The molecular characteristics could supplement the staging system of pT2/T3N0M0 esophageal squamous cell carcinoma: a translational study based on a cohort with over 20 years of follow-up.

OBJECTIVE: This study aimed to construct a model based on 23 enrolled molecules to evaluate prognoses of pT2/3N0M0 esophageal squamous cell carcinoma (ESCC) patients with up to 20 years of follow-up. METHODS: The lasso-Cox model was used to identify the candidate molecule. A nomogram was conducted to develop the survival model (molecular score, MS) based on the molecular features. Cox regression and Kaplan-Meier analysis were used in this study. The concordance index (C-index) was measured to compare the predicted ability between different models. The primary endpoint was overall survival (OS). RESULTS: A total of 226 patients and 23 proteins were enrolled in this study. Patients were classified into high-risk (MS-H) and low-risk (MS-L) groups based on the MS score of 227. The survival curves showed that the MS-L cohort had better 5-year and 10-year survival rates than the MS-H group (5-year OS: 51.0% vs. 8.0%; 10-year OS: 45.0% vs. 5.0%, all p < 0.001). Furthermore, multivariable analysis confirmed MS as an independent prognostic factor after eliminating the confounding factors (Hazard ratio 3.220, p < 0.001). The pT classification was confirmed to differentiate ESCC patients' prognosis (Log-rank: p = 0.029). However, the combination of pT and MS could classify survival curves evidently (overall p < 0.001), which showed that the prognostic prediction efficiency was improved significantly by the combination of the pT and MS than by the classical pT classification (C-index: 0.656 vs. 0.539, p < 0.001). CONCLUSIONS: Our study suggested an MS for significant clinical stratification of T2/3N0M0 ESCC patients to screen out subgroups with poor prognoses. Besides, the combination of pT staging and MS could predict survival more accurately for this cohort than the pT staging system alone.

Vaccinia virus induces EMT-like transformation and RhoA-mediated mesenchymal migration.

The emerging outbreak of monkeypox is closely associated with the viral infection and spreading, threatening global public health. Virus-induced cell migration facilitates viral transmission. However, the mechanism underlying this type of cell migration remains unclear. Here we investigate the motility of cells infected by vaccinia virus (VACV), a close relative of monkeypox, through combining multi-omics analyses and high-resolution live-cell imaging. We find that, upon VACV infection, the epithelial cells undergo epithelial-mesenchymal transition-like transformation, during which they lose intercellular junctions and acquire the migratory capacity to promote viral spreading. After transformation, VACV-hijacked RhoA signaling significantly alters cellular morphology and rearranges the actin cytoskeleton involving the depolymerization of robust actin stress fibers, leading-edge protrusion formation, and the rear-edge recontraction, which coordinates VACV-induced cell migration. Our study reveals how poxviruses alter the epithelial phenotype and regulate RhoA signaling to induce fast migration, providing a unique perspective to understand the pathogenesis of poxviruses.

Lamin A/C and Vimentin as a Coordinated Regulator during Amoeboid Migration in Microscale Confined Microenvironments.

Cell migration occurs in confined microenvironments, which plays a vital role in the process of tumor metastasis. However, it is challenging to study their behaviors in vivo. Here we developed a cell squeeze system that can be scaled down to micrometers to mimic native physical confined microenvironments, wherein degrees of surface adhesion and mechanical constraints could be manipulated in order to investigate cell-migrating behaviors. Based on the microscale cell squeeze system, we found the synergistic role of lamin A/C and vimentin in cell transition and migration under strong confinement. The dynamic variations in lamin A/C and vimentin expression establish a positive feedback loop in response to confinement, effectively promoting amoeboid migration by modulating nuclear deformability while ensuring cell viability. This work shed light on modulating cell response to microenvironments by altering the expression of lamin A/C and/or vimentin, which may be a more efficient way of inhibiting cancer metastasis.

The complete plastome of Spondias dulcis (Anacardiaceae): an edible deciduous tree species from South America.

Spondias dulcis is a deciduous tree in the family Anacardiaceae. The species originates in South America and now is widely cultivated in tropical areas due to its edible fruits. In this study, we find that the length of the complete plastome of S. dulcis is 162,256 bp. It includes 35 tRNA genes and eight rRNA genes, 86 protein-coding genes and totally 130 genes. The complete plastome of S. dulcis includes a small single-copy (SSC) region of 18,459 bp, a large single-copy (LSC) region of 89,353 bp, and two inverted repeats (IRs) regions of 27,222 bp. The total G/C content of S. dulcis is 37.7%. It shows that S. dulcis is closely related to S. mombin within Anacardiaceae. It will contribute to the conservation genetics of S. dulcis and the phylogenetic studies in Anacardiaceae.

