Identification of WUSCHEL-related homeobox gene and truncated small peptides in transformation efficiency improvement in Eucalyptus.

BACKGROUND: The WUSCHEL-related Homeobox (WOX) genes, which encode plant-specific homeobox (HB) transcription factors, play crucial roles in regulating plant growth and development. However, the functions of WOX genes are little known in Eucalyptus, one of the fastest-growing tree resources with considerable widespread cultivation worldwide. RESULTS: A total of nine WOX genes named EgWOX1-EgWOX9 were retrieved and designated from Eucalyptus grandis. From the three divided clades marked as Modern/WUS, Intermediate and Ancient, the largest group Modern/WUS (6 EgWOXs) contains a specific domain with 8 amino acids: TLQLFPLR. The collinearity, cis-regulatory elements, protein-protein interaction network and gene expression analysis reveal that the WUS proteins in E. grandis involve in regulating meristems development and regeneration. Furthermore, by externally adding of truncated peptides isolated from WUS specific domain, the transformation efficiency in E. urophylla × E. grandis DH32-29 was significant enhanced. The transcriptomics data further reveals that the use of small peptides activates metabolism pathways such as starch and sucrose metabolism, phenylpropanoid biosynthesis and flavonoid biosynthesis. CONCLUSIONS: Peptides isolated from WUS protein can be utilized to enhance the transformation efficiency in Eucalyptus, thereby contributing to the high-efficiency breeding of Eucalyptus.

Efficacy and safety of janagliflozin monotherapy in Chinese patients with type 2 diabetes mellitus inadequately controlled on diet and exercise: A multicentre, randomized, double-blind, placebo-controlled, Phase 3 trial.

AIMS: To evaluate the efficacy and safety of janagliflozin, a selective renal sodium-glucose cotransporter-2 inhibitor, as monotherapy in drug-naive Chinese patients with type 2 diabetes mellitus (T2DM). MATERIALS AND METHODS: This Phase 3 trial included a 24-week, multicentre, randomized, double-blind, placebo-controlled period, followed by a 28-week extension period. A total of 432 patients with glycated haemoglobin (HbA1c) levels ≥7.0% (53 mmol/mol) and ≤10.5% (91 mmol/mol) were randomized (1:1:1) to receive once-daily placebo, 25 mg or 50 mg janagliflozin. After 24 weeks, patients on placebo were switched and re-randomized (1:1) to 25 mg or 50 mg janagliflozin, whereas patients on janagliflozin maintained the initial therapy. The primary endpoint was change from baseline in HbA1c after 24 weeks. RESULTS: At Week 24, the placebo-adjusted least squares mean changes in HbA1c were -0.80% (95% confidence interval [CI] -0.98% to -0.62%)/-8.7 mmol/mol (95% CI -10.7 mmol/mol to -6.8 mmol/mol) and -0.88% (95% CI -1.06% to -0.70%)/-9.6 mmol/mol (95% CI -11.6 mmol/mol to -7.7 mmol/mol), respectively (P < 0.001 for both). A higher proportion of patients achieved HbA1c <7.0% (53 mmol/mol) with janagliflozin 25 mg and janagliflozin 50 mg compared with placebo (47.2%, 49.3%, and 23.5%, respectively). Both janagliflozin doses significantly decreased fasting plasma glucose, 2-hour postprandial glucose, body weight and systolic blood pressure, as well as increased high-density lipoprotein (HDL) cholesterol and insulin sensitivity compared with placebo (P < 0.05 for all). The trends in improvement of these variables were sustained during the 28-week extension period. Overall incidences of adverse events were 67.8%, 71.5% and 60.7% with janagliflozin 25 mg, janagliflozin 50 mg and placebo, respectively. The incidence of urinary tract infections and genital fungal infections was low. No severe hypoglycaemia or ketoacidosis occurred. CONCLUSIONS: Janagliflozin 25 mg and 50 mg monotherapy once-daily effectively improved glycaemic control, reduced body weight and blood pressure, improved HDL cholesterol and insulin sensitivity, and was generally well tolerated.

A Strategy to Enhance the Ferroelectric Behavior of MOF-802(Hf) via Doping Zr4+ Ions.

