Characterization and engineering of peroxisome targeting sequences for compartmentalization engineering in Pichia pastoris.

Peroxisomal compartmentalization has emerged as a highly promising strategy for reconstituting intricate metabolic pathways. In recent years, significant progress has been made in the peroxisomes through harnessing precursor pools, circumventing metabolic crosstalk, and minimizing the cytotoxicity of exogenous pathways. However, it is important to note that in methylotrophic yeasts (e.g. Pichia pastoris), the abundance and protein composition of peroxisomes are highly variable, particularly when peroxisome proliferation is induced by specific carbon sources. The intricate subcellular localization of native proteins, the variability of peroxisomal metabolic pathways, and the lack of systematic characterization of peroxisome targeting signals have limited the applications of peroxisomal compartmentalization in P. pastoris. Accordingly, this study established a high-throughput screening method based on ß-carotene biosynthetic pathway to evaluate the targeting efficiency of PTS1s (Peroxisome Targeting Signal Type 1) in P. pastoris. First, 25 putative endogenous PTS1s were characterized and 3 PTS1s with high targeting efficiency were identified. Then, directed evolution of PTS1s was performed by constructing two PTS1 mutant libraries, and a total of 51 PTS1s (29 classical and 22 noncanonical PTS1s) with presumably higher peroxisomal targeting efficiency were identified, part of which were further characterized via confocal microscope. Finally, the newly identified PTS1s were employed for peroxisomal compartmentalization of the geraniol biosynthetic pathway, resulting in more than 30% increase in the titer of monoterpene compared with when the pathway was localized to the cytosol. The present study expands the synthetic biology toolkit and lays a solid foundation for peroxisomal compartmentalization in P. pastoris.

Biomimetic and Wound Microenvironment-Modulating PEGylated Glycopolypeptide Hydrogels for Arterial Massive Hemorrhage and Wound Prohealing.

Despite great progress in the hydrogel hemostats and dressings, they generally lack resistant vascular bursting pressure and intrinsic bioactivity to meet arterial massive hemorrhage and proheal wounds. To address the problems, we design a kind of biomimetic and wound microenvironment-modulating PEGylated glycopolypeptide hydrogels that can be easily injected and gelled in ∼10 s. Those glycopolypeptide hydrogels have suitable tissue adhesion of ∼20 kPa, high resistant bursting pressure of ∼150 mmHg, large microporosity of ∼15 µm, and excellent biocompatibility with ∼1% hemolysis ratio and negligible inflammation. They performed better hemostasis in rat liver and rat and rabbit femoral artery bleeding models than Fibrin glue, Gauze, and other hydrogels, achieving fast arterial hemostasis of <20 s and lower blood loss of 5-13%. As confirmed by in vivo wound healing, immunofluorescent imaging, and immunohistochemical and histological analyses, the mannose-modified hydrogels could highly boost the polarization of anti-inflammatory M2 phenotype and downregulate pro-inflammatory tumor necrosis factor-α to relieve inflammation, achieving complete full-thickness healing with thick dermis, dense hair follicles, and 90% collagen deposition. Importantly, this study provides a versatile strategy to construct biomimetic glycopolypeptide hydrogels that can not only resist vascular bursting pressure for arterial massive hemorrhage but also modulate inflammatory microenvironment for wound prohealing.

A dominant variant in apoptosis-related gene XKR8 is relevant to hereditary auditory neuropathy.