Integrated microfluidic-SERS for exosome biomarker profiling and osteosarcoma diagnosis.

Osteosarcoma is one of the most frequent primary sarcoma of bone among adolescents. Early diagnosis of osteosarcoma is the key factor to achieve high survival rate of patients. Nevertheless, traditional histological biopsy is highly invasive and associated with the risk of arousing tumor spread. Herein, we develop a method integrating microfluidics and surface-enhanced Raman spectroscopy (SERS) to isolate plasma-derived exosomes and profile multiple exosomal biomarkers for the diagnosis of osteosarcoma. The method showed highly efficient isolation of exosomes directly from human plasma and can profile exosomes based on protein biomarkers, with the detection limit down to 2 exosomes per µL. The whole assay can be performed in 5 h and only consumed 50 µL of plasma for one analysis. With the method, we analyzed the level of three protein biomarkers, i.e., CD63, vimentin (VIM) and epithelial cell adhesion molecule (EpCAM), on plasma-derived exosomes from 20 osteosarcoma patients and 20 heathy controls. Significantly higher levels of CD63, VIM and EpCAM were observed on plasma exosomes from the osteosarcoma patients compared to the healthy controls. Based on the level of the exosomal biomarkers, a classification model was built for the rapid diagnosis of osteosarcoma, with the sensitivity, specificity and accuracy of 100%, 90% and 95%, respectively. The proposed method does not require complex operations nor expensive equipment, and has great promise in clinical diagnosis of cancer as a liquid biopsy technique.

Effects of Soundscape on Flow State during Diabolo Exercise.

"Soundscape" is defined as "an acoustic environment as perceived or experienced and/or understood by a person or people, in context." The research focuses on the physical properties of sound, paying attention to the relationship between the soundscape and people. Sports provide a comprehensive context, including the athletes, their behavior, the environment, spectators, and other elements. Soundscape in sports has many important functions, such as indicating the movement process, promoting positive emotions, and enhancing the entertainment atmosphere. It is also related to the physical and mental health of people who are exercising. As a technical sport with multidimensional soundscape qualities, diabolo is very popular among older adults in China due to its easy portability and operation. In order to explore the influence mechanism of the soundscape on the mental health of older players and better guide the shaping of soundscapes at sports venues, this paper focuses on soundscape perception and proposes a two-channel (direct and indirect) model of the influence of the diabolo soundscape on the psychological perception of older players. At the same time, we introduce the flow state as an indicator to evaluate mental health, and use the Short Flow State Scale (SFSS) to measure the flow state. By setting up a control experiment using the diabolo with and without sound and using the two-channel (direct and indirect) influence model to compare the differences in flow state scores under the two states, we analyzed the influence mechanism of the diabolo soundscape on the flow state of elderly groups. The results show that the scores of various flow state dimensions and total flow scores in the silent state of diabolo exercise are significantly lower than those in the sound state (p < 0.05 and p < 0.001), and the main differences are in the three dimensions of unambiguous feedback, sense of control, and autotelic experience. The two-channel influence model can effectively explain the differences in older adults' flow state, which provides a new comprehensive perspective to study the effect of the soundscape in sports on people's mental health.

Topological and enzymatic analysis of human Alg2 mannosyltransferase reveals its role in lipid-linked oligosaccharide biosynthetic pathway.

N-glycosylation starts with the biosynthesis of lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum (ER). Alg2 mannosyltransferase adds both the α1,3- and α1,6-mannose (Man) onto ManGlcNAc2-pyrophosphate-dolichol (M1Gn2-PDol) in either order to generate the branched M3Gn2-PDol product. The well-studied yeast Alg2 interacts with ER membrane through four hydrophobic domains. Unexpectedly, we show that Alg2 structure has diverged between yeast and humans. Human Alg2 (hAlg2) associates with the ER via a single membrane-binding domain and is markedly more stable in vitro. These properties were exploited to develop a liquid chromatography-mass spectrometry quantitative kinetics assay for studying purified hAlg2. Under physiological conditions, hAlg2 prefers to transfer α1,3-Man onto M1Gn2 before adding the α1,6-Man. However, this bias is altered by an excess of GDP-Man donor or an increased level of M1Gn2 substrate, both of which trigger production of the M2Gn2(α-1,6)-PDol. These results suggest that Alg2 may regulate the LLO biosynthetic pathway by controlling accumulation of M2Gn2 (α-1,6) intermediate.

