On the regeneration of fish scales: structure and mechanical behavior.

Fish scales serve as a dermal armor that provides protection from physical injury. Owing to a number of outstanding properties, fish scales are inspiring new concepts for layered engineered materials and next-generation flexible armors. Although past efforts have primarily focused on the structure and mechanical behavior of ontogenetic scales, the structure-property relationships of regenerated scales have received limited attention. In the present study, common carp (Cyprinus carpio) acquired from the wild were held live in an aquatic laboratory at 10°C and 20°C. Ontogenetic scales were extracted from the fish for analysis, as well as regenerated scales after approximately 1 year of development and growth. Their microstructure was characterized using microscopy and Raman spectroscopy, and the mechanical properties were evaluated in uniaxial tension to failure under hydrated conditions. The strength, strain to fracture and toughness of the regenerated scales were significantly lower than those of ontogenetic scales from the same fish, regardless of the water temperature. Scales that regenerated at 20°C exhibited significantly higher strength, strain to fracture and toughness than those regenerated at 10°C. The regenerated scales exhibited a highly mineralized outer layer, but no distinct limiting layer or external elasmodine; they also possessed a significantly lower number of plies in the basal layer than the ontogenetic scales. The results suggest that a mineralized layer develops preferentially during scale regeneration with the topology needed for protection, prior to the development of other qualities.

The relation of low levels of bone mineral density with coronary artery calcium and mortality.

Osteoporosis and atherosclerosis are two prevalent major healthcare concerns that frequently coexist. The clinical outcome of 5590 consecutive subjects who underwent coronary artery calcium (CAC) scanning and thoracic bone mineral density (BMD) measurement was assessed. A significant link between low BMD levels and CAC with increased risk of mortality in both genders across ethnicities noted. INTRODUCTION: While a relation of CAC with lower levels of BMD reported previously; it is unclear whether low levels of BMD would be an independent risk factor for CAC and mortality. This study investigated the relation of BMD levels with CAC and mortality in both genders across ethnicities. METHODS: This study consisted of 5590 consecutive at-risk subjects without known coronary artery disease (CAD), age 57 ± 12, and 69% male, who underwent non-enhanced cardiac computed tomography, and were followed for mean of 8 years. The subjects' CAC (Agatston score) and thoracic BMD levels (mg/cm3) were measured. CAC stratified based on the severity to CAC 0, 1-100, 101-400, and 400+. Low-BMD levels defined as BMD levels below median (180 mg/cm3). Physician verified that all-cause mortality was assessment hard-endpoint. Multivariate regression analysis, adjusted for age, gender, and other cardiovascular risk factors, was used to assess the relationship between BMD and CAC. RESULTS: The BMD levels were proportionally lowering with the severity of CAC in both genders, especially in postmenopausal women (p < 0.05). The risk of each standard deviation reduce in BMD levels increased with the severity of CAC, as compared to CAC = 0 across ethnicities (p < 0.05). Low BMD levels were an independent predictor of mortality and event-free survival rate decreased from 99% in those within normal BMD levels to 93% in those with low BMD levels (p = 0.0001). Furthermore, a significant link between low BMD levels and CAC > 0 with increased risk of mortality was noted (p = 0.0001). The relative risk of death was 2.8, 5.9, and 14.3-folds higher in CAC 1-100, 101-400, and 400+ with low BMD levels, compared to CAC = 0 and within normal BMD levels, respectively (p < 0.05). CONCLUSIONS: The lower BMD levels are independently associated with the severity of CAC that predicts mortality.

[Clinical and genetic characteristics of 9 rare cases with coexistence of dual genetic diagnoses].