MOF ferroelectrics, as a crucial member of molecular ferroelectrics, have shown intriguing advantages owing to the designability of structures and tunability of physicochemical properties, which make them an appealing group of ferroelectric materials. However, the weak ferroelectric property still is a huge challenge for further development. Here, a series of Zr-doped MOF-802(Hf)s were successfully synthesized through doping Zr4+ ions into the parent MOF-802(Hf) to improve ferroelectric properties. The well-shaped P-E hysteresis loops of Zr-doped MOF-802(Hf)s illustrate their ferroelectricity, and ferroelectric properties are effectively enhanced compared with the parent MOF-802(Hf). What's more, remanent polarization reaches 0.511 µC/cm2 when the concentration of Zr4+ ions is 5%, which is 5 times higher than that of the parent MOF-802(Hf) and is on par with some perovskite ferroelectrics. The increased ferroelectric performance is attributed to the enhanced polarity of the whole structure triggered by lattice distortion when Hf4+ ions of the parent MOF-802(Hf) are substituted by Zr4+ ions. As far as we know, this is the first report on Hf-MOF exhibiting improved ferroelectric behaviors through doping metal ions into lattice nodes. This work demonstrates that introducing the second metal ions into lattice nodes of MOFs is an efficacious approach for exploiting MOF ferroelectrics with superior performance.

Effective Enhancement of the Ferroelectric Performance of Polar Co-Gallate MOF by Doping M2+ Ions (M = Mg, Mn, Ni) into Framework Nodes.

MOF ferroelectrics have been demonstrated to be a promising candidate owing to various structures and controllable properties. However, weak ferroelectricity hampers their boom. Herein, a convenient strategy, doping metal ions into the framework nodes of parent MOF, is adopted to enhance ferroelectric performance. A series of M-doped Co-Gallate (M = Mg, Mn, Ni) were synthesized to improve ferroelectric properties. The electrical hysteresis loop demonstrated its ferroelectric behaviors, exhibiting obviously improved ferroelectric properties compared with the parent Co-Gallate. The remanent polarization was enhanced by two times for Mg-doped Co-Gallate, six times for Mn-doped Co-Gallate, and four times for Ni-doped Co-Gallate. The promoted ferroelectric performances are ascribed to the enhanced polarity of the overall structure triggered by framework distortion. Intriguingly, ferroelectric behaviors increase in the order Mg < Ni < Mn, displaying the same tendency as the difference value in the ionic radius between Co2+ ions and M2+ metal ions (M = Mg, Mn, Ni). These results demonstrate doping of metal ions is a valid strategy to enhance ferroelectric performances, which may serve as a guide in modulating ferroelectric behaviors.

Genome-Wide Analysis of the FBA Subfamily of the Poplar F-Box Gene Family and Its Role under Drought Stress.

F-box proteins are important components of eukaryotic SCF E3 ubiquitin ligase complexes, which specifically determine protein substrate proteasomal degradation during plant growth and development, as well as biotic and abiotic stress. It has been found that the FBA (F-box associated) protein family is one of the largest subgroups of the widely prevalent F-box family and plays significant roles in plant development and stress response. However, the FBA gene family in poplar has not been systematically studied to date. In this study, a total of 337 F-box candidate genes were discovered based on the fourth-generation genome resequencing of P. trichocarpa. The domain analysis and classification of candidate genes revealed that 74 of these candidate genes belong to the FBA protein family. The poplar F-box genes have undergone multiple gene replication events, particularly in the FBA subfamily, and their evolution can be attributed to genome-wide duplication (WGD) and tandem duplication (TD). In addition, we investigated the P. trichocarpa FBA subfamily using the PlantGenIE database and quantitative real-time PCR (qRT-PCR); the results showed that they are expressed in the cambium, phloem and mature tissues, but rarely expressed in young leaves and flowers. Moreover, they are also widely involved in the drought stress response. At last, we selected and cloned PtrFBA60 for physiological function analysis and found that it played an important role in coping with drought stress. Taken together, the family analysis of FBA genes in P. trichocarpa provides a new opportunity for the identification of P. trichocarpa candidate FBA genes and elucidation of their functions in growth, development and stress response, thus demonstrating their utility in the improvement of P. trichocarpa.

Design, synthesis and structure-activity relationship studies of pyrido[2,3-d]pyrimidin-7-ones as potent Janus Kinase 3 (JAK3) covalent inhibitors.