How Physical Factors Coordinate Virus Infection: A Perspective From Mechanobiology.

Pandemics caused by viruses have threatened lives of thousands of people. Understanding the complicated process of viral infection provides significantly directive implication to epidemic prevention and control. Viral infection is a complex and diverse process, and substantial studies have been complemented in exploring the biochemical and molecular interactions between viruses and hosts. However, the physical microenvironment where infections implement is often less considered, and the role of mechanobiology in viral infection remains elusive. Mechanobiology focuses on sensation, transduction, and response to intracellular and extracellular physical factors by tissues, cells, and extracellular matrix. The intracellular cytoskeleton and mechanosensors have been proven to be extensively involved in the virus life cycle. Furthermore, innovative methods based on micro- and nanofabrication techniques are being utilized to control and modulate the physical and chemical cell microenvironment, and to explore how extracellular factors including stiffness, forces, and topography regulate viral infection. Our current review covers how physical factors in the microenvironment coordinate viral infection. Moreover, we will discuss how this knowledge can be harnessed in future research on cross-fields of mechanobiology and virology.

ErbB4 regulate extracellular dopamine through the p38 MAPK signaling pathway.

ErbB4 loss-of-function in catecholaminergic neurons induces catecholamine dyshomeostasis. Despite ErbB4's significant role in neuropathology, the signaling pathways that regulate these changes are still widely unknown. In this study, we attempt to identify the downstream pathway of ErbB4 that regulates catecholamine homeostasis. The SH-SY5Y human neuroblastoma cell line was used as the in vitro model for catecholaminergic neurons. Western blotting, enzyme-linked immunosorbent assay, and pharmacological and genetic manipulations by agonist/antagonist or small interference RNA were used to investigate the relationship between ErbB4 and extracellular catecholamines. We confirmed that ErbB4 is abundantly expressed in undifferentiated and retinoic acid-differentiated catecholaminergic cells from the SH-SY5Y cell line. ErbB4 inhibition increase the ratio of phosphorylated p38 to total p38 in SH-SY5Y human neuroblastoma cells. Consistent with previous in vivo observations in mice, ErbB4 deficiency led to increases in extracellular dopamine and norepinephrine levels. However, the resulting increase in extracellular dopamine, but not norepinephrine, could be suppressed by p38 inhibitor SB202190. Our results suggest that both extracellular dopamine and norepinephrine homeostasis could be regulated by ErbB4 in human catecholaminergic cells, and ErbB4 may regulate extracellular dopamine, but not norepinephrine, through the p38 MAPK signaling pathway, thus indicating different regulatory pathways of dopamine and norepinephrine by ErbB4 in catecholaminergic neurons.

[Advanced Nitrogen Removal Characteristics of Low Carbon Source Municipal Wastewater Treatment via Partial-denitrification Coupled with ANAMMOX].

Partial-denitrification coupled with ANAMMOX is a novel biological nitrogen removal technology, which is expected to significantly reduce the external carbon source dosage for advanced nitrogen removal from municipal wastewater. In this study, ANAMMOX sludge was inoculated to investigate advanced nitrogen removal performance and sludge characteristics in a partial-denitrification/ANAMMOX reactor. The results showed that inoculation of ANAMMOX sludge could quickly start the partial-denitrification/ANAMMOX reactor. The effluent total nitrogen concentrations were (4.82±1.84) mg·L-1 with a chemical oxygen demand of 2.19±0.08. Sludge particles larger than 0.20 mm accounted for 86.16% in the reactor. This meant that granular sludge was formed, which was conducive to good retention of ANAMMOX bacteria in the reactor. The external carbon source dosage and the oxygen requirement for nitrification can be reduced by applying partial-denitrification coupled with ANAMMOX to advanced nitrogen removal from the effluent of secondary clarifier in municipal wastewater treatment plants.

Electrically precise control of the spin polarization of electronic transport at the single-molecule level.

Compared with the conventional magnetic means (such as ferromagnetic contacts), controlling a spin current by electrical methods could largely reduce the energy consumption and dimensions of nano-devices, which has become a focus of research in spintronics. Inspired by recent progress in the synthesis of an iron-based metal-organic nanostructure, we investigate the spin-dependent electronic transport of the molecule of Fe3-terpyridine-phenyl-phenyl-terpyridine-Fe3 (Fe3-TPPT-Fe3) through first-principles calculations, and propose a three-terminal device without ferromagnetics. By applying a gate voltage, not only the spin polarization can be switched between 100% and -100% to achieve a dual-spin filter, but also its fine regulation can be realized, where the transmission with any ratio of spin-up to spin-down electron numbers is achievable. Analysis shows that the particular transmission spectra are the key mechanism, where two peaks reside discretely on both sides of the Fermi level with opposite spins. Such a feature is found to be robust to the number of Fe atoms and TPPT chain length, suggesting that it is an intrinsic feature of such systems and very conducive to practical applications. The electrical control (such as an electric field) of spin polarization is realized at the single-molecule level, showing great application potential.

