[Retrospective clinical study on cryopreservation-free integrated autologous hematopoietic stem cell transplantation model for newly diagnosed multiple myeloma].

Objective: To explore the efficacy and safety of cryopreservation-free integrated autologous hematopoietic stem cell transplantation (HSCT) model for patients with multiple myeloma. Methods: A total of 96 patients with newly diagnosed multiple myeloma (NDMM) between July 31, 2020, and December 31, 2022, were retrospectively analyzed, of which 41 patients in the observation group received integrated non-cryopreserved transplantation mode. After hematopoietic stem cells were mobilized and collected, melphalan was started immediately for pre-transplant conditioning, and non-cryopreserved grafts from the medical blood transfusion refrigerator were directly injected intravenously into the patient within 24-48 h after the melphalan conditioning. The control group consisted of 55 patients who received traditional transplantation mode. After hematopoietic stem cells were collected, stem cell cryopreservation was performed in liquid nitrogen, and then the transplant plans were started at the right time. All patients received mobilization of autologous hematopoietic stem cells using the G-CSF combined with the plerixafor. Results: ① A total of 34 patients (82.9% ) with VGPR plus CR in the observation group were significantly higher than 33 patients (60.0% ) in the control group (P=0.016). ②Compared with the control group, the incidence of grade 1 oral mucosal inflammation was higher in the observation group (P<0.001) ; however, the incidence of grades 2 and 3 oral mucosal inflammation was lower (P=0.004, P=0.048), and neither group experienced grade 4 or above oral mucosal inflammation. The incidence of grade 1 diarrhea was higher in the observation group (P=0.002), whereas the incidence of grade 3 diarrhea was lower (P=0.007). No statistically significant difference was observed in the incidence of grade 4 diarrhea (P=0.506), and neither group experienced grade 5 diarrhea. ③ The incidence of bacterial infection in the observation group was lower than that in the control group (34.1% vs 65.5%, P=0.002), whereas no statistically significant difference was observed in the incidence of fungal infection (29.3% vs 31.4%, P=0.863) and viral infection (4.88% vs 3.64%, P=0.831). ④No statistically significant difference was observed in the implantation time of granulocytes and platelets between the observation and control groups [10 (8-20) days vs 11 (8-17) days, P=0.501; 13 (10-21) days vs 15 (10-20) days, P=0.245]. ⑤ All patients did not receive lenalidomide treatment 100 days post-transplantation. At 30 days post-transplantation, the CTL, NK, and Th cell counts in the observation group were lower than those in the control group (P<0.001, P=0.002, P=0.049), and the NKT cell counts were higher than those in the control group (P=0.024). At 100 days post-transplantation, the CTL, NKT, and Th cell counts in the observation group were higher than those in the control group (P=0.025, P=0.011, P=0.007), and no statistically significant difference in NK cell counts was observed between the two groups (P=0.396). ⑥ The median follow-up was 18 (4-33) months. The overall 2-year survival rates of the observation and control groups post-transplantation were 91.5% and 78.2%, respectively (P=0.337). The recurrence-free survival rates were 85.3% and 77.6%, respectively (P=0.386), and the cumulative recurrence rates were 9.8% and 16.9%, respectively (P=0.373) . Conclusion: In NDMM, the cryopreservation-free integrated autologous HSCT model can achieve similar therapeutic effects as traditional transplantation models, with lower rates of severe mucosal inflammation and infection compared with traditional transplantation models.

[Genotype-phenotype analysis of Fabry disease caused by GLA gene variation in a pedigree].