BACKGROUND: Auditory neuropathy is an unusual type of hearing loss. At least 40% of patients with this disease have underlying genetic causes. However, in many hereditary auditory neuropathy cases, etiology remains undetermined. METHODS: We collected data and blood samples from a four-generation Chinese family. After excluding relevant variants in known deafness-related genes, exome sequencing was conducted. Candidate genes were verified by pedigree segregation, transcript/protein expression in the mouse cochlea, and plasmid expression studies in HEK 293T cells. Moreover, a mutant mouse model was generated and underwent hearing evaluations; protein localization in the inner ear was also assessed. RESULTS: The clinical features of the family were diagnosed as auditory neuropathy. A novel variant c.710G > A (p.W237X) in apoptosis-related gene XKR8 was identified. Genotyping of 16 family members confirmed the segregation of this variant with the deafness phenotype. Both XKR8 mRNA and XKR8 protein were expressed in the mouse inner ear, predominantly in regions of spiral ganglion neurons; Moreover, this nonsense variant impaired the surface localization of XKR8 in cells. Transgenic mutant mice exhibited late-onset auditory neuropathy, and their altered XKR8 protein localization in the inner ear confirmed the damaging effects of this variant. CONCLUSIONS: We identified a variant in the XKR8 gene that is relevant to auditory neuropathy. The essential role of XKR8 in inner ear development and neural homeostasis should be explored.

Modulating photothermal properties by integration of fined Fe-Co in confined carbon layer of SiO2 nanosphere for pollutant degradation and solar water evaporation.

Environmental governance by photothermal materials especially for the separation of organic pollutants and regeneration of freshwater afford growing attention owing to their special solar-to-heat properties. Here, we construct a special functional nanosphere composed of an internal silica core coated by a thin carbon layer encapsulated plasmonic bimetallic FeCo2O4 spinel (SiO2@CoFe/C) by a facile self-assembled approach and tuned calcination. Through combining the advantage of bimetallic Fe-Co and carbon layer, this obtained nanosphere affords improved multiple environmental governing functions including peroxymonosulfate (PMS) activation to degrade pollutants and photothermal interfacial solar water evaporation. Impressively, fined bimetal (FeCo) species (20 nm) acted as main catalytic substance were distributed on the N-doping carbon thin layer, which favors electron transfer and reactive accessibility of active metals. The increasing treatment temperature of catalysts caused the optimization of the surface active metal species and tuning catalytic properties in the AOPs. Besides, the incorporation of Co in the SiO2@CoFe/C-700 could enable the improved PMS activation efficiency compared to SiO2@Fe/C-700 and the mixed SiO2@Co/C-700 and SiO2@Fe/C-700, hinting a synergetic promotion effect. The bimetal coupled catalyst SiO2@CoFe/C-700 affords enhanced photothermal properties compared to SiO2@Co/C-700. Furthermore, photothermal catalytic PMS activation using optimal SiO2@CoFe/C-700 was further explored in addressing stubborn pollutants including oxytetracycline, sulfamethoxazole, 2, 4-dichlorophenol, and phenol. The free radical quenching control suggests that both the sulfate radical, hydroxyl radical, superoxide radical, and singlet oxygen species are involved in the degradation, while the hydroxyl radical and singlet oxygen play a dominant role. Furthermore, the implementation of a solar-driven interfacial water evaporation model using SiO2@CoFe/C-700 was further studied to obtain freshwater regeneration (1.26 kg m-2 h-1, 76.81% efficiency), indicating the comprehensive ability of the constructed nanocomposites for treating complicated environmental pollution including organics removal and freshwater regeneration.

Construction of ajmalicine and sanguinarine de novo biosynthetic pathways using stable integration sites in yeast.

Yeast cell factories have been increasingly employed for producing plant-derived natural products. Unfortunately, the stability of plant natural product biosynthetic pathway genes, particularly when driven by the same sets of promoters and terminators, remains one of the biggest concerns for synthetic biology. Here we profile genomic loci flanked by essential genes as stable integration sites in a genome-wide manner, for stable maintenance of multigene biosynthetic pathways in yeast. We demonstrate the application of our yeast integration platform in the construction of sanguinarine (24 expression cassettes) and ajmalicine (29 expression cassettes) de novo biosynthetic pathways for the first time. Moreover, we establish stable yeast cell factories that can produce 119.2 mg L-1 heteroyohimbine alkaloids (containing 61.4 mg L-1 ajmalicine) in shake flasks, representing the highest titer of monoterpene indole alkaloids (MIAs) ever reported and promising the complete biosynthesis of other high-value MIAs (such as vinblastine) for biotechnological applications.