Aberrantly activated Janus kinase 3 (JAK3) has been constantly detected in various immune disorders and hematopoietic cancers, suggesting its potential of being an attractive therapeutic target for these indications. Clinical benefits of drugs selectively targeting JAK3 versus pan-JAK inhibitors remain unclear. In this study, we report the design and synthesis of a new series of JAK3 covalent inhibitors with a pyrido[2,3-d]pyrimidin-7-one scaffold. After the extensive SAR study, compound 10f emerged to be the most potent JAK3 inhibitor with an IC50 value of 2.0 nM. It showed excellent selectively proliferation inhibitory activity against U937 cells harboring JAK3 M511I mutation, while remained weakly active to the other tested cancer cells. Compound 10f also dose-dependently inhibited the phosphorylation of JAK3 and its downstream signal STAT5 in U937 cells. Taken together, 10f may serve as a promising tool molecule for treating cancers with aberrantly activated JAK3.

Updated Confirmatory Diagnosis for Mucopolysaccharidoses in Taiwanese Infants and the Application of Gene Variants.

Mucopolysaccharidosis (MPS) is a lysosomal storage disease caused by genetic defects that result in deficiency of one specific enzyme activity, consequently impairing the stepwise degradation of glycosaminoglycans (GAGs). Except for MPS II, the other types of MPS have autosomal recessive inheritance in which two copies of an abnormal allele must be present in order for the disease to develop. In this study, we present the status of variant alleles and biochemistry results found in infants suspected of having MPS I, II, IVA, and VI. A total of 324 suspected infants, including 12 for MPS I, 223 for MPS II, 72 for MPS IVA, and 17 for MPS VI, who were referred for MPS confirmation from newborn screening centers in Taiwan, were enrolled. In all of these infants, one specific enzyme activity in dried blood spot filter paper was lower than the cut-off value in the first blood sample, as well asin a second follow-up sample. The confirmatory methods used in this study included Sanger sequencing, next-generation sequencing, leukocyte enzyme fluorometric assay, and GAG-derived disaccharides in urine using tandem mass spectrometry assays. The results showed that five, nine, and six infants had MPS I, II, and IVA, respectively, and all of them were asymptomatic. Thus, a laboratory diagnosis is extremely important to confirm the diagnosis of MPS. The other infants with identified nucleotide variations and reductions in leukocyte enzyme activities were categorized as being highly suspected cases requiring long-term and intensive follow-up examinations. In summary, the final confirmation of MPS depends on the most powerful biomarkers found in urine, i.e., the quantification of GAG-derived disaccharides including dermatan sulfate, heparan sulfate, and keratan sulfate, and analysis of genetic variants can help predict outcomes and guide treatment.

Significantly Enhancing the Lithium Ionic Conductivity of Metal-Organic Frameworks via a Postsynthetic Modification Strategy.

Metal-organic frameworks (MOFs), due to their possessing a porous structure, are potential candidates for solid-state ionic conduction materials. Moreover, uncoordinated carboxylic acid groups (-COOH) of MOFs can be used as postsynthetic modification sites, which are favorable for lithium ion exchange. Herein, we synthesized a unique multiple carboxylic zinc metal-organic framework (Zn-MOF-COOH) containing uncoordinated carboxylic acid groups. Zn-MOF-COOLi was synthesized through deprotonation using LiOH via a straightforward acid-base reaction at room temperature (RT), thereby exhibiting better good electrochemical properties. The lithium ionic conductivity (σ) increased from 1.81 × 10-5 to 1.65 × 10-4 S·cm-1, lithium ion transference number (tLi+) rose from 0.67 to 0.77, and the electrochemical window improved from 2.0-5.5 to 1.5-6.5 V. This work offers a new strategy to improve the σ of MOFs and a new perspective toward manufacturing of high-performance solid-state ionic conduction materials.

Stable Europium-based Metal-Organic Frameworks for Naked-eye Ultrasensitive Detecting Fluoroquinolones Antibiotics.