Objective: To investigate the clinical phenotype and genetic characteristics of patients with Fabry disease caused by a GLA variant, IVS4+919G>A. Methods: It was a prospective study. Fabry disease screening was conducted among high-risk population in Ninghai from October 2021 to August 2023. Those children with decreased α-galactosidase enzyme activity<2.40 µmol/(L·h) or elavated Lyso-GL-3 level>1.10 µg/L in dried blood spot (DBS) method underwent GLA genetic testing for diagnosis confirmation. Meanwhile, family screening was carried out. A proband and his family members diagnosed with Fabry disease were research subjects. The clinical and genetic characteristics of patients with Fabry disease caused by the GLA variant (IVS4+919G>A) were analyzed. Results: The female proband aged 9.8 years with pain in both lower limbs as the initial symptom was found to have a heterozygous GLA variant IVS4+919G>A among 102 patients. In family screening, there were 4 family members (proband's father, elder sister, elder male cousin and elder female cousin) with Fabry disease and a family member (proband's fifth aunt) with a GLA variant. Among these 4 diagnosed family members, the elder male cousin of the proband, a boy aged 13.2 years had a heterozygous GLA variant, IVS4+919G>A with intermittent pain in both lower limbs as the initial symptom. The proband's father had knee joint pain. The proband's elder sister had decreased vision and his elder female cousin had no obvious symptoms. The proband's fifth aunt with a GLA variant had decreased vision. Conclusions: High-risk screening in children and family screening are helpful for early diagnosis and treatment of Fabry disease. Neuropathic pain may be a early symptom in children with Fabry disease caused by the GLA variant, IVS4+919G>A.

[Measurement of corneal nerve fiber parameters in patients with Parkinson's disease].

Objective: To analyze the characteristic changes of corneal nerve fibers in patients with Parkinson's disease (PD) by corneal confocal microscopy (CCM) and investigate the association of corneal nerve fiber parameters with disease severity and motor symptoms. Methods: Forty-two patients with PD were recruited from the Department of Neurology, Henan University People's Hospital from June 2018 to October 2019. Meanwhile, 40 healthy controls who visited the hospital for physical examination at the same period were enrolled. Corneal nerve fibers in both eyes of all participants were detected by using CCM. The differences of corneal nerve fibers were comparatively analyzed between PD group and healthy controls. Associations of corneal nerve parameters with clinical characteristics such as course of disease, Hoehn and Yahr stage (H-Y stage), unified Parkinson disease rating scale (UPDRS), levodopa equivalent daily dosage (LEDD) were analyzed by using partial correlations. The receiver operating characteristic (ROC) curve was used to analyze the capability of corneal nerve fibers for distinguishing patients with PD from healthy controls. Results: Corneal nerve fiber density (CNFD) in PD group ((19±3)/mm2) was significantly decreased compared with healthy controls ((28±4)/mm2) (t=10.798, P<0.001). However, corneal nerve branch density (CNBD) was significantly increased in PD group ((25±11)/mm2) compared with healthy controls ((18±6)/mm2) (t=-3.427, P=0.001). Meanwhile, corneal nerve fiber length (CNFL) was decreased in PD group ((11.0±2.5) mm/mm2) in comparison with healthy controls ((12.5±1.6) mm/mm2) (t=3.139, P=0.002). ROC curve analysis revealed that CNFD could discriminate PD patients from healthy controls, with an area under the curve of 0.961 3 (95%CI: 92.42-99.84, P<0.000 1). CNFD was negatively correlated with H-Y stage and UPDRS-Ⅲ (r=-0.501 and -0.399, both P<0.05). CNBD was significantly negatively associated with H-Y stage, UPDRS-Ⅲ and UPDRS-Total (r=-0.622, -0.394 and -0.354, respectively, all P<0.05). CNFL was negatively correlated with H-Y stage, UPDRS-Ⅲ and UPDRS-total (r=-0.574, -0.484 and -0.422, respectively, all P<0.05). Conclusion: Small nerve fiber injuries exist in PD patients. Corneal nerve fibers negatively correlates with motor symptoms. CNFD have a good discriminative power to distinguish PD patients from healthy controls and may serve as a marker for PD.

The diagnostic and prognostic values of serum and urinary kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin in sepsis induced acute renal injury patients.