Multivalent Polypeptide and Tannic Acid Cooperatively Iron-Coordinated Nanohybrids for Synergistic Cancer Photothermal Ferroptosis Therapy.

Owing to having a unique mechanism to kill cancer cells via the membrane accumulation of lipid peroxide (LPO) and the downregulation of glutathione peroxidase-4 (GPX-4), the ferroptosis therapy (FT) of tumors based on the Fenton reaction of iron nanoparticles has been receiving much attention in the past decade; however, there are some hurdles including the uncontrollable release of iron ions, slower kinetics of the intracellular Fenton reaction, and poor efficacy of FT that need to be overcome. Considering cooperative coordination of a multivalent thiol-pendant polypeptide ligand with iron ions, we put forward a facile strategy for constructing the iron-coordinated nanohybrid of methacryloyloxyethyl phosphorylcholine-grafted polycysteine/iron ions/tannic acid (i.e., PCFT), which could deliver a higher concentration of iron ions into cells. The dynamic and unsaturated coordination in PCFT is favorable for the intracellular stimuli-triggered release and fast Fenton reaction to realize efficient FT, while its intrinsic photothermia would boost the Fenton reaction to induce a synergistic effect between FT and photothermal therapy (PTT). Both immunofluorescence analyses of reactive oxygen species (ROS) and LPO confirmed that the intracellular Fenton reaction resulted in efficient FT, during which process the photothermia greatly boosted ferroptosis, and the Western blot assay corroborated that the expression level of GPX-4 was downregulated by FT and highly degraded by the photothermia to induce synergistic PTT-FT in vitro. Excitingly, by a single intravenous dose of PCFT plus one NIR irradiation, in vivo PTT-FT treatment completely eradicated 4T1 tumors without skin scar and tumor recurrence for 16 days, demonstrating prominent antitumor efficacy, as evidenced by the GPX-4, H&E, and TUNEL assays.

Exercise Rehabilitation Training in Patients With Pulmonary Hypertension: A Review.

BACKGROUND: Pulmonary hypertension (PH) has a high morbidity and mortality. Despite the existing disease-targeted therapy, most patients with PH continue to suffer from difficulty in breathing, exercise intolerance, and reduced quality of life. Recently, a large body of research results has suggested that exercise rehabilitation training (ERT) seems to be a beneficial, safe, and cost-effective treatment for patients with PH. However, knowledge gaps still exist for a uniformly accepted ERT protocol, the modality, duration, intensity, and frequency of ERT in PH. AIM: The purpose of this review is to summarise the existing research evidence and knowledge, aiming to strengthen clinicians' awareness of the application of ERT in patients with PH. METHODS AND RESULTS: PubMed databases were systematically searched for eligible studies. Twelve (12) randomised controlled trials and other important studies documenting effectiveness, safety, and adverse events of ERT are summarised. Additionally, the modality, duration, intensity, and frequency of various types of ERT and future research directions are discussed. CONCLUSIONS: In summary, ERT is generally effective and safe for PH patients as an adjuvant treatment to disease-specific therapy. It can improve the exercise capacity and tolerance, skeletal and respiratory muscle performance, cardiopulmonary function, and quality of life of PH patients. RECOMMENDATIONS: In view of the occurrence of a small number of adverse events, we currently recommend a combination of in-hospital and home-based ERT under close supervision. In the future, more multi-centre randomised controlled studies are needed to evaluate the effectiveness and feasibility of long-term, community, or home-based ERT, as well as to explore the molecular mechanism behind it.

Identification of novel metabolic engineering targets for S-adenosyl-L-methionine production in Saccharomyces cerevisiae via genome-scale engineering.