Using 17ß-estradiol heparin-poloxamer thermosensitive hydrogel to enhance the endometrial regeneration and functional recovery of intrauterine adhesions in a rat model.

Mechanical damage or infection to the endometrium can lead to the formation of adhesions in the uterine cavity, which may result in reduced reproductive outcome and/or pregnancy complications. The prognosis of this disease is poor due to few effective treatments and the complex environment of endometrium. Heparin-Poloxamer Hydrogel (HP hydrogel) is a nontoxic and biodegradable biomaterial, which has been commonly used as a sustained-release delivery system. In this study, we applied a mini-endometrial curette to scrape the endometrium of rats to mimic the process of curettage in patients. After the establishment of IUA model in rats, we injected the thermo-sensitive hydrogel(E2-HP hydrogel) into the injured uterine cavity and evaluated the therapeutic effect of E2-HP hydrogel on the recovery of IUA. Our results showed that E2-HP hydrogel can significantly facilitate the regeneration of injured endometrium along with inhibiting the cell apoptosis in IUA model. Furthermore, we revealed that E2-HP hydrogel on the recovery of IUA was closely associated with the upregulation of kisspeptin through activating the ERK1/2 and MAPKs p38 pathways. In conclusion, E2-HP hydrogel can effectively transfer E2 into the injured endometrium and it can be considered as a promising therapeutic method for the women with intrauterine adhesions.

Reconstitution of the lipid-linked oligosaccharide pathway for assembly of high-mannose N-glycans.

The asparagine (N)-linked Man9GlcNAc2 is required for glycoprotein folding and secretion. Understanding how its structure contributes to these functions has been stymied by our inability to produce this glycan as a homogenous structure of sufficient quantities for study. Here, we report the high yield chemoenzymatic synthesis of Man9GlcNAc2 and its biosynthetic intermediates by reconstituting the eukaryotic lipid-linked oligosaccharide (LLO) pathway. Endoplasmic reticulum mannosyltransferases (MTases) are expressed in E. coli and used for mannosylation of the dolichol mimic, phytanyl pyrophosphate GlcNAc2. These recombinant MTases recognize unique substrates and when combined, synthesize end products that precisely mimic those in vivo, demonstrating that ordered assembly of LLO is due to the strict enzyme substrate specificity. Indeed, non-physiological glycans are produced only when the luminal MTases are challenged with cytosolic substrates. Reconstitution of the LLO pathway to synthesize Man9GlcNAc2 in vitro provides an important tool for functional studies of the N-linked glycoprotein biosynthesis pathway.

Metformin Promotes Regeneration of the Injured Endometrium Via Inhibition of Endoplasmic Reticulum Stress-Induced Apoptosis.

Intrauterine adhesion (IUA) is now recognized as one of the most common diseases in reproductive-age women. Metformin, a well-known frontline oral antidiabetic drug, has been found effective in numerous different diseases. The aim of this study was to determine the effect of metformin on reducing adhesions in an animal model of IUA. Sprague-Dawley rats were randomized into 4 groups: sham operation, control, metformin-treated for 7 days, and metformin-treated for 14 days. To establish the IUA model, mechanical injury to the endometria of rats was induced with a mini curette. Metformin was injected intraperitoneally after surgery. A significant amelioration in both the number of glands and the fibrotic area, compared to those of the control group, was detected 14 days after metformin intervention. The expression levels of antigen KI-67 and vascular endothelial growth factor were increased at 7 and 14 days after treatment. However, the transforming growth factor-ß expression was decreased at 14 days after treatment. Endoplasmic reticulum stress-related apoptosis proteins (glucose-regulated protein 78, caspase-12, and CCAAT/enhancer binding protein (EBP) homologous protein) were downregulated after metformin treatment. Moreover, we determined that the effect of metformin was related to the inhibition of endoplasmic reticulum stress-induced apoptosis via the Phosphatidylinositol 3 kinase (PI3K)/Protein kinase B (AKT) and Extracellular regulated protein kinases1/2 pathways. In conclusion, metformin can attenuate the adhesion and promote the regeneration of the endometrium of the IUA rat, and metformin may serve as a novel therapeutic strategy for IUA patients.

