicable to the Chinese population.

Phagocytosis by endothelial cells inhibits procoagulant activity of platelets of essential thrombocythemia in vitro.

BACKGROUND: Essential thrombocythemia (ET) is characterized by thrombocytosis with increased platelet number and persistent activation. The mechanisms of thrombosis and the fate of these platelets are not clear. The aim of the present study is to explore the phagocytosis of platelets of ET patients by endothelial cells (ECs) in vitro and its relevance to the procoagulant activity (PCA). METHODS: Phosphatidylserine (PS) exposure on platelets was detected by flow cytometry. Phagocytosis of the platelets by ECs was performed using flow cytometry, confocal microscopy, and electron microscopy. The PCA of platelets was evaluated by coagulation time and purified coagulation complex assays. RESULTS: The PS exposure on platelets in ET patients is higher than that in healthy controls. The PS-exposed platelets are highly procoagulant and lactadherin reduced 80% of the PCA by blockade of PS. When cocultured, the platelets of ET patients were sequestered by ECs in a time-dependent fashion. Lactadherin enhanced phagocytosis by bridging the PS on activated platelets and the integrin αvß3 on ECs, and P-selectin played at least a partial role in this process. Furthermore, factor Xa and prothrombinase activity of PS-exposed platelets were decreased after incubation with ECs. CONCLUSION: Our results suggest that phagocytic clearance of platelets by ECs occurs in ET patients, thus representing a novel mechanism to remove activated platelets from the circulation; lactadherin and phagocytosis could cooperatively limit the thrombophilia in ET patients.

The photocatalytic degradation of chloramphenicol with electrospun Bi2O2CO3-poly(ethylene oxide) nanofibers: the synthesis of crosslinked polymer, degradation kinetics, mechanism and cytotoxicity.

Insoluble poly(ethylene oxide) (PEO) nanofibers were synthesized by adding pentaerythrotol triacrylate (PETA) into precursor solutions prior to electrospinning, and then the obtained fibers were exposed to an electron beam (EB) irradiation. Bi2O2CO3 was incorporated into these fibers to extend their photocatalytic properties. Studies confirmed that EB irradiation induced characteristic changes in PEO and led to the formation of a crosslinked structure, from which we optimized the irradiation dose of fibers as 210 kGy. The optimum PEO/Bi2O2CO3 membranes achieved 99.5% CPL degradation within 60 min, and we also proposed the possible degradation pathways of CPL in this study. Besides, all the water samples and extracts of nanomaterials showed no cytotoxicity on L-929 cells. The subtle variations in the cell viability of treated and untreated water samples could be due to the toxic intermediates arising from the photocatalytic process. Therefore, this photocatalyst-polymer membrane can be considered as a biocompatible composite system that can change the solubility of a polymer and also act as a highly efficient photocatalyst for organic wastewater treatments.

Innervation of the tibial epiphysis through the intercondylar foramen.

The periosteum and mineralized bone are innervated by nerves that sense pain. These include both myelinated and unmyelinated neurons with either free nerve endings or bearing nociceptors. Parasympathetic and sympathetic autonomic nerves also innervate bone. However, little is known about the route sensory nerves take leaving the epiphyses of long bones at the adult knee joint. Here, we used transgenic mice that express fluorescent Venus protein in Schwann cells (Sox10-Venus mice) to visualize myelinated and unmyelinated nerves in the tibial epiphysis. Immunofluorescence to detect a pan-neuronal marker and the sensory neuron markers calcitonin gene-related peptide (CGRP) and tropomyosin receptor kinase A (TrkA) also revealed Schwann cell-associated sensory neurons. Foramina in the intercondylar area of the tibia were conserved between rodents and primates. Venus-labeled fibers were detected within bone marrow of the proximal epiphysis, exited through foramina along with blood vessels in the intercondylar area of the tibia, and joined Venus-labeled fibers of the synovial membrane and meniscus. These data suggest that innervation of the subchondral plate and trabecular bone within the tibial epiphysis carries pain signals from the knee joint to the brain through intercondylar foramina.

Glycolipids: Essential regulator of neuro-inflammation, metabolism and gliomagenesis.

Gene knockout mice of glycosyltransferases have clearly showed roles of their products in the bodies, while there are examples where phenotype of knockout was much less severe than expected probably due to functional redundancy. The most striking novel finding obtained from ganglioside-deficient mice was that progressive inflammatory reaction took place, leading to neurodegeneration. In particular, dysfunction of complement-regulatory proteins due to deteriorated architecture of lipid rafts seemed to be essential mechanisms for the inflammation. Furthermore, roles of gangliosides in neurons were demonstrated by neuron-specific transgenic of B4galnt1 with genetic background of B4galnt1 deficiency. From study of gene knockout mice of St8sia1, new roles of b-series gangliosides in leptin secretion from adipocytes, and roles of a-series gangliosides in leptin receptor, ObR in hypothalamus were demonstrated, leading to apparent intact balance of energy. Essential roles of b-series gangliosides in malignant properties of gliomas were also shown, suggesting their roles in the regulation of inflammation and proliferation in nervous tissues. How to apply these findings for the control of newly discovered patients with ganglioside deficiency remains to be investigated. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa.