OBJECTIVE: The kidney injury molecule-1 (uKIM-1) and neutrophil gelatinase-associated lipocalin (uNGAL, sNGAL) have been demonstrated to be diagnostic biomarkers for acute kidney injury (AKI) in a variety of diseases. However, both of them were not well validated in sepsis patients with acute kidney injury. PATIENTS AND METHODS: This was a prospective and observational study which was performed in the three intensive care units of the Beijing Chao-Yang Hospital. Over a 12-month period, 174 patients (70 sepsis patients, 69 sepsis with AKI and 35 controls) were enrolled. Blood and urinary specimens were collected at admission as soon as possible (within 24 hours) and KIM-1 and NGAL levels were tested. RESULTS: Levels of uKIM-1, uNGAL, sNGAL were significantly higher in the sepsis patients who developed AKI compared to those sepsis with no-AKI (0.88 ng/ml (0.37, 2.14) vs. 1.21 ng/ml (0.67, 3.26) p=0.003, 63.54 ng/ml (21.66, 125.45) vs. 249.85 ng/ml (86.60, 585.97) p<0.001, and 108.08 ng/ml (67.74, 212.22) vs. 200.01 ng/ml (102.76, 300.77) p=0.001, respectively). sKIM-1 also had significant differences between the two groups (83.98 pg/ml (54.00,147.08) vs. 193.41 pg/ml (106.90, 430.60) p<0.001). The four biomarkers (uKIM-1, sKIM-1, uNGAL, sNGAL) all could be predictive for AKI, and the areas under the receiver operating characteristic curves (AUROC) were 0.607, 0.754, 0.768, 0.658, respectively. The uNGAL was an independent risk factor for septic AKI, and the AUROC was 0.768 (95% CI: 0.689 to 0.835). The uNGAL and sNGAL were related to the prognosis of sepsis. CONCLUSIONS: Our results showed that NGAL was a promising biomarker of septic AKI. Like the uKIM-1, the sKIM-1 could early predict the occurrence of septic AKI too, but both of them did not have the predictive value in judging the severity of AKI and the prognosis of sepsis.

[Depletion of PRDM9 inhibited the osteogenic differentiation potential of periodontal ligament stem cells].

Objective: To investigate the effect of PR domain zinc finger protein 9 (PRDM9), one of the histone methylated transferases, on osteogenic differentiation ability of periodontal ligament mesenchymal stem cells (PDLSC). Methods: PDLSC with PRDM9 gene knocked down by PRDM9 shRNA using recombinant lentiviral vector were allocated into the PRDM9sh group, and the transfected shRNA was as the control group. The gene expression efficiency was evaluated by reverse transcription polymerase chain reaction (RT-PCR). Alkaline phosphatase activity (ALP), alizarin red staining, mineralization and osteocalcin, which belongs to osteogenic differentiation markers detected by RT-PCR and Western blotting to detect the osteogenic differentiation ability of stem cells from periodontal ligaments in vitro. In vivo, PRDM9sh and control group cells was transplanted into the dorsal dermal to explore the osteogenesis. The area percentage of new osteogenic tissue was calculated by image pro software and statistically analyzed. Results: RT-PCR results showed that the relative expression of PRDM9 gene in PRDM9sh (0.460±0.017) was significantly lower than that in control group (1.000±0.107) (P<0.05). The results of ALP activity determined at 5 days postinduction in a significant decrease in PRDM9sh cells (0.762±0.063) compared with control group (1.225±0.058) (P<0.01). Alizarin red staining induced by osteogenesis at 2 weeks and 3 weeks showed that the staining of PRDM9sh was significantly lighter than that in control group. Quantitative calcium analysis results showed that the calcium ion concentration induced by osteogenesis at 2 weeks and 3 weeks [(0.071±0.004), (0.075±0.001)] in PRDM9sh was significantly lower than that in control group at 2 weeks and 3 weeks [(0.282±0.006), (0.485+0.004)] (P<0.01). RT-PCR results showed that the relative expression of osteocalcin mRNA in PRDM9sh (1.059±0.148) was significantly lower than that in control group at 2 weeks (2.542±0.190) (P<0.01). Western blotting results showed that osteocalcin expression in PRDM9sh was significantly lower than that in control group at 1 and 2 weeks after osteogenesis induction. Animal transplantation experiments results indicated that PRDM9 significantly inhibited the osteogenesis of PDLSC in vivo, and the proportion of osteogenic area calculated showed that the osteogenic capacity of PRDM9sh [(3.8±2.41)%] was significantly lower than that in control group [(24.54±7.06)%](P<0.05). Conclusions: Depletion of PRDM9 repressed the osteogenic differentiation of stem cells from periodontal ligament in vitro and in vivo.