S-Adenosyl-L-methionine (SAM) is an important intracellular metabolite and widely used for treatment of various diseases. Although high level production of SAM had been achieved in yeast, novel metabolic engineering strategies are needed to further enhance SAM production for industrial applications. Here genome-scale engineering (GSE) was performed to identify new targets for SAM overproduction using the multi-functional genome-wide CRISPR (MAGIC) system, and the effects of these newly identified targets were further validated in industrial yeast strains. After 3 rounds of FACS screening and characterization, numerous novel targets for enhancing SAM production were identified. In addition, transcriptomic and metabolomic analyses were performed to investigate the molecular mechanisms for enhanced SAM accumulation. The best combination (upregulation of SNZ3, RFC4, and RPS18B) improved SAM productivity by 2.2-fold and 1.6-fold in laboratory and industrial yeast strains, respectively. Using GSE of laboratory yeast strains to guide industrial yeast strain engineering presents an effective approach to design microbial cell factories for industrial applications.

Employing DIALux to relieve machine-learning training data collection when designing indoor positioning systems.

We propose and demonstrate using the DIALux software with our proposed linear-regression machine-learning (LRML) algorithm for designing a practical indoor visible light positioning (VLP) system. Experimental results reveal that the average position errors and error distributions of the model trained via the DIALux simulation and trained via the experimental data match with each other. This implies that the training data can be generated in DIALux if the room dimensions and LED luminary parameters are available. The proposed scheme could relieve the burden of training data collection in VLP systems.

Cloning and characterization of a panel of mitochondrial targeting sequences for compartmentalization engineering in Saccharomyces cerevisiae.

Mitochondrion is generally considered as the most promising subcellular organelle for compartmentalization engineering. Much progress has been made in reconstituting whole metabolic pathways in the mitochondria of yeast to harness the precursor pools (i.e., pyruvate and acetyl-CoA), bypass competing pathways, and minimize transportation limitations. However, only a few mitochondrial targeting sequences (MTSs) have been characterized (i.e., MTS of COX4), limiting the application of compartmentalization engineering for multigene biosynthetic pathways in the mitochondria of yeast. In the present study, based on the mitochondrial proteome, a total of 20 MTSs were cloned and the efficiency of these MTSs in targeting heterologous proteins, including the Escherichia coli FabI and enhanced green fluorescence protein (EGFP) into the mitochondria was evaluated by growth complementation and confocal microscopy. After systematic characterization, six of the well-performed MTSs were chosen for the colocalization of complete biosynthetic pathways into the mitochondria. As proof of concept, the full α-santalene biosynthetic pathway consisting of 10 expression cassettes capable of converting acetyl-coA to α-santalene was compartmentalized into the mitochondria, leading to a 3.7-fold improvement in the production of α-santalene. The newly characterized MTSs should contribute to the expanded metabolic engineering and synthetic biology toolbox for yeast mitochondrial compartmentalization engineering.

Pseudotear Sign of the Anterior Horn of the Meniscus.

PURPOSE: To observe the morphology of the transverse geniculate ligament of the knee (TGL) by magnetic resonance imaging (MRI) and to analyze the cause of the pseudotear sign of the anterior horn of the meniscus caused by the TGL. METHODS: Patients who underwent MRI examination of the knee joint in the orthopaedics department of our hospital from July 2016 to August 2019 were identified. The occurrence rate, length, width, thickness, cross-sectional shape, pattern, appearance, and position relative to the anterior horn of the lateral and medial meniscus and anatomical variations were observed by multiplane and multisequence MRI. The frequency and cause of the pseudotear sign also were observed. RESULTS: The data of 101 patients were analyzed. Among them, 60 were male, and 41 were female. The average age was 42.01 (18-75) years. The occurrence rate of the TGL was 67.3% (68/101), the average length was 38.75 ± 3.56 mm, the median coronal diameter was 1.79 ± 0.60 mm, the median sagittal diameter was 1.88 ± 0.35 mm, and the cross-sectional morphology was mostly oval and round. There were 5 types of TGL connection to the anterior horn of the medial meniscus: type 1, located at the front edge; type 2, located at the upper front edge; type 3, located at the upper edge; type 4, located at the back upper edge; and type 5, was located at the back edge of the anterior horn of the medial meniscus. There was only one type of TGL insertion into the anterior horn of the lateral meniscus, located at the anterior superior edge of the anterior horn of the lateral meniscus. There were 4 cases of the pseudotear sign in the anterior horn of the meniscus, 3 in the lateral meniscus and 1 in the medial meniscus. The pseudotear sign of the anterior horn of the meniscus caused by the TGL was observed at a rate of 5.88% (4/68). CONCLUSIONS: In MRI examination of the knee, the anterior horn of the meniscus sometimes shows a pseudotear sign. According to the shape and route of the TGL on MRI and the direction and position of the pseudotear sign of the anterior horn of the meniscus, true and false tears of the anterior horn of the meniscus can be identified. LEVEL OF EVIDENCE: Level III, diagnostic study (retrospective, noncomparative, observational case series without a consistently applied reference "gold" standard).