Fluoroquinolone antibiotic (FQ) residues, such as ciprofloxacin (CIP) and ofloxacin (OFLX), have aroused public concerns owing to their serious impact in environmental water or food fields which influence human health. A facile and high-performance sensory method for detecting FQs is highly desirable for practical requirements. Herein, we have presented a luminescent Eu-MOF with unique 2D (4-c) {44.62}-connected topology, which holds the outstanding fluorescent property and excellent chemical stability in aqueous solution for 15 days. Thus, Eu-MOF can be considered as a highly sensitive chemo-sensor for sensing CIP and OFLX with different fluorescent color conversion (red changes to green for OFLX and to blue for CIP) and a low detection limit of 0.693 and 0.802 ppb, respectively. Furthermore, the mechanism of sensing CIP and OFLX was exposed to the photoinduced electron transfer (PET) and dynamic quenching process, as evaluated by DFT calculations and fluorescence lifetime decay measurements. Our work first reports a simple and efficient strategy for recognizing CIP and OFLX with a special luminescence color-change phenomenon based on MOF materials, serving as a meaningful guide for researchers in beneficial applications.

Recent Advances on Fabrication of Polymeric Composites Based on Multicomponent Reactions for Bioimaging and Environmental Pollutant Removal.

As the core of polymer chemistry, manufacture of functional polymers is one of research hotspots over the past several decades. Various polymers are developed for diverse applications due to their tunable structures and unique properties. However, traditional step-by-step preparation strategies inevitably involve some problems, such as separation, purification, and time-consuming. The multicomponent reactions (MCRs) are emerging as environmentally benign synthetic strategies to construct multifunctional polymers or composites with pendant groups and designed structures because of their features, such as efficient, fast, green, and atom economy. This mini review summarizes the latest advances about fabrication of multifunctional fluorescent polymers or adsorptive polymeric composites through different MCRs, including Kabachnik-Fields reaction, Biginelli reaction, mercaptoacetic acid locking imine reaction, Debus-Radziszewski reaction, and Mannich reaction. The potential applications of these polymeric composites in biomedical and environmental remediation are also highlighted. It is expected that this mini-review will promote the development preparation and applications of functional polymers through MCRs.

Star Identification Based on Multilayer Voting Algorithm for Star Sensors.

This paper describes the multilayer voting algorithm, a novel autonomous star identification method for spacecraft attitude determination. The proposed algorithm includes two processes: an initial match process and a verification process. In the initial match process, a triangle voting scheme is used to acquire candidates of the detected stars, in which the triangle unit is adopted as the basic voting unit. During the identification process, feature extraction is implemented, and each triangle unit is described by its singular values. Then the singular values are used to search for candidates of the imaged triangle units, which further improve the efficiency and robustness of the algorithm. After the initial match step, a verification method is applied to eliminate incorrect candidates from the initial results and then outputting the final match results of the imaged stars. Experiments show that our algorithm has more robustness to position noise, magnitude noise, and false stars than the other three algorithms, the identification speed of our algorithm is largely faster than the geometric voting algorithm and optimized grid algorithm. However, it takes more memory, and SVD also seems faster.

[Exploration of Distress Symptoms and Medical Interventions in Patients With Terminal Cancer in Hospice Shared Care].

BACKGROUND: Hospice shared care is a model of care widely used in patients with terminal cancer. Appropriate interventions to improve related symptoms should be provided during disease progression through the end of life. PURPOSE: The purpose of this study was to explore the related symptoms and to compare symptom severity before and after the implementation of hospice shared care and medical care interventions. METHODS: Fifty patients with terminal cancer were enrolled in this longitudinal, quasi-experimental research. Inclusion criteria included having an expected life span of < 6 months and agreeing to enter hospice shared care. RESULTS: The three most-frequently noted physical symptoms were, in rank order: "pain", "weakness", and "dyspnea". In terms of severity, "pain" was the most severe, followed by "weakness" and "disturbance of sleep". The three most-frequently noted psychological symptoms were, in rank order: "depression", "worry about the disease", and "afraid of dragging others down". In terms of severity, "depression" was the most severe, followed by "anxiety" and "worry about the disease". The frequency and severity of the top-3 social and spiritual distress symptoms were, in rank order: "unfinished wish", "economic difficulties", and "painlessness". During the study period, the severity of physical symptoms improved gradually, while the severity of psychological symptoms improved significantly. The top-3 items in the original medical team`s hospice-care interventions were, in rank order: "tube care", "laboratory test", and "wound care" in the non-pharmacological category; "symptom control medication", "antibiotic injection", and "intravenous fluid infusion" in the pharmacological category; and "cognitive clarification of the prognosis condition", "do not conduct resuscitation discussions and signing", and "emotional stress consoling" in the problem-coordination and interview categories. The interventions implemented by the hospice specialist team included "emotional stress interview", "team communication", and "cognitive clarification of prognosis condition" in the problem coordination and interview categories, and "massage", "consultation", and "nursing advice" in the non-pharmacological category. CONCLUSIONS / IMPLICATIONS FOR PRACTICE: The symptoms identified in this study provide clinical staff with a reference for the rapid assessment of patients with terminal-stage cancer. Manpower from various professional fields are committed to providing diversified services in the care teams, which positively affect the control of related symptoms. The experience presented in this article may be used as a reference to promote the hospice shared care model.