[Correlation between ultrasonographic optic nerve sheath diameter and intracranial pressure].

Objective: To evaluate the association of the ultrasonographic optic nerve sheath diameter (ONSD) and intracranial pressure (ICP), and the feasibility of ultrasonographic ONSD in predicting high ICP. Methods: A prospective study. The outpatients who planned to measure ICP by lumbar puncture in Department of Neurology, Xuanwu Hospital, Capital Medical University were selected from January 2011 to May 2012. All the retrobulbar ONSD measurement with B-scan ultrasound was performed just before lumbar puncture. When high ICP was defined as ICP more than 200 mmH2O(1 mmH2O=0.009 8 kPa), the participants were divided into the high ICP group and the normal ICP group. The Pearson correlation coefficient analysis was used to analyze the correlation between ICP and postbulbar ONSD measurements. The difference in ONSD was compared between the high ICP and normal ICP groups with the t test. The receiver operating characteristic (ROC) curve was used to calculate the cutoff value of mean ONSD and evaluate the sensitivity and specificity of the method. Results: A total of 130 participants were involved in this study. There were 71 males and 59 females, aged (38±14) years.The mean ICP was (209.84±79.99) mmH2O. The mean ONSD was (5.68±0.78) mm in the right eyes, (5.78±0.78) mm in the left eyes, and (5.73±0.71) mm in both eyes. The ICP had a significant correlation with ONSD in the right eyes (r=0.54, P<0.001), ONSD in the left eyes (r=0.56, P<0.001) and ONSD in both eyes (r=0.60, P<0.001), but no correlation with age (r=-0.14, P=0.114) and gender (r=0.20, P=0.817). The ONSD in the high ICP group (n=65) was (6.11±0.66) mm, (6.22±0.56) mm and (6.17±0.50) mm in the right eyes, left eyes, and both eyes, respectively. Compared with the ONSD in the normal ICP group (n=65), which was (5.26±0.64) mm in the right eyes, (5.34±0.72) mm in the left eyes and (5.30±0.62) mm in both eyes, there was a significantly enlarged ONSD in the high ICP group (t=-7.507, -7.778, -8.779, all P<0.001). The ROC analysis showed the ONSD of 5.6 mm was the best cutoff value with a sensitivity of 86% and a specificity of 71% for identifying high ICP. Conclusions: There is a significantly positive correlation between ICP and postbulbar ONSD measured by ultrasound. This non-invasive method may be an alternative approach to predicting the ICP value of patients whose ICP measurement via lumbar puncture is at high risk. However, it can not replace the direct ICP measurement with the invasive method. (Chin J Ophthalmol, 2018, 54: 683-687).

[Retrospective analysis of 13 cases of nocardiosis].

Objective: To evaluate the clinical features, chest radiological manifestations, microbiological examination and treatments of nocardial disease. Methods: A retrospective study was conducted to analyze the data of patients with nocardial infection admitted to Beijing Chaoyang Hospital from January 2010 to January 2016. Results: The 13 patients, 6 males and 7 females, aged (51±17) years. Twelve cases were diagnosed with pulmonary nocardiosis, and 1 with disseminated nocardial infection. Most of these patients had complications: autoimmune diseases in 3 (2 with autoimmune hemolytic anemia and 1 with systemic lupus erythematosus), and bronchiectasis in 6 patients. The most common symptoms were cough, expectoration and fever. The main manifestations of CT scans included nodules or masses, bronchiectasis, ground glass opacity, cavity and pleural thickening. Six cases were confirmed by sputum smear microscopy, 4 by bronchoalveolar lavage, 2 by percutaneous lung biopsy and 1 by renal abscess puncture. After diagnosis, antibiotics such as Co-trimoxazole, amikacin, cephalosporins, imipenem, minomycin, or linezolid were used, and the 13 patients were all cured and discharged. Conclusions: Pulmonary nocardiosis was the most common clinical presentation of nocardial infection. Cough, expectoration and fever were the most common symptoms. The main findings of CT scans were nodules or masses, bronchiectasis, ground glass opacity, cavity and pleural thickening. The diagnosis of nocardiosis was not easy because of the non-specific clinical presentations and difficult culture of nocardia spp. Thus, high clinical suspicion of nocardiosis is necessary for earlier diagnosis and treatment.

