Comprehensive assessment of 11 de novo HiFi assemblers on complex eukaryotic genomes and metagenomes.

Pacific Biosciences (PacBio) HiFi sequencing technology generates long reads (>10 kbp) with very high accuracy (<0.01% sequencing error). Although several de novo assembly tools are available for HiFi reads, there are no comprehensive studies on the evaluation of these assemblers. We evaluated the performance of 11 de novo HiFi assemblers on (1) real data for three eukaryotic genomes; (2) 34 synthetic data sets with different ploidy, sequencing coverage levels, heterozygosity rates, and sequencing error rates; (3) one real metagenomic data set; and (4) five synthetic metagenomic data sets with different composition abundance and heterozygosity rates. The 11 assemblers were evaluated using quality assessment tool (QUAST) and benchmarking universal single-copy ortholog (BUSCO). We also used several additional criteria, namely, completion rate, single-copy completion rate, duplicated completion rate, average proportion of largest category, average distance difference, quality value, run-time, and memory utilization. Results show that hifiasm and hifiasm-meta should be the first choice for assembling eukaryotic genomes and metagenomes with HiFi data. We performed a comprehensive benchmarking study of commonly used assemblers on complex eukaryotic genomes and metagenomes. Our study will help the research community to choose the most appropriate assembler for their data and identify possible improvements in assembly algorithms.

Antihypertensive treatment during pregnancy induces long-term changes in gut microbiota and the behaviors of the attention deficit hyperactivity disorder offspring.

The pathogenesis of attention-deficit/hyperactivity disorder (ADHD) has not been fully elucidated. Gestational hypertension could double the probability of ADHD in the offspring, while the initial bacterial communication between the mother and offspring has been associated with psychiatric disorders. Thus, we hypothesize that antihypertensive treatment during pregnancy may abate the impairments in neurodevelopment of the offspring. To test this hypothesis, we chose Captopril and Labetalol, to apply to pregnant spontaneously hypertensive rat (SHR) dams and examined the outcomes in the male offspring. Our data demonstrated that maternal treatment with Captopril and Labetalol had long-lasting changes in gut microbiota and behavioral alterations, including decreased hyperactivity and increased curiosity, spatial learning and memory in the male offspring. Increased diversity and composition were identified, and some ADHD related bacteria were found to have the same change in the gut microbiota of both the dam and offspring after the treatments. LC-MS/MS and immunohistochemistry assays suggested elevated expression of brain derived neurotrophic factor (BDNF) and dopamine in the prefrontal cortex and striatum of offspring exposed to Captopril/ Labetalol, which may account for the improvement of the offspring's psychiatric functions. Therefore, our results support the beneficial long-term effects of the intervention of gestational hypertension in the prevention of ADHD.

H2S aids osmotic stress resistance by S-sulfhydration of melatonin production-related enzymes in Arabidopsis thaliana.

KEY MESSAGE: Hydrogen sulfide closed Arabidopsis thaliana stomata by increasing the transcription of melatonin-producing enzymes and the post-translational modification levels to combat osmotic stress. Hydrogen sulfide (H2S) and melatonin (MEL) reportedly have similar functions in many aspects of plant growth, development and stress response. They regulate stomatal movement and enhance drought resistance. However, their physiological relationship is not well understood. Here, their crosstalk involved in osmotic stress resistance in Arabidopsis thaliana was studied. Exogenous H2S and MEL closed stomata under normal or osmotic stress conditions and increased the relative water contents of plants under osmotic stress conditions. At the same time, exogenous H2S and MEL responded to osmotic stress by increasing the content of proline and soluble sugar, and reducing malondialdehyde (MDA) content and relative conductivity. Using mutants in the MEL-associated production of serotonin N-acetyltransferase (snat), caffeic acid O-methyltransferase (comt1) and N-acetylserotonin methyltransferase (asmt), we determined that H2S was partially dependent on MEL to close stomata. Additionally, the overexpression of ASMT promoted stomatal closure. Exogenous H2S increased the transcription levels of SNAT, ASMT and COMT1. Furthermore, exogenous H2S treatments increased the endogenous MEL content significantly. At the post-translational level, H2S sulfhydrated the SNAT and ASMT, but not COMT1, enzymes associated with MEL production. Thus, H2S appeared to promote stomatal closure in response to osmotic stress by increasing the transcription levels of MEL synthesis-related genes and the sulfhydryl modification of the encoded enzymes. These results increased our understanding of H2S and MEL functions and interactions under osmotic stress conditions.