Hybrid Fiber-Optic Sensing Integrating Brillouin Optical Time-Domain Analysis and Fiber Bragg Grating for Long-Range Two-Parameter Measurement.

Distributed fiber sensing (DFS) can provide real-time signals and warnings. The entire length of fiber optic cable can act as a sensing element, but the accuracy is sometimes limited. On the other hand, point-to-point fiber sensing (PPFS) is usually implemented using one or more fiber Bragg gratings (FBGs) at specific positions along with the fiber for the monitoring of specific parameters (temperature, strain, pressure, and so on). However, the cost becomes expensive when the number of FBGs increases. A hybrid fiber sensing scheme is thus proposed, combining the advantages of DFS and PPFS. It is based on a Brillouin optical time-domain analysis (BOTDA) fiber system with additional FBGs embedded at certain positions where it is necessary to detect specific parameters. The hybrid fiber sensing system has the advantages of full sensing coverage at essential locations that need to be carefully monitored. In our work, the test results showed that the proposed system could achieve a sensing distance of 16 km with the single-mode fiber with a 2 m spatial resolution. For FBG parameter measurements, the temperature variation was 52 °C, from 25 °C to 77 °C, with a temperature sensitivity of 23 pm/°C, and the strain was from 0 to 400 µÎµ, with a strain sensitivity of 0.975 pm/µÎµ, respectively, using two FBGs.

PLA2G10 facilitates the cell-cycle progression of soft tissue leiomyosarcoma cells at least by elevating cyclin E1/CDK2 expression.

Soft tissue leiomyosarcoma (STLMS) is a major histological subtype of adult sarcoma. Although the molecular mechanisms ofLMS have been gradually revealed, no valid therapeutic targets have been identified. In this study, we performed a systematic screening to explore relapse-associated genes in STLMS, using data from The Cancer Genome Atlas-Sarcoma (TCGA-SARC). Then, we investigated the functional role of the gene with the best relapse-prediction value in STLMS by both in-vitro and in-vivo studies. Results showed that AMH and PLA2G10 were two genes with area under curve (AUC) values higher than 0.80 in ROC analysis when detecting relapse. Patients in the high AMH or PLA2G10 expression group had significantly worse relapse-free survival (RFS) compared to the respective low expression group. PLA2G10 was highly expressed in STLMS, but not in other sarcoma subtypes. PLA2G10 overexpression promoted SK-LMS-1 cell growth and G1/S transition, while PLA2G10 knockdown slowed the growth and resulted in G1 phase arrest. PLA2G10 overexpression markedly increased the expression of CDK2 and cyclin E1, but did not influence CDK4, CDK6, cyclin D1, CDK1 or cyclin A expression. PLA2G10 overexpression enhanced SK-LMS-1 cell-derived xenograft tumor growth in nude mice, while PLA2G10 inhibition slowed the growth. Mutation of two critical catalyzing amino acid residues (p.H88A and p.D89A) abrogated the capability of PLA2G10 to catalyze the production of arachidonic acid (AA), and also canceled the regulatory effects on cyclin E1 and CDK2 expression, as well as G1/S transition. In conclusion, PLA2G10 was a specific relapse-associated gene in STLMS. It facilitated the cell-cycle progression of STLMS cells at least by elevating the expression of cyclin E1 and CDK2. The hydrolytic activity was crucial for its oncogenic properties.