Diagnostic value of microRNAs in breast cancer: a meta-analysis.

OBJECTIVE: To explore the value of diagnosis accuracy of aberrant microRNAs in breast cancer. MATERIALS AND METHODS: We searched PubMed, Embase, EBSCO and the Cochrane Library, accessing to the case of articles about microRNA expression in breast carcinoma patients after literature screening and quality assessment, extracting data from included studies and using Stata 14.0 analysis data for meta-analysis. RESULTS: 14 English studies met the inclusion criteria. After meta-analysis for included studies obtained high sensitivity and specificity and diagnostic odds ratio, the combined OR value is 17.96 (95% CI: 11.42-28.42), sensitivity is 0.85 (95% CI: 0.81-0.88), specificity is 0.77 (95% CI: 0.69-0.82), diagnostic odds ratio is 18 (95% CI: 12-29), operating characteristic area under the curve is 0.88 (95% CI: 0.85-0.91). CONCLUSIONS: The microRNAs can be used as a clinical auxiliary reference index for diagnosis of breast cancer.

PTX3 expression in the plasma of elderly ACI patients and its relationship with severity and prognosis of the disease.

OBJECTIVE: This study aims to investigate the expression of pentraxin3 (PTX3) in elderly patients with acute cerebral infarction (ACI) and to analyze the relationship of PTX3 with the severity and prognosis of ACI. PATIENTS AND METHODS: Between June 2014 and August 2015, 96 elderly patients with first-onset of ACI admitted to our institution were enrolled in the present study. Also, 70 healthy elderly subjects were included as controls in this study. Levels of PTX3, C-reactive protein (CRP), tumor necrosis factor (TNF)-α, homocysteine (Hcy), fibrinogen (FIB), total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were determined in all patients both pre-therapeutically and 30 days post-therapeutically. Moreover, the severity of ACI was evaluated using the National Institute of Health stroke scale (NIHSS), and the prognosis was evaluated using the Modified Rankin Scale (mRS). The differences in the levels of above parameters were compared between groups, and the relationship between PTX3 and above biochemical parameters as well as the scores were investigated. RESULTS: PTX3 levels in the plasma of ACI patients were significantly higher than those of healthy controls (p < 0.05). Compared to low score group, mRS and levels of all parameters except Hcy and HDL-C were significantly increased in the patients of the high score group. In addition, plasma levels of HDL-C in the patients of the high score group were significantly lower than those in the low score group (p < 0.05). Biochemical parameters, NIHSS scores and mRS scores were significantly higher in the patients of the high concentration group than the low concentration group (p < 0.05), while no significant differences were observed in the plasma HDL-C levels between these two groups (p > 0.05). NIHSS scores and levels of all the biochemical parameters except HDL-C were significantly lower in the patients of the good prognosis group than in the patients of the poor prognosis group. HDL-C levels were significantly higher in the good prognosis group than in the poor prognosis group (p < 0.05). PTX3 levels were positively correlated with levels of CRP, TNF-α, Hcy, TC, TG and LDL-C as well as NIHSS score and mRS score, respectively, (r = 0.814, 0.682, 0.704, 0.726, 0.699, 0.734, 0.746, 0.753, p = 0.008, 0.043, 0.034, 0.027, 0.036, 0.024, 0.021, 0.019). However, no significant correlations were observed between FIB levels and HDL-C levels (r = 0.326, 0.626, p = 0.392, 0.071). CONCLUSIONS: Plasma levels of PTX3 are significantly elevated in elderly ACI patients, and the levels increase along with the exacerbation of the disease and deterioration in the prognosis. Also, PTX3 levels are positively correlated with levels of inflammatory markers as well as lipids. Therefore, PTX3 can be used as a biomarker for predicting and evaluating clinical conditions of ACI in elderly patients.