Objective: To analyze the clinical and genetic characteristics of pediatric patients with dual genetic diagnoses (DGD). Methods: Clinical and genetic data of pediatric patients with DGD from January 2021 to February 2022 in Peking University First Hospital were collected and analyzed retrospectively. Results: Among the 9 children, 6 were boys and 3 were girls. The age of last visit or follow-up was 5.0 (2.7,6.8) years. The main clinical manifestations included motor retardation, mental retardation, multiple malformations, and skeletal deformity. Cases 1-4 were all all boys, showed myopathic gait, poor running and jumping, and significantly increased level of serum creatine kinase. Disease-causing variations in Duchenne muscular dystrophy (DMD) gene were confirmed by genetic testing. The 4 children were diagnosed with DMD or Becker muscular dystrophy combined with a second genetic disease, including hypertrophic osteoarthropathy, spinal muscular atrophy, fragile X syndrome, and cerebral cavernous malformations type 3, respectively. Cases 5-9 were clinically and genetically diagnosed as COL9A1 gene-related multiple epiphyseal dysplasia type 6 combined with NF1 gene-related neurofibromatosis type 1, COL6A3 gene-related Bethlem myopathy with WNT1 gene-related osteogenesis imperfecta type XV, Turner syndrome (45, X0/46, XX chimera) with TH gene-related Segawa syndrome, Chromosome 22q11.2 microduplication syndrome with DYNC1H1 gene-related autosomal dominant lower extremity-predominant spinal muscular atrophy-1, and ANKRD11 gene-related KBG syndrome combined with IRF2BPL gene-related neurodevelopmental disorder with regression, abnormal movement, language loss and epilepsy. DMD was the most common, and there were 6 autosomal dominant diseases caused by de novo heterozygous pathogenic variations. Conclusions: Pediatric patients with coexistence of double genetic diagnoses show complex phenotypes. When the clinical manifestations and progression are not fully consistent with the diagnosed rare genetic disease, a second rare genetic disease should be considered, and autosomal dominant diseases caused by de novo heterozygous pathogenic variation should be paid attention to. Trio-based whole-exome sequencing combining a variety of molecular genetic tests would be helpful for precise diagnosis.

[Clinical follow-up analysis of nusinersen in the disease-modifying treatment of pediatric spinal muscular atrophy].

Objective: To explore the clinical efficacy of disease-modifying drug nusinersen on children with spinal muscular atrophy. Methods: The baseline and longitudinal clinical data of 15 children who were treated with nusinersen in the Children's Hospital, Zhejiang University School of Medicine from October 2019 to October 2021 were retrospectively collected. The general data (gender, age, genotype, and clinical classification, etc.), motor function, nutritional status, scoliosis and respiratory function were analyzed. Wilcoxon rank-sum test was used for comparing multi-system conditions before and after treatment. Results: The age of 15 cases (7 males, 8 females) was 6.8 (2.8, 8.3) years, with 2 cases of type 1, 6 cases of type 2, and 7 cases of type 3 respectively, and the course of disease was 55.0 (21.0, 69.0) months. After 9.0 (9.0, 24.0) months of treatment, the motor function scale evaluations of the Hammersmith neurological examination section 2 (13.0 (7.0, 23.0) vs. 18.0 (10.0, 25.0) scores, Z=-2.67, P=0.018) of 15 children, the Hammersmith functional motor scale expanded (38.0 (18.5, 45.5) vs. 42.0 (23.0, 51.0) scores, Z=-2.38, P=0.018), and the revised upper limb module (27.0 (19.5, 32.0) vs. 33.0 (22.5, 35.5) scores, Z=-2.52, P=0.012) of children with type 2 and 3 had significantly improved. Thirteen patients achieved clinically significant motor function improvement, and 2 of them had kept stable scale scores. Subjective reports also indicated that the muscle strength and daily exercise ability of these children improved after treatment, and no serious adverse reactions were reported. Supplemented by the multi-disciplinary team management, the levels of some indicators such as Cobbs angle of scoliosis and forced vital capacity all had significantly improved (all P<0.05). Conclusions: Nusinersen can improve the motor function of patients with 5q spinal muscular atrophy, which is also proved safe to be used in children. The drug treatment supplemented by the multi-disciplinary team management is helpful to improve the multi-system function of the children with spinal muscular atrophy.

[Clinical follow-up analysis of multidisciplinary treatment of children with spinal muscular atrophy].