Oral immunization of BALB/c mice with recombinant Helicobacter pylori antigens and double mutant heat-labile toxin (dmLT) induces prophylactic protective immunity against H. pylori infection.

Helicobacter pylori infection and associated diseases remain a major public health problem worldwide. Much effort has been made over the last several decades in vaccine development, but there is no licensed vaccine on the market. We have previously reported that oral immunization with H. pylori lysates and double mutant heat-labile toxin (dmLT) affords prophylactic protection against H. pylori infection in mice. In the present study, we investigated the effects of oral immunization with recombinant H. pylori protein antigens (NAP/UreA/UreB) and dmLT on H. pylori challenge in BALB/c mice. We found that oral immunization with candidate antigens and dmLT significantly reduced the gastric colonization of H. pylori 6 weeks after challenge, as compared to unimmunized mice. Moreover, the subunit vaccine appeared to provide a better protection than the bacterial lysate vaccine. The immunized mice showed enhanced antigen-specific lymphocyte proliferation, and serum IgG and mucosal IgA responses. Furthermore, the immunization increased the proportion of CD4+ IL-17+ lymphocytes in spleen and mesenteric lymph nodes, and enhanced the production of IL-17, IL-16, IL-6 and TNF-α in lymphocyte culture supernatants. Taken together, our results suggest that oral vaccination with recombinant H. pylori antigens (NAP/UreA/UreB) and dmLT confers more effective prophylactic protection against H. pylori infection than whole bacterial lysates in BALB/c mice. The reduction of H. pylori colonization was associated with the induction of antigen-specific Th17 and local mucosal IgA immune responses.

Intriguing pH-modulated Luminescence Chameleon System based on Postsynthetic Modified Dual-emitting Eu3+@Mn-MOF and Its Application for Histidine Chemosensor.

Due to the disruption by other nonanalyte factors, single-emission probes have been limited in complicated detecting systems. In this work, a pH-modulated luminescence chameleon system based on lanthanide-based MOF (Eu3+@Mn-MOF), with stable structure and miraculous dual-emitting fluorescent properties, was synthesized by a postsynthetic modification (PSM) strategy of a simple hydrothermal and agitation method. Amazingly, not only can the Eu3+@Mn-MOF emit a broad emission at 500 nm attributed to the ligand-based fluorescence emission but it can also exhibit the characteristic emission of Eu3+ ions responding to the antenna effect. Moreover, the Eu3+@Mn-MOF displays an interesting luminescence color transition between acidic and basic solutions. Inspired by this phenomenon, a pH-modulated luminescence chameleon system was first constructed and employed to detect histidine, a kind of basic amino acid for a variety of biological matters, causing a unique fluorescence signal of the ratio-dependent color to change from yellow to light pink which differs from the color change of other water-soluble amino acids. Therefore, Eu3+@Mn-MOF can be as a practical pH-modulated luminescence chameleon system chemsensor for sensing histidine with low detection limit, high sensitivity, and rapid sensing time. In conclusion, the postsynthetic modified Eu3+@Mn-MOF has outstanding applications in the fields of chemical detection and human health.

Taiwan National Newborn Screening Program by Tandem Mass Spectrometry for Mucopolysaccharidoses Types I, II, and VI.