Brief reports: TRPM7 Senses mechanical stimulation inducing osteogenesis in human bone marrow mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are multipotential stem cells residing in the bone marrow. Several studies have shown that mechanical stimulation modulates MSC differentiation through mobilization of second messengers, but the mechanism of mechanotransduction remains poorly understood. In this study, using fluorescence and laser confocal microcopy as well as patch-clamp techniques, we identified the transient receptor potential melastatin type 7 (TRPM7) channel as the key channel involved in mechanotransduction in bone marrow MSCs. TRPM7 knockdown completely abolished the pressure-induced cytosolic Ca(2+) increase and pressure-induced osteogenesis. TRPM7 directly sensed membrane tension, independent of the cytoplasm and the integrity of cytoskeleton. Ca(2+) influx through TRPM7 further triggered Ca(2+) release from the inositol trisphosphate receptor type 2 on the endoplasmic reticulum and promoted NFATc1 nuclear localization and osteogenesis. These results identified a central role of TRPM7 in MSC mechanical stimulation-induced osteogenesis.

Molecular cloning, sequence analysis, prokaryotic expression, and function prediction of foot-specific peroxidase in Hydra magnipapillata Chinese strain.

The cDNA sequence of foot-specific peroxidase PPOD1 from the Chinese strain of Hydra magnipapillata was cloned by reverse transcription-polymerase chain reaction. The cDNA sequence contained a coding region with an 873-bp open reading frame, a 31-bp 5'-untranslated region, and a 36-bp 3'-untranslated region. The structure prediction results showed that PPOD1 contains 10.34% of α-helix, 38.62% of extended strand, 12.41% of ß-turn, and 38.62% of random coil. The structural core was α-helix at the N terminus. The GenBank protein blast server showed that PPOD1 contains 2 fascin-like domains. In addition, high-level PPOD1 activity was only present in the ectodermal epithelial cells located on the edge of the adhesive face of the basal disc, and that these cells extended lamellipodia and filopodia when the basal disc was tightly attached to a glass slide. The fascin-like domains of Hydra PPOD1 might contribute to the bundling of the actin filament of these cells, and hence, the formation of filopodia. In conclusion, these cells might play an important role in strengthening the adsorbability of the basal disc to substrates.

Detecting early breast tumour by finite element thermal analysis.

Thermography has been proved to be an effective technique for indicating breast disease abnormalities or risks. However, the abnormalities might not express clearly due to various factors, such as when a small tumour is located in a deep region, or environmental influences that make breast disease difficult to find. This study aims to solve these problems for early detection of breast tumour. A three-dimensional breast model is presented to investigate the relationship between an embedded tumour and the surface temperature distribution. Then a subtraction technique is used to enhance the thermal signature of breast tumour. It was showed that the surface thermal characteristics of a small tumour even in a deep region could be found easily by this method. Furthermore, it was also found that the surface thermal characteristics of tumour obscured due to environmental cooling effect can be clearly displayed. The results are very useful for analysing breast thermograms.

Potential sources of carcinogenic heterocyclic amines in the Chinese diet: results from a 24-h dietary recall study in Singapore.