Objective: To analyze the follow-up and clinical effect of multidisciplinary treatment on the children with spinal muscular atrophy (SMA). Methods: The clinical data including nutritional status, respiratory function, bone health and motor function of 45 children with SMA who received multidisciplinary management 1-year follow-up in the Children's Hospital, Zhejiang University School of Medicine from July 2019 to October 2021 were retrospectively collected. Comparisons before and after management were performed using paired-samples t-test or Wilcoxon rank-sum test, etc. Results: The age of 45 patients (25 boys and 20 girls) was 50.4 (33.6, 84.0) months at the enrollment, with 6 cases of type 1, 22 cases of type 2, and 17 cases of type 3 respectively. After the multidisciplinary management, the cases of SMA patients with malnutrition decreased from 22 to 12 (P=0.030), the level of vitamin D were significantly increased ((45±17) vs. (48±14) nmol/L, t=-4.13, P<0.001). There was no significant difference in the forced vital capacity %pred, the forced expiratory volume at 1 second %pred, and the peak expiratory flow %pred ((76±19)% and (76±21)%, (81±18)% and (79±18)%, (81±21)% and (78±17)%; t=-0.24, 1.36, 1.21; all P>0.05). The Cobbs angle of scoliosis also improved significantly (8.0°(0°, 13.0°) vs. 10.0°(0°, 18.5°), Z=-3.01, P=0.003). The Hammersmith functional motor scale expanded scores of children with SMA type 2 and type 3 both showed significant elevation (11.0 (8.0, 18.0) vs. 11.0 (5.0, 18.5) scores, 44.0 (36.5, 53.0) vs. 44.0 (34.0, 51.5) scores, Z=2.44, 3.11, P=0.015, 0.002). Conclusion: Multidisciplinary management is beneficial for delaying the progression of the multi-system impairments of SMA patients, such as malnutrition, restrictive ventilation dysfunction and scoliosis.

TGF-ß induced epithelial-mesenchymal transition in an advanced cervical tumor model by 3D printing.

An advanced in vitro cervical tumor model was established by 3D printing to study the epithelial-to-mesenchymal transition (EMT), which is a very important stage of dissemination of carcinoma leading to metastatic tumors. A HeLa/hydrogel grid construct composed of gelatin, alginate, Matrigel and HeLa cells was fabricated by forced extrusion in a layer-by-layer fashion. HeLa cells rapidly proliferated, formed spheroids and presented tumorigenic characteristic in the 3D-printed structure. With the supplement of TGF-ß, aggregated HeLa cells started to disintegrate, and some of them changed into fibroblast-like spindle morphology, which indicated that EMT was induced. The down-regulation of epithelial marker E-cadherin, and up-regulation of mesenchymal markers such as snail, vimentin and N-cadherin were all observed in the 3D-printed model, and performed differently in 3D and 2D models. The TGF-ß induced EMT was inhibited by the treatment of disulfiram and EMT pathway inhibitor C19 in a dose dependent manner, showing great potential for future studies of a therapeutic program towards cervical tumor metastasis.

Discovery of a novel, selective, and orally bioavailable class of thrombin inhibitors incorporating aminopyridyl moieties at the P1 position.

A novel class of thrombin inhibitors incorporating aminopyridyl moieties at the P1 position has been discovered. Four of these thrombin inhibitors (13b,c,e and 14d) showed nanomolar potency (Ki 0.8-12 nM), 300-1500-fold selectivity for thrombin compared with trypsin, and good oral bioavailability (F = 40-76%) in rats or dogs. The neutral P1 was expected to increase metabolic stability and oral absorption. Identification of this novel aminopyridyl group at P1 was a key step in our search for a clinical candidate.

Inhibition of thrombin by peptides containing Lysyl-alpha-keto carbonyl derivatives.