Increased a-series gangliosides positively regulate leptin/Ob receptor-mediated signals in hypothalamus of GD3 synthase-deficient mice.

Gangliosides are widely involved in the regulation of cells and organs. However, little is known about their roles in adipose tissues and hypothalamus. In GD3 synthase-knockout (GD3S KO) mice, deletion of b-series gangliosides resulted in the reduction of serum leptin due to disturbed secretion from adipocytes. To examine whether leptin signals altered, leptin/leptin receptor (ObR)-mediated signaling in hypothalamus was analyzed. Hypothalamus of GD3S KO mouse showed increased expression of GM1 and GD1a, and increased activation of ObR-mediated signals such as pSTAT3 and c-Fos. Leptin stimulation of hypothalamus-derived N-41 cells and their transfectants with GD3S cDNA showed that a-series gangliosides positively regulate leptin/ObR-mediated signals. Co-precipitation analysis revealed that ObR interacts with a-series gangliosides with increased association by leptin stimulation. In brown adipose tissues (BAT) of GD3S KO mice, their weights and adipocyte numbers were increased, and BAT markers such as PGC1α and UCP-1 were also up-regulated. These results suggested that leptin/ObRb-mediated signals were enhanced in hypothalamus of GD3S KO mice due to increased a-series gangliosides, leading to the apparently similar features of energy expenditure between the KO and wild type mice.

Sialylation converts arthritogenic IgG into inhibitors of collagen-induced arthritis.

Rheumatoid arthritis (RA)-associated IgG antibodies such as anti-citrullinated protein antibodies (ACPAs) have diverse glycosylation variants; however, key sugar chains modulating the arthritogenic activity of IgG remain to be clarified. Here, we show that reduced sialylation is a common feature of RA-associated IgG in humans and in mouse models of arthritis. Genetically blocking sialylation in activated B cells results in exacerbation of joint inflammation in a collagen-induced arthritis (CIA) model. On the other hand, artificial sialylation of anti-type II collagen antibodies, including ACPAs, not only attenuates arthritogenic activity, but also suppresses the development of CIA in the antibody-infused mice, whereas sialylation of other IgG does not prevent CIA. Thus, our data demonstrate that sialylation levels control the arthritogenicity of RA-associated IgG, presenting a potential target for antigen-specific immunotherapy.

Indolic uremic solutes enhance procoagulant activity of red blood cells through phosphatidylserine exposure and microparticle release.

Increased accumulation of indolic uremic solutes in the blood of uremic patients contributes to the risk of thrombotic events. Red blood cells (RBCs), the most abundant blood cells in circulation, may be a privileged target of these solutes. However, the effect of uremic solutes indoxyl sulfate (IS) and indole-3-acetic acid (IAA) on procoagulant activity (PCA) of erythrocyte is unclear. Here, RBCs from healthy adults were treated with IS and IAA (mean and maximal concentrations reported in uremic patients). Phosphatidylserine (PS) exposure of RBCs and their microparticles (MPs) release were labeled with Alexa Fluor 488-lactadherin and detected by flow cytometer. Cytosolic Ca(2+) ([Ca(2+)]) with Fluo 3/AM was analyzed by flow cytometer. PCA was assessed by clotting time and purified coagulation complex assays. We found that PS exposure, MPs generation, and consequent PCA of RBCs at mean concentrations of IS and IAA enhanced and peaked in maximal uremic concentrations. Moreover, 128 nM lactadherin, a PS inhibitor, inhibited over 90% PCA of RBCs and RMPs. Eryptosis or damage, by indolic uremic solutes was due to, at least partially, the increase of cytosolic [Ca(2+)]. Our results suggest that RBC eryptosis in uremic solutes IS and IAA plays an important role in thrombus formation through releasing RMPs and exposing PS. Lactadherin acts as an efficient anticoagulant in this process.

b-Series gangliosides crucially regulate leptin secretion in adipose tissues.

Gangliosides are widely involved in the regulation of cells and organs. However, little is known about their roles in leptin secretion from adipose tissues. Genetic deletion of b-series gangliosides resulted in the marked reduction of serum leptin. Expression analysis of leptin revealed that leptin accumulated in the adipose tissues of GD3 synthase-knockout (GD3S KO) mice. Analysis of primary cultured stromal vascular fractions (SVF) derived from GD3S KO mice revealed that leptin secretion was reduced, although leptin amounts in cells were increased compared with those of wild type. Interestingly, addition of b-series gangliosides to the culture medium of differentiated SVF resulted in the restoration of leptin secretion. Results of methyl-ß-cyclodextrin treatment of differentiated 3T3-L1 cells as well as immunocytostaining of leptin and caveolin-1 suggested that b-series gangliosides regulate the leptin secretion from adipose tissues in lipid rafts.