OBJECTIVE: Heterocyclic amines (HCAs) from high-temperature cooking of meat have been linked to increased cancer incidence in Western populations, but data on the sources of HCAs in Asian diets are scarce. Our aim was to identify potential sources of HCAs in the Chinese diet, and to provide the basis for efforts to quantify dietary exposure to these compounds. DESIGN AND SETTING: We conducted 24-h dietary recall interviews among 986 Chinese men and women in Singapore, who were a randomly selected subpopulation of participants from the Singapore Chinese Health Study, a population-based cohort. Details of all foods and beverages consumed by each subject in the past 24 h were recorded, and information on meat type, cooking method and portion size were abstracted from all meat-containing dishes, and gram weight equivalents computed. RESULTS: The mean meat intake per person was 103.0 g/day (standard deviation 74.2), of which 97.2% was fresh meat. Fish (38.0%), pork (30.6%), and poultry (21.0%) accounted for 89.6% of meat consumed. Patterns of meat consumption and cooking methods differed markedly from Western populations. Documented high-temperature cooking methods, combined with stir-frying, accounted for 44.3% of fish, 35.1% of pork and 25.6% of poultry consumed. Specifically, potentially significant sources of HCAs were pan-fried fish and barbecued pork. CONCLUSIONS: Our results identify the potential sources of HCA in the Chinese diet, highlight aspects which are relevant to HCA formation and intake, and call for novel approaches to estimating individual exposure to dietary HCAs in this and similar populations.

The signaling mechanism of Arabidopsis CRY1 involves direct interaction with COP1.

Dark-grown transgenic Arabidopsis seedlings expressing the C-terminal domains (CCT) of the cryptochrome (CRY) blue light photoreceptors exhibit features that are normally associated only with light-grown seedlings, indicating that the signaling mechanism of Arabidopsis CRY is mediated through CCT. The phenotypic properties mediated by CCT are remarkably similar to those of the constitutive photomorphogenic1 (cop1) mutants. Here we show that Arabidopsis cryptochrome 1 (CRY1) and its C-terminal domain (CCT1) interacted strongly with the COP1 protein. Coimmunoprecipitation studies showed that CRY1 was bound to COP1 in extracts from both dark- and light-grown Arabidopsis. An interaction also was observed between the C-terminal domain of Arabidopsis phytochrome B and COP1, suggesting that phytochrome signaling also proceeds, at least in part, through direct interaction with COP1. These findings give new insight into the initial step in light signaling in Arabidopsis, providing a molecular link between the blue light receptor, CRY1, and COP1, a negative regulator of photomorphogenesis.

An Arabidopsis circadian clock component interacts with both CRY1 and phyB.

Most organisms, from cyanobacteria to mammals, use circadian clocks to coordinate their activities with the natural 24-h light/dark cycle. The clock proteins of Drosophila and mammals exhibit striking homology but do not show similarity with clock proteins found so far from either cyanobacteria or Neurospora. Each of these organisms uses a transcriptionally regulated negative feedback loop in which the messenger RNA levels of the clock components cycle over a 24-h period. Proteins containing PAS domains are invariably found in at least one component of the characterized eukaryotic clocks. Here we describe ADAGIO1 (ADO1), a gene of Arabidopsis thaliana that encodes a protein containing a PAS domain. We found that a loss-of-function ado1 mutant is altered in both gene expression and cotyledon movement in circadian rhythmicity. Under constant white or blue light, the ado1 mutant exhibits a longer period than that of wild-type Arabidopsis seedlings, whereas under red light cotyledon movement and stem elongation are arrhythmic. Both yeast two-hybrid and in vitro binding studies show that there is a physical interaction between ADO1 and the photoreceptors CRY1 and phyB. We propose that ADO1 is an important component of the Arabidopsis circadian system.

The C termini of Arabidopsis cryptochromes mediate a constitutive light response.

Cryptochrome blue light photoreceptors share sequence similarity to photolyases, flavoproteins that mediate light-dependent DNA repair. However, cryptochromes lack photolyase activity and are characterized by distinguishing C-terminal domains. Here we show that the signaling mechanism of Arabidopsis cryptochrome is mediated through the C terminus. On fusion with beta-glucuronidase (GUS), both the Arabidopsis CRY1 C-terminal domain (CCT1) and the CRY2 C-terminal domain (CCT2) mediate a constitutive light response. This constitutive photomorphogenic (COP) phenotype was not observed for mutants of cct1 corresponding to previously described cry1 alleles. We propose that the C-terminal domain of Arabidopsis cryptochrome is maintained in an inactive state in the dark. Irradiation with blue light