OBJECTIVE: To evaluate the initial cutoff values, rates of screen positives, and genotypes for the large-scale newborn screening program for multiple mucopolysaccharidoses (MPS) in Taiwan. STUDY DESIGN: More than 100 000 dried blood spots were collected consecutively as part of the national Taiwan newborn screening programs. Enzyme activities were measured by tandem mass spectrometry from dried blood spot punches. Genotypes were obtained when a second newborn screening specimen again had a decreased enzyme activity. Additional clinical evaluation was then initiated based on enzyme activity and/or genotype. RESULTS: Molecular genetic analysis for cases with low enzyme activity revealed 5 newborns with pathogenic alpha-L-iduronidase mutations, 3 newborns with pathogenic iduronate-2-sulfatase mutations, and 1 newborn was a carrier of an arylsulfatase B mutation. Several variants of unknown pathogenic significance were also identified, most likely causing pseudodeficiency. CONCLUSIONS: The highly robust tandem mass spectrometry-based enzyme assays for MPS-I, MPS-II, and MPS-VI allow for high-throughput newborn screening for these lysosomal storage disorders. Optimized cutoff values combined with second tier testing could largely eliminate false-positive results. Accordingly, newborn screening for these lysosomal storage disorders is possible.

Fabrication of a Luminescence-Silent System Based on a Post-Synthetic Modification Cd-MOFs: A Highly Selective and Sensitive Turn-on Luminescent Probe for Ascorbic Acid Detection.

A unique three-dimensional luminescent metal-organic framework (Cd-MOFs), [Cd(tpbpc)2]·2H2O·DMF (Htpbpc = 4'-[4,2';6',4″]-terpyridin-4'-yl-biphenyl-4-carboxylic acid; DMF = dimethylformamide), was synthesized and structurally characterized; it exhibits excellent luminescent property and structural stability in aqueous solutions. Interestingly, an unparalleled luminescence-silent system CrO42-@Cd-MOFs was successfully fabricated by postsynthetic modification of metal-organic frameworks. This luminescence-silent system represents a highly selective and sensitive turn-on luminescent responding to ascorbic acid. First, this advanced fluorescent sensor displays excellent performance for CrO42- ions with a quenching of fluorescence intensity originating from fluorescence resonance energy transfer (FRET) mechanism. What's more, the fluorescent intensity of CrO42-@Cd-MOFs system can be recovered (turn-on) for sensing ascorbic acid because of the elimination of FRET process. Such a novel fabrication strategy should offer the guidance to develop various MOFs-implicated luminescence-silent systems as "turn-on" sensors for detection of specific chemicals.

Elastic bending of variable curvature mirrors: validation of a simplified analytical method.

This work explores the variable curvature mirror's (VCM) elastic bending rules through modeling it as a thin elastic plate with an exponential thickness distribution actuated with a uniform pressure under simply supported boundary conditions. By using the small-parameter method, the general analytical expression of a plate's deflection is worked out. The results calculated by the analytical solution are compared to the finite element analysis of a VCM model with the same specific parameters. We demonstrate that the two have a good correlation with the each other. This analytical solution is an effective way to predict a VCM's deflection.

Recent Advances and Progress on Melanin-like Materials and Their Biomedical Applications.

Melanins are well-known biopolymers that are ubiquitous in nature, distributed widely in microorganisms, plants, and animals, and play significant physiological roles. They are mostly biopolymers formed from phenolic compounds by polymerization via quinones. Poly(dopamine) (PDA), a melanin-like material, is similar in structure and properties to eumelanin and has attracted considerable interest for various types of biological applications. This review outlines the recent advances in the structure and synthesis of PDA and discusses applications of PDA in many biological fields, such as biological imaging, photothermal therapy, and drug delivery systems. The purpose of this review is to give a brief overview of the synthesized procedures, structure, biomedical applications, and prospects of melanin-like materials.

Recent Advances and Future Prospects of Aggregation-induced Emission Carbohydrate Polymers.

Aggregation-induced emission (AIE) is an abnormal phenomenon that has sparked great attention for diverse applications in different fields. In particular, the fabrication and biological imaging applications of AIE-active fluorescent organic nanoparticles (FONs) have become a focus in the emerging and promising fields. A large number of AIE-active polymeric nanoprobes have recently been fabricated through different strategies. The advances and progress in this direction have also recently been summarized by some groups. However, the fabrication and biomedical applications of AIE-active FONs based on carbohydrate polymers and AIE-active dyes are quite rare and limited. In this feature article, the recently reported AIE-active FONs with different structures and applications based on AIE-active dyes and carbohydrate polymers are highlighted, and the major current limitations and development tendencies are also discussed.