Several H-N-Me-D-Phe-Pro-Lysyl-alpha-keto carbonyl derivatives were shown to be potent thrombin inhibitors (Ki 0.2 to 27 nM). The inhibitory potencies of these compounds toward tissue plasminogen activator, plasmin and factor Xa were minimal; however, substantial cross-reactivity versus trypsin was observed (Ki values from 0.5 to 1500 nM). Inhibition of thrombin by alpha-keto carbonyl compounds appeared to occur via a one-step reversible reaction. The alpha-keto carbonyl inhibitors bound thrombin with a second order rate constant (k1 1-4 microM-1s-1) that was 10-100-fold slower than that expected for a diffusion-controlled reaction. Certain alpha-keto carbonyl inhibitors were as potent (on a weight basis) as hirudin when evaluated in a rat arterial thrombosis model. The modest oral bioavailability (10-19%) in rats demonstrated for three of the alpha-keto carbonyl thrombin inhibitors suggests the possibility that alpha-keto amide containing thrombin inhibitors may have utility as orally-active antithrombotic agents.

Coronary artery motion during the cardiac cycle and optimal ECG triggering for coronary artery imaging.

RATIONALE AND OBJECTIVES: Our purpose was to investigate the motion characteristics of the coronary arteries and determine optimal electrocardiographic (ECG) trigger time during the cardiac cycle to minimize motion artifacts. METHODS: Contrast-enhanced multislice movie studies of electron beam tomography (EBT) images were performed on 70 subjects. The EBT datasets, which covered an entire cardiac cycle at 58-ms intervals, were acquired for a short-axis view of the heart with ECG triggering. The pixel values along x and y axes were measured at multiple intervals during the cardiac cycle to establish the motion distance and velocity of three major coronary arteries. RESULTS: Coronary artery motion varied greatly throughout the cardiac cycle in three major coronary arteries and increased with the patient's baseline heart rate. The greatest and lowest velocities of coronary arterial movement during the cardiac cycle were determined. Based on the lowest velocity of right coronary artery movement during the cardiac cycle, the optimal ECG trigger times were located at approximately 35% (31.4%-37.6%) or 70% (68.7%-71.4%) of the R-R interval in patients whose resting heart rate was < or =70 beats per minute (bpm); at 50% (47.2%-61.1%) of the R-R interval in the 71- to 100-bpm group; and at 55% (52.8%-59.1%) of the R-R interval in the >100-bpm group. Our data demonstrated that the motion characteristics of the left circumflex artery were quite similar to those of the right coronary artery and that the left anterior descending coronary artery had no significant differences in motion throughout the cardiac cycle. A minimum scan speed of 35.4 to 75.5 ms per slice is needed to completely diminish cardiac motion artifacts (in-plane coronary artery motion with <1-mm displacement). CONCLUSIONS: For coronary artery screening, the optimal ECG trigger time should be determined according to the patient's heart rate, thus greatly reducing motion and motion artifacts during 100-ms acquisitions.

Laser plasma spectroscopy of Al-Cu-Fe quasicrystal an d alloy.

Electron number density and temperature were determined from laser-induced plasmas produced by irradiating Al-Cu-Fe targets of a quasicrystal and of an alloy of similar composition. The Al(I) atomic emission spectra of the two systems were measured as a function of the distance from the target and of the time delay after laser irradiation. Differences of plasma characteristics were observed for laser ablation of quasicrystal and alloy targets, and the results were interpreted on the basis of different plasma formation mechanisms for the two systems.

The effect of maternal infection on cognitive development and hippocampus neuronal apoptosis, proliferation and differentiation in the neonatal rats.