Activation of Paraventricular Melatonin Receptor 2 Mediates Melatonin-Conferred Cardioprotection Against Myocardial Ischemia/Reperfusion Injury.

Previous studies have shown that melatonin (Mel) can effectively ameliorate myocardial ischemia/reperfusion (MI/R) injury, but the mechanism is yet to be fully elucidated. Mel receptors are expressed in the paraventricular nucleus (PVN), which is also involved in regulating cardiac sympathetic nerve activity. The aim of this study was to examine whether Mel receptors in the PVN are involved in the protective effects of Mel against MI/R injury. The results of quantitative polymerase chain reaction, western blot, and immunofluorescence assays indicated that Mel receptor 2 (MT2) expression in the PVN was upregulated after MI/R. Intraperitoneal administration of Mel significantly improved post-MI/R cardiac function and reduced the infarct size, whereas shRNA silencing of MT2 in the PVN partially blocked this effect. Intraperitoneal administration of Mel reduced sympathetic nerve overexcitation caused by MI/R, whereas shRNA silencing of MT2 in the PVN partially diminished this effect. Furthermore, enzyme-linked immunosorbent assay and western blot results indicated that intraperitoneal administration of Mel lowered the levels of inflammatory cytokines in the PVN after MI/R injury, whereas the application of sh-MT2 in the PVN reduced this effect of Mel. Mel significantly reduced the levels of NF-κB after astrocyte oxygen and glucose deprivation/reoxygenation injury, and this effect was offset when MT2 was silenced. The above experimental results suggest that MT2 in the PVN partially mediated the protective effects of Mel against MI/R injury, and its underlying mechanisms may be related to postactivation amelioration of PVN inflammation and reduction of cardiac sympathetic nerve overexcitation.

Ultrasonography for the Prediction of High-Volume Lymph Node Metastases in Papillary Thyroid Carcinoma: Should Surgeons Believe Ultrasound Results?

BACKGROUND: Lymph node metastasis (LNM) often occurs in papillary thyroid carcinoma (PTC); the efficacy of ultrasound for predicting high-volume lymph node metastases (LNMs) in patients with PTC remains unexplored. METHODS: The medical records of 2073 consecutive PTC patients were reviewed. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated to evaluate the efficacy of ultrasound. Risk factors for LNM/high-volume LNMs and lymph node involvement on ultrasound (usLNM) were identified by univariate and multivariate analyses. RESULTS: Of all the patients, 936 (45.2%) patients had LNMs, and 254 (12.3%) patients had high-volume LNMs. The sensitivity of ultrasound for detecting LNM/high-volume LNMs was 27.9% and 63.8%, respectively; the specificity was 93.1% and 90.3%, respectively. The NPV for ultrasound in detecting high-volume LNMs was 94.7%. In multivariate analysis, male sex (OR = 2.108, p < 0.001), tumor diameter > 1.0 cm (OR = 2.304, p < 0.001) and usLNM (+) (OR = 12.553, p < 0.001) were independent clinical risk factors for high-volume LNMs. Tumor diameter > 1 cm (OR = 3.036, p < 0.001) and male sex (OR = 1.642, p < 0.001) were independent clinical risk factors for usLNM; a skilled sonographer (OR = 1.121, p = 0.358) was not significantly associated with usLNM. CONCLUSIONS: Lymph node involvement found by ultrasound has great predictive value for high-volume LNMs; the NPV is very high for patients without lymph node involvement on ultrasound. The ultrasound results do not appear to be influenced by the experience of the sonographer.

Infusion of Melatonin Into the Paraventricular Nucleus Ameliorates Myocardial Ischemia-Reperfusion Injury by Regulating Oxidative Stress and Inflammatory Cytokines.