Photoresponsive Polypeptide-Glycosylated Dendron Amphiphiles: UV-Triggered Polymersomes, OVA Release, and In Vitro Enhanced Uptake and Immune Response.

Efficient therapeuic proteins' delivery into mammalian cells and subcellular transport (e.g., fast escape from endolysosomes into cytoplasm) are two key biological barriers that need to be overcome for antigen-based immunotherapy and related biomedical applications. For those purposes, we designed a novel kind of photoresponsive polypeptide-glycosylated poly(amidoamine) (PAMAM) dendron amphiphiles (PGDAs), and their synthesis, UV-responsive self-assembly, and triggered ovalbumin (OVA) release have been fully investigated. The highly anisotropic PGDA4 with a glycosylated second-generation PAMAM dendron self-assembled into stable polypeptide vesicles (polymersomes) within 20-50 wt % water, which exhibited UV-responsive reassembly, dynamic binding with a lectin of concanavalin A, and an accelerated OVA release in vitro. Moreover, upon 365 nm UV irradiation, the self-assembled polymersomes of those glycopolypeptides were transformed into micellar aggregates in aqueous solution at pH 7.4 but disassembled completely at pH 5. The OVA-loaded polymersomes could efficiently deliver OVA into RAW264.7 cells and achieve enhanced endolysosomes escape upon UV irradiation, as revealed by flow cytometry and confocal laser scanning microscopy (CLSM). Furthermore, the enzyme-linked immunosorbent assay (ELISA) showed that the blank sugar-coated polypeptidosomes activated a high level of tumor necrosis factor α (TNF-α) of 468 pg/mL, playing a better role of immune adjuvant for activating the macrophages. Upon the UV irradiation with a dose of 3 J/cm2, the OVA-loaded polymersomes could further stimulate RAW264.7 and enhance the TNF-α level by about 45%. Consequently, this work provides a versatile platform to construct photosensitive and sugar-coated polymersomes of glycopolypeptides that have potential applications for protein delivery, immune adjuvant, and antigen-based immunotherapy.

Cardiac manifestations of COVID-19 in Shenzhen, China.

PURPOSE: The coronavirus disease 2019 (COVID-19) outbreak has become a global public health concern; however, relatively few detailed reports of related cardiac injury are available. The aims of this study were to compare the clinical and echocardiographic characteristics of inpatients in the intensive-care unit (ICU) and non-ICU patients. METHODS: We recruited 416 patients diagnosed with COVID-19 and divided them into two groups: ICU (n = 35) and non-ICU (n = 381). Medical histories, laboratory findings, and echocardiography data were compared. RESULTS: The levels of myocardial injury markers in ICU vs non-ICU patients were as follows: troponin I (0.029 ng/mL [0.007-0.063] vs 0.006 ng/mL [0.006-0.006]) and myoglobin (65.45 µg/L [39.77-130.57] vs 37.00 µg/L [26.40-53.54]). Echocardiographic findings included ventricular wall thickening (12 [39%] vs 1 [4%]), pulmonary hypertension (9 [29%] vs 0 [0%]), and reduced left-ventricular ejection fraction (5 [16%] vs 0 [0%]). Overall, 10% of the ICU patients presented with right heart enlargement, thickened right-ventricular wall, decreased right heart function, and pericardial effusion. Cardiac complications were more common in ICU patients, including acute cardiac injury (21 [60%] vs 13 [3%]) (including 2 cases of fulminant myocarditis), atrial or ventricular tachyarrhythmia (3 [9%] vs 3 [1%]), and acute heart failure (5 [14%] vs 0 [0%]). CONCLUSION: Myocardial injury marker elevation, ventricular wall thickening, pulmonary artery hypertension, and cardiac complications including acute myocardial injury, arrhythmia, and acute heart failure are more common in ICU patients with COVID-19. Cardiac injury in COVID-19 patients may be related more to the systemic response after infection rather than direct damage by coronavirus.