Structural and functional analysis of Alg1 beta-1,4 mannosyltransferase reveals the physiological importance of its membrane topology.

In eukaryotes, the biosynthesis of a highly conserved dolichol-linked oligosaccharide (DLO) precursor Glc3Man9GlcNAc2-pyrophosphate-dolichol (PP-Dol) begins on the cytoplasmic face of the endoplasmic reticulum (ER) and ends within the lumen. Two functionally distinguished heteromeric glycosyltransferase (GTase) complexes are responsible for the cytosolic DLO assembly. Alg1, a ß-1, 4 mannosyltransferase (MTase) physically interacts with Alg2 and Alg11 proteins to form the multienzyme complex which catalyzes the addition of all five mannose to generate the Man5GlcNAc2-PP-Dol intermediate. Despite the fact that Alg1 plays a central role in the formation of the multi-MTase has been confirmed, the topological information of Alg1 including the molecular mechanism of membrane association are still poorly understood. Using a combination of bioinformatics and biological approaches, we have undertaken a structural and functional study on Alg1 protein, in which the enzymatic activities of Alg1 and its variants were monitored by a complementation assay using the GALpr-ALG1 yeast strain, and further confirmed by a liquid chromatography-mass spectrometry-based in vitro quantitative assay. Computational and experimental evidence confirmed Alg1 shares structure similarity with Alg13/14 complex, which has been defined as a membrane-associated GT-B GTase. Particularly, we provide clear evidence that the N-terminal transmembrane domain including the following positively charged amino acids and an N-terminal amphiphilic-like α helix domain exposed on the protein surface strictly coordinate the Alg1 orientation on the ER membrane. This work provides detailed membrane topology of Alg1 and further reveals its biological importance at the spatial aspect in coordination of cytosolic DLO biosynthesis.

Creation of a rabbit model for intrauterine adhesions using electrothermal injury.

The pathogenesis and therapeutic treatment of intrauterine adhesions (IUAs) remain unsolved, highlighting the need for stable and effective experimental animal models. In this study, uterine electrocoagulation of twenty-one female New Zealand White rabbits was carried out to establish an IUA model. As rabbits have two completely separate uterine horns, each rabbit had its own internal control: one uterine horn was given an electrothermal injury (Group A, n=21), and the contralateral uterine horn received no treatment and served as the control (Group B, n=21). The endometrial morphology, number of endometrial glands, area of endometrial fibrosis, and number of implanted fetuses were compared between the two groups. In Group A, the numbers of endometrial glands on Days 7 and 14 and the number of implanted fetuses were significantly lower than those in Group B (P<0.05, P<0.05, and P<0.01, respectively), while the ratio of the area with endometrial stromal fibrosis to the total endometrial area was significantly increased (P<0.01). These results suggest that this method of electrothermal injury is effective for the establishment of a rabbit IUA model between 7 and 14 d after surgery.

Alternative routes for synthesis of N-linked glycans by Alg2 mannosyltransferase.

Asparagine ( N)-linked glycosylation requires the ordered, stepwise synthesis of lipid-linked oligosaccharide (LLO) precursor Glc3Man9GlcNAc2-pyrophosphate-dolichol (Glc3Man9Gn2-PDol) on the endoplasmic reticulum. The fourth and fifth steps of LLO synthesis are catalyzed by Alg2, an unusual mannosyltransferase (MTase) with two different MTase activities; Alg2 adds both an α1,3- and α1,6-mannose onto ManGlcNAc2-PDol to form the trimannosyl core Man3GlcNAc2-PDol. The biochemical properties of Alg2 are controversial and remain undefined. In this study, a liquid chromatography/mass spectrometry-based quantitative assay was established and used to analyze the MTase activities of purified yeast Alg2. Alg2-dependent Man3GlcNAc2-PDol production relied on net-neutral lipids with a propensity to form bilayers. We further showed addition of the α1,3- and α1,6-mannose can occur independently in either order but at differing rates. The conserved C-terminal EX7E motif, N-terminal cytosolic tail, and 3 G-rich loop motifs in Alg2 play crucial roles for these activities, both in vitro and in vivo. These findings provide insight into the unique bifunctionality of Alg2 during LLO synthesis and lead to a new model in which alternative, independent routes exist for Alg2 catalysis of the trimannosyl core oligosaccharide.-Li, S.-T., Wang, N., Xu, X.-X., Fujita, M., Nakanishi, H., Kitajima, T., Dean, N., Gao, X.-D. Alternative routes for synthesis of N-linked glycans by Alg2 mannosyltransferase.