Melatonin, the receptors for which are abundant in the hypothalamic paraventricular nucleus (PVN), can protect the heart from myocardial ischemia-reperfusion (MI/R) injury. The aim of this study was to determine whether the infusion of melatonin into the PVN protects the heart from MI/R injury by suppressing oxidative stress or regulating the balance between proinflammatory cytokines and anti-inflammatory cytokines in MI/R rats. Male Sprague-Dawley rats were treated with a bilateral PVN infusion of melatonin. MI/R operation was performed 1 week after infusion. At the end of the third week after the infusion, all the rats were euthanized. This was followed by immunohistochemistry and immunofluorescence studies of the rats. MI/R rats showed larger infarct size, increased left ventricular (LV) end-diastolic volume, and decreased LV ejection fraction and LV fractional shortening. Moreover, MI/R rats had a higher level of norepinephrine in the plasma, heart, and PVN; higher PVN levels of reactive oxygen species, NOX2, NOX4, IL-1ß, and NF-κB activity; and lower PVN levels of copper/zinc superoxide dismutase (Cu/Zn-SOD) and IL-10 compared with the sham group. Melatonin infusion in PVN reduced LV end-diastolic volume, norepinephrine, reactive oxygen species, NOX2, NOX4, IL-1ß, and NF-κB activity, and increased LV ejection fraction, LV fractional shortening, Cu/Zn-SOD, and IL-10. Overall, these results suggest that the infusion of melatonin ameliorates sympathetic nerve activity and MI/R injury by attenuating oxidative stress and inflammatory cytokines in the PVN of MI/R rats.

MCSS: microbial community simulator based on structure.

De novo assembly plays a pivotal role in metagenomic analysis, and the incorporation of third-generation sequencing technology can significantly improve the integrity and accuracy of assembly results. Recently, with advancements in sequencing technology (Hi-Fi, ultra-long), several long-read-based bioinformatic tools have been developed. However, the validation of the performance and reliability of these tools is a crucial concern. To address this gap, we present MCSS (microbial community simulator based on structure), which has the capability to generate simulated microbial community and sequencing datasets based on the structure attributes of real microbiome communities. The evaluation results indicate that it can generate simulated communities that exhibit both diversity and similarity to actual community structures. Additionally, MCSS generates synthetic PacBio Hi-Fi and Oxford Nanopore Technologies (ONT) long reads for the species within the simulated community. This innovative tool provides a valuable resource for benchmarking and refining metagenomic analysis methods. Code available at: https://github.com/panlab-bio/mcss.

RAfilter: an algorithm for detecting and filtering false-positive alignments in repetitive genomic regions.

Telomere to telomere (T2T) assembly relies on the correctness of sequence alignments. However, the existing aligners tend to generate a high proportion of false-positive alignments in repetitive genomic regions which impedes the generation of T2T-level reference genomes for more important species. In this paper, we present an automatic algorithm called RAfilter for removing the false-positives in the outputs of existing aligners. RAfilter takes advantage of rare k-mers representing the copy-specific features to differentiate false-positive alignments from the correct ones. Considering the huge numbers of rare k-mers in large eukaryotic genomes, a series of high-performance computing techniques such as multi-threading and bit operation are used to improve the time and space efficiencies. The experimental results on tandem repeats and interspersed repeats show that RAfilter was able to filter 60%-90% false-positive HiFi alignments with almost no correct ones removed, while the sensitivities and precisions on ONT datasets were about 80% and 50% respectively.

PEGylated Cisplatin Nanoparticles for Treating Colorectal Cancer in a pH-Responsive Manner.

Colorectal cancer (CRC) is a common malignant tumor, and its incidence ranks third and mortality rate ranks second in the world. Cisplatin cannot target CRC cells and has notable toxicity, which significantly limits its clinical application. The emerging PEGylated nanodrug delivery system can improve circulation time and enhance tumor targeting. In this study, the HA-mPEG-Cis NPs were synthesized by self-assembly, which can target CD44-positive CRC cells and dissolve the PEG hydration layer responsive to the weakly acidic tumor environment. The average hydrodynamic diameter of HA-mPEG-Cis NPs was 48 nm with the polydispersity index of 0.13. The in vitro cisplatin release was in a pH-responsive manner. The HA-mPEG-Cis NPs group showed the highest apoptosis rate (25.1%). The HA-mPEG-Cis NPs exhibited antitumor efficacy via the PI3K/AKT/mTOR signaling pathway. The HA-mPEG-Cis NPs showed the lowest tumor volume and weight among all the groups in CT26 cell-bearing mouse model. The HA-mPEG-Cis nanodrug delivery system not only increases the stability and circulation time but also reduces the side effects of loaded cisplatin. Overall, the in vitro and in vivo experiments confirmed the satisfied antitumor efficacy of HA-mPEG-Cis NPs. Therefore, this study provides a rational design for application of pH-responsive HA-mPEG-Cis nanodrug delivery system in the future.

Identification and functional characterization of a cystathionine ß-lyase (CBL) enzyme for H2S production in Arabidopsis thaliana.