First case of COVID-19 complicated with fulminant myocarditis: a case report and insights.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has been demonstrated to be the cause of pneumonia. Nevertheless, it has not been reported as the cause of acute myocarditis or fulminant myocarditis. CASE PRESENTATION: A 63-year-old male was admitted with pneumonia and cardiac symptoms. He was genetically confirmed as having COVID-19 according to sputum testing on the day of admission. He also had elevated troponin I (Trop I) level (up to 11.37 g/L) and diffuse myocardial dyskinesia along with a decreased left ventricular ejection fraction (LVEF) on echocardiography. The highest level of interleukin-6 was 272.40 pg/ml. Bedside chest radiographs showed typical ground-glass changes indicative of viral pneumonia. Laboratory test results for viruses that cause myocarditis were all negative. The patient conformed to the diagnostic criteria of the Chinese expert consensus statement for fulminant myocarditis. After receiving antiviral therapy and mechanical life support, Trop I was reduced to 0.10 g/L, and interleukin-6 was reduced to 7.63 pg/mL. Moreover, the LVEF of the patient gradually recovered to 68%. The patient died of aggravation of secondary infection on the 33rd day of hospitalization. CONCLUSION: COVID-19 patients may develop severe cardiac complications such as myocarditis and heart failure. This is the first report of COVID-19 complicated with fulminant myocarditis. The mechanism of cardiac pathology caused by COVID-19 needs further study.

The variation degree of coagulation function is not responsible for extra risk of hemorrhage in gestational diabetes mellitus.

BACKGROUND: Gestational diabetes mellitus (GDM) is characterized as glucose intolerance of any degree that begins or first diagnosed during pregnancy. It possesses a higher risk of haemorrhage, which may be caused by the coagulation dysfunction. However, there has been no study focus on how coagulation state changes in the progress of GDM pregnancy. Our study is aimed to assess the association of coagulation function and haemorrhage in GDM. METHODS: A total of 662 subjects (273 from a population-based study and 389 from a prospective cohort study) were selected to measure mean platelet volume (MPV), platelet distribution width (PDW), platelet (PLT), thrombocytocrit (PCT), prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and fibrinogen (FIB). All pregnant individuals were divided into normal glucose tolerance (NGT) controls and GDM patients diagnosed between the 24th and 28th weeks of gestation. RESULTS: Compared with NGT controls, GDM females showed shortened PT, shortened APTT, and increased blood FIB levels, while the platelet parameters MPV, PDW, PLT, and PCT remained unchanged in mid-pregnancy. By late pregnancy, the platelet parameters MPV, PDW, and PCT were increased in the GDM group compared with the NGT group, while PT and APTT were unchanged. CONCLUSIONS: The GDM group was hypercoagulable compared with the NGT group rather than hypocoagulable as predicted, but still within the normal range. Therefore, our findings demonstrate that the variation degree of coagulation function is not responsible for extra risk of hemorrhage in GDM, and prevention of hemorrhage should focus on other causes.

In vivo Oxygen Condition of Human Nasal Inferior Turbinate-Derived Stem Cells in Human Nose.

BACKGROUND AND OBJECTIVE: Human nasal inferior turbinate-derived stem cells (hNTSCs) have been considered as a potent and useful source for regenerative medicine. To most effectively mimic the native environment of inferior turbinate could be very effective to hNTSCs biology. Thus, the purpose of this study was to evaluate partial pressure of oxygen (ppO2) and temperature in inferior turbinate. METHODS: Ten patients were enrolled who underwent endoscopic endonasal transsphenoidal skull base tumor surgery between January 2014 and December 2015. The commercially available OxyLab pO2 monitor gauges the ppO2 and temperature using a fluorescence quenching technique. Also, hNTSCs were isolated from 10 patients and cultivated under hypercapnic condition (5, 10, and 15%) to mimic hypoxic intranasal conditions. RESULTS: The measured oxygen concentration in submucosa tissue was higher than that at the surface of the inferior turbinate and the temperature in submucosa tissue was higher than the value at the surface of inferior turbinate. The patterns of proliferation were significantly different according to hypercapnic cultivation conditions and there were statistically significant decreased proliferation rates after the exposure of higher CO2 over a period of 5 days. CONCLUSIONS: Intranasal turbinate tissue showed the hypoxia state in concordance with the result of the other tissues or organs. However, indirectly induced hypoxia influenced the influence on the hNTSCs proliferation negatively. Further study is needed to mimic the real hypoxic state, but our results could be used to optimize the culture environment of hNTSCs, thereby producing the stem cells for regenerative therapies.