Many epidemiological reports stated a strong association between maternal infection and development of cerebral palsy, which is a major cause of cognitive impairment. The pathophysiological mechanism of intrauterine inflammation is complex. Recently, it was demonstrated that inflammation has a modulating effect on adult neurogenesis. In this study, we discovered the effect of maternal infection to hippocampal neuronal apoptosis, proliferation and differentiation, and cognitive development in the developing brains of neonatal rats. Morris water maze test was used to assess learning and memory. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to determine neuronal apoptosis, immunostaining was conducted to assess neurogenesis, and Western blot for extracellular signal-regulated kinase (ERK), cyclic AMP responsive element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF) expression in the hippocampus. Results demonstrated that maternal infection increased neuronal apoptosis and significantly impaired spatial learning and memory ability. Maternal infection significantly increased cell proliferation, accompanied by an increased expression of ERK (P3-P7), CREB (P3-P7) and BDNF (P3). On P28, there was no significant difference of cell survival and differentiation in two groups. These results suggest that variation in ERK activity and subsequent expression of its downstream targets, including CREB and BDNF might contribute, at least partially, to modulation of inflammation related cell proliferation, survival and differentiation. Maternal infection increased hippocampal neuronal apoptosis and affected cell proliferation and differentiation in neonatal rats, which may be regarded as an etiological factor in cognitive development impairment.

Arteriographic demonstration of both kinking at the origin and compression between the great vessels of an anomalous right coronary artery arising in common with a left coronary artery from above the left sinus of Valsalva.

Angled angiographic views demonstrated two areas of significant systolic narrowing in an anomalous right coronary artery arising in common with a left coronary artery from above the left sinus of Valsalva: 1) an ostial stenosis due to kinking as the anomalous artery turned sharply to the right after its origin from the aorta; 2) compression of the proximal segment as it coursed between the aorta and pulmonary artery. Appropriate angiographic studies to evaluate the presence of these changes may help to elucidate their significance.

Total biventricular volume and total left ventricular volume by ultrafast computed tomography: prediction of left ventricular mass.

Since LV hypertrophy is an independent predictor of increased cardiac mortality, a simple noninvasive measure of LV mass would be a valuable screening tool. A retrospective assessment of 50 UFCT studies was performed. Measurements of LV mass, TBV, and TLV were determined in each case. There was excellent correlation between TBV and LVM (R = 0.93) and between TLV and LV mass (R = 0.92). Also, there was minimal interobserver variability between two independent observers for estimates of both TBV (R = 0.995) and TLV (R = 0.99). Thus these preliminary data indicate that LV mass may be estimated from TBV or TLV and that these tools may assist in the identification of patients potentially at high risk for future coronary events, as indicated by the presence of increased LV mass and coronary atherosclerosis.

Mechanism-based inhibition of a mutant Escherichia coli ribonucleotide reductase (cysteine-225----serine) by its substrate CDP.

The B1 subunit of Escherichia coli ribonucleotide reductase (EC 1.17.4.1) has been overexpressed using the pT7-5/pGP1-2 system developed by Tabor and Richardson [Tabor, S. & Richardson, C. (1985) Proc. Natl. Acad. Sci. USA 82, 1074-1078]. This method has allowed the preparation of two mutant B1 subunits in which two of the four thiols postulated to be within the active site of the enzyme, Cys-225 and Cys-759, have been changed to serines. Incubation of the [Ser225]B1 mutant with the B2 subunit, [U-14C]CDP, and the allosteric effector ATP results in production of cytosine, destruction of the tyrosyl radical in B2, radiolabeling of the protein, and cleavage of the B1 subunit into two pieces of 26 and 61.5 kDa. This process is independent of the identity of reductant. The [Ser759]B1 mutant reduces CDP in the presence of thioredoxin/thioredoxin reductase at 7.7% the rate of wild-type B1. When dithiothreitol is utilized as reductant, however, the rate of CDP reduction with [Ser759]B1 is identical to that observed with wild type.

Reaction pathway for inhibition of blood coagulation factor Xa by tick anticoagulant peptide.