Sulfide or sulfur metabolism plays an important role in the growth and development of plants. Cystathionine ß-lyase (CBL) is an important enzyme in methionine synthesis, but a comprehensive understanding of CBL functions is limited. As the third gasotransmitter, hydrogen sulfide (H2S) plays important physiological roles in plants. In this study, we found that the endogenous H2S content in Arabidopsis thaliana cbl mutants was lower than that in the wild type. Under PEG-based osmotic stress conditions, the H2S contents of CBL-overexpression (OE-CBL) plants increased significantly compared with the wild type. Additionally, the OE-CBL plants increased their tolerance to osmotic stress by increasing the transcription levels of drought-related genes and their relative water-loss rates. Compared with cbl and wild type, OE-CBL plants resisted drought stress by significantly closing their stomata, resulting in improved survival rates. Root tip-bending experiments showed that CBL overexpression relieved osmotic, heavy metal and cold stresses in Arabidopsis. The recombinant CBL activity in vitro revealed that CBL produced H2S using L-cysteine as a substrate. Thus, CBL had a very strong cysteine desulfhydrase activity that could produce endogenous H2S using L-cysteine as a substrate, and it played an important role in plant abiotic stress resistance.

Identification and Analysis of Hub Genes in Diabetic Cardiomyopathy: Potential Role of Cytochrome P450 1A1 in Mitochondrial Metabolism and STZ-Induced Myocardial Dysfunction.

Diabetic cardiomyopathy (DCM) is a primary cause of death in diabetic patients; however, its molecular mechanism is not yet clear, and there is no uniform standard for diagnosis. The aim of this study is to discover the pathogenesis and potential therapeutic targets of DCM through screening and analysis of differentially expressed genes (DEGs) in heart ventricles of DCM, and to testify the role of key hub genes in DCM-induced myocardial dysfunction. Datasets GSE4745 and GSE6880 were downloaded from the GEO database. The difference analysis, visual analysis, cluster analysis and enrichment analysis were performed by using R language, python scripts and bioinformatics software followed by the construction of protein-protein interaction (PPI) network to obtain hub genes. The DCM models were established by streptozocin (STZ) injection to the male mice. The cardiac function and the expressions of hub genes were examined by using echocardiography and real-time quantitative poly-merase chain reaction (RT-qPCR), followed by multiple statistical analyses. Bioinformatic results indicate that mitochondrial dysfunction, disturbed lipid metabolism and decreased collagen synthesis are the main causes of the DCM development. In particular, the hub gene Cyp1a1 that encodes Cytochrome P450 1A1 (CYP4501A1) enzyme has the highest connectivity in the interaction network, and is associated with mitochondrial homeostasis and energy metabolism. It plays a critical role in the oxidation of endogenous or exogenous substrates. Our RT-qPCR results confirmed that ventricular Cyp1a1 mRNA level was nearly 12-fold upregulated in DCM model compared to normal control, which was correlated with abnormal cardiac function in diabetic individuals. CYP4501A1 protein expression in mitochondria was also increased in diabetic hearts. However, we found no significant changes in collagen expressions in cardiac ventricles of mice with DCM. This study provided compact data support for understanding the pathogenesis of DCM. CYP4501A1 might be considered as a potential candidate targeting for DCM therapy. Follow-up animal and clinical verifications need to be further explored.

Risk factors for high-volume lymph node metastases in cN0 papillary thyroid microcarcinoma.