NIR-Responsive Polypeptide Nanocomposite Generates NO Gas, Mild Photothermia, and Chemotherapy to Reverse Multidrug-Resistant Cancer.

Multidrug resistance (MDR) of cancers that results from overexpression of a P-glycoprotein (P-gp) transporter mainly causes chemotherapy (CT) failure and hinders clinical transitions of current polypeptide nanomedicines. Herein, a novel polypeptide nanocomposite PNOC-PDA that integrates heat-sensitive NO gas delivery and photothermal conversion attributes can overcome MDR and maximize CT; meanwhile the optimized CT and intracellular high-concentration NO gas can assist a mild photothermal therapy (PTT) to eradicate cancer cells. The triple therapies produced a superior and synergistic effect on MDR-reversal and killing MCF-7/ADR in vitro, and the P-gp expression level was downregulated to 46%, as confirmed by means of MTT, Western blot, flow cytometry, and confocal laser scanning microscopy. Significantly, by using one intravenous injection of PNOC-PDA/DOX and a single near-infrared irradiation, the triple therapies of mild PTT, NO gas therapy, and CT achieved complete MCF-7/ADR tumor ablation without skin damage, scarring, and tumor recurrence within 30 days. This work provides a versatile method for the fabrication of NIR-responsive polypeptide nanocomposite with intrinsic photothermal conversion and NO-releasing attributes, opening up a new avenue for reversing MDR in tumors.

Elevated Kallikrein-binding protein in diabetes impairs wound healing through inducing macrophage M1 polarization.

BACKGROUND: The accumulation of M1-polarized macrophages and excessive inflammation are important in the pathogenesis of diabetic foot ulcer (DFU). However, the underlying mechanism of DFU pathogenesis and the crucial regulators of DFU are less well known. Our previous study reported that kallikrein-binding protein (KBP), an angiogenesis inhibitor, was significantly upregulated in diabetic patients compared to its levels in controls. The effects of KBP on monocyte chemotaxis and macrophage M1 polarization were elucidated in this study. METHODS: Plasma KBP levels and monocyte counts were assessed by ELISA and flow cytometry. Wound closure rates in different groups were monitored daily. The phenotype and recruitment of macrophages were measured by real-time PCR, western blot and immunofluorescence assays. The expression of Notch and NF-κB signalling pathway members was determined by the methods mentioned above. ChIP and dual-luciferase reporter gene assays were employed to explore the binding and transcriptional regulation of Hes1 and iNOS. RESULTS: We found that plasma KBP levels and circulating monocytes were elevated in diabetic patients compared to those in nondiabetic controls, and both were higher in diabetic patients with DFU than in diabetic patients without DFU. KBP delayed wound healing in normal mice; correspondingly, KBP-neutralizing antibody ameliorated delayed wound healing in diabetic mice. Circulating monocytes and macrophage infiltration in the wound were upregulated in KBP-TG mice compared to those in control mice. KBP promoted the recruitment and M1 polarization of macrophages. Mechanistically, KBP upregulated iNOS by activating the Notch1/RBP-Jκ/Hes1 signalling pathway. Hes1 downregulated CYLD, a negative regulator of NF-κB signalling, and then activated the IKK/IκBα/NF-κB signalling pathway. CONCLUSIONS: Our findings demonstrate that KBP is the key regulator of excessive inflammation in DFUs and provide a novel target for DFU therapy.