The reaction pathway for inhibition of human factor Xa (fXa) by recombinant tick anticoagulant peptide (rTAP) was studied by stopped-flow fluorometry. In the presence of the fluorogenic substrate N-tert-butyloxycarbonyl-L-isoleucyl-L-glutamylglycyl-L-arginyl-7-amido-4 - methylcoumarin (B-IEGR-AMC) and under pseudo-first-order conditions, inhibition appears to occur via a two-step process. Initially, a weak enzyme-inhibitor complex forms with a dissociation constant (Ki) of 68 +/- 6 microM. The initial complex then rearranges to a more stable fXa-rTAP complex with a rate constant (k2) of 123 +/- 5 s-1. The apparent second-order rate constant (k2/Ki) describing formation of the stable complex is (1.8 +/- 0.2) x 10(6) M-1 s-1. Studies of the reaction of rTAP with fXa in the presence of the fluorescent active-site probe p-amino-benzamidine (P) revealed a reaction pathway wherein rTAP initially binds to the fXa-P complex in a two-step process prior to displacing P from the active site. These results indicate that rTAP can bind fXa via a site distinct from the active site (an exosite). The subsequent displacement of P from the active site of fXa by rTAP exhibits a dependence on the concentration of P, indicating that rTAP is locked into the active site in a third step.(ABSTRACT TRUNCATED AT 250 WORDS)

Variation of heart rate and electrocardiograph trigger interval during ultrafast computed tomography.

OBJECTIVES: Electrocardiographic (ECG) trigger records obtained during cardiac ultrafast computed tomography (UFCT) scanning were analyzed to estimate the variability in heart rate and ECG trigger interval to develop a protocol that would allow the development of better ECG triggering software. METHODS: One-hundred-eighteen patients underwent cardiac UFCT imaging for diagnostic purposes. All subjects were divided into three groups according to the heart rate and ECG trigger condition. Thirty slices were obtained in the high-resolution volume mode for each patient. RESULTS: A decrease in heart rate and ECG trigger interval was found during image acquisition of the first four slices in all three groups. The nadir of the heart rate occurred during acquisition of the 4th slice, 5.3, 3.5, and 5.6 beats per minute less than the initial heart rate in groups 1, 2, and 3 respectively, with a 6.9%, 2.8%, and 5.0% shorter ECG trigger interval (p < .001, p = .08, p < .05, respectively). From the 4th to the 30th slices, heart rate and ECG trigger interval progressively increased, but less variability was found in the last 20 slices in all three groups. CONCLUSIONS: Significant variation in heart rate and ECG trigger interval was seen during 30-level cardiac UFCT imaging, especially during image acquisition of the first four slices (approximately 1-6 seconds after breatholding). This can result in scanning during the suboptimal phase of the cardiac cycle by the current UFCT triggering software. A delay in the initiation of scanning to approximately 6 to 10 seconds after breatholding would result in imaging during a time when the heart rate is relatively stable, and a smaller variability in ECG trigger interval occurs. Recalculation of the required delay before each heart beat may improve the precision of ECG triggering.

Characterization of C439SR1, a mutant of Escherichia coli ribonucleotide diphosphate reductase: evidence that C439 is a residue essential for nucleotide reduction and C439SR1 is a protein possessing novel thioredoxin-like activity.

Ribonucleotide reductase from Escherichia coli catalyzes the conversion of nucleotides to deoxynucleotides. Cysteine 439 is proposed to be the protein radical on R1 which initiates the reduction reaction by cleavage of the 3' carbon-hydrogen bond of the nucleotide (Mao et al., 1992a,b). C439 is thus proposed to be essential for catalysis. The C439S mutant of R1 (C439SR1) was prepared. The structure of this mutant was determined to be similar to wt-R1, based on identical CD spectra, isolation via an affinity column specific for the allosteric binding domain, binding of the substrate GDP, and competition with R1 for binding to R2. Preparations of C439SR1 are contaminated with low levels of wt-R1 due to the expression system. The wt-R1 in these preparations can be specifically inactivated by the stoichiometric mechanism-based inhibitor, 2'-azido-2'-deoxyuridine 5'-diphosphate. The activity of the resulting C439SR1 was shown to be less than 0.03% that of the corresponding wt-R1. This is the lower limit of detection with the present assay method. Thus C439 appears to be essential for catalysis. During these studies an unexpected activity of the C439SR1 was uncovered. Its additional cysteines, presumably C754 and C759, appear to function as a thioredoxin with the wt-R1, even though it is incapacitated with respect to nucleotide reduction.