BACKGROUND: Lymph node metastasis (LNM) often occurs in clinical lymph node negative (cN0) papillary thyroid microcarcinoma (PTMC). The risk factors for LNM, especially for high-volume LNM, were investigated in this study. METHODS: The medical records of 1,974 consecutive PTMC patients admitted to the Peking Union Medical College Hospital (PUMCH) from 2013 to 2015 were reviewed. Their clinicopathological features were collected. Univariate and multivariate analyses were performed to identify the risk factors for LNM/high-volume LNM. RESULTS: Of all the patients, cervical lymph node metastases were detected in 690 patients (34.95%), and high-volume LNM was detected in 75 patients (3.80%). The results of univariate analysis revealed that sex, age, chronic thyroiditis, multifocality, and tumor diameter were significantly correlated with LNM (P<0.05) and that sex, age, multifocality, and tumor diameter were significantly correlated with high-volume LNM (P<0.05). Multivariate logistic regression analysis demonstrated that male sex [odds ratio (OR) =1.657, P<0.001], multifocality (OR =1.601, P<0.001), and tumor diameter >0.5 cm (OR =1.770, P<0.001) were independent risk factors for LNM; age of 40-59 years old (OR =0.427, P<0.001), age ≥60 years old (OR =0.291, P<0.001), and chronic thyroiditis (OR =0.562, P<0.001) were independent protective factors for LNM. For high-volume LNM, male sex (OR =2.250, P=0.002), tumor diameter >0.5 cm (OR =3.664, P=0.013) and multifocality (OR =2.034, P=0.004) were independent risk factors, whereas age ≥40 years old (OR =0.240, P<0.001) was an independent protective factor. CONCLUSIONS: Lymph node metastases are common in cN0 PTMC, whereas high-volume LNM is rare. Active surveillance may be reasonable for patients with tumor diameter ≤0.5 cm, age ≥40 years old, female sex and isolated lesions.

Azorhizobium caulinodans Transmembrane Chemoreceptor TlpA1 Involved in Host Colonization and Nodulation on Roots and Stems.

Azorhizobium caulinodans ORS571 is a motile soil bacterium that interacts symbiotically with legume host Sesbania rostrata, forming nitrogen-fixing root and stem nodules. Bacterial chemotaxis plays an important role in establishing this symbiotic relationship. To determine the contribution of chemotaxis to symbiosis in A. caulinodans ORS571-S. rostrata, we characterized the function of TlpA1 (transducer-like protein in A. caulinodans), a chemoreceptor predicted by SMART (Simple Modular Architecture Research Tool), containing two N-terminal transmembrane regions. The tlpA1 gene is located immediately upstream of the unique che gene cluster and is transcriptionally co-oriented. We found that a ΔtlpA1 mutant is severely impaired for chemotaxis to various organic acids, glycerol and proline. Furthermore, biofilm forming ability of the strain carrying the mutation is reduced under certain growth conditions. Interestingly, competitive colonization ability on S. rostrata root surfaces is impaired in the ΔtlpA1 mutant, suggesting that chemotaxis of the A. caulinodans ORS571 contributes to root colonization. We also found that TlpA1 promotes competitive nodulation not only on roots but also on stems of S. rostrata. Taken together, our data strongly suggest that TlpA1 is a transmembrane chemoreceptor involved in A. caulinodans-S. rostrata symbiosis.

GPER inhibits diabetes-mediated RhoA activation to prevent vascular endothelial dysfunction.

The effect of estrogen receptors on diabetes-induced vascular dysfunction is critical, but ambiguous. Individuals with diabetic vascular disease may require estrogen receptor-specific targeted therapy in the future. The G protein-coupled estrogen receptor (GPER) has beneficial effects on vascular function. However, its fundamental mechanisms are unclear. The RhoA/Rho-kinase pathway contributes to diabetic vascular complications, whereas estrogen can suppress Rho-kinase function. Thus, we assumed that GPER inhibits diabetes-mediated RhoA activation to prevent vascular dysfunction. We further investigated the underlying mechanisms involved in this process. Vascular endothelial cells and ex vivo cultured ovariectomized (OVX) C57BL/6 mouse aortae were treated with high glucose (HG) alone or in combination with GPER agonist (G1). G1 treatment was also administered to OVX db/db mice for 8 weeks. An ex-vivo isovolumic myograph was used to analyze the endothelium-dependent vasodilation and endothelium-independent contraction of mouse aortae. Apoptosis, oxidative stress, and inflammation were attenuated in G1-pretreated vascular endothelial cells. G1 significantly decreased the phosphorylation of inhibitory endothelial nitric oxide (NO) synthase residue threonine 495 (eNOS Thr495), inhibited RhoA expression, and increased NO production. Additionally, G1 rescued the impaired endothelium-dependent relaxation and inhibited RhoA activation in the thoracic aorta of OVX db/db mice and ex-vivo cultured OVX C57BL/6 mouse aortae treated with HG. Estrogens acting via GPER could protect vascular endothelium, and GPER activation might elicit ERα-independent effect to inhibit RhoA/Rho-kinase pathway. Additionally, GPER activation might reduce vascular smooth muscle contraction by inhibiting RhoA activation. Thus, the results of the present study suggest a new therapeutic paradigm for end-stage vascular dysfunction by inhibiting RhoA/Rho-kinase pathway via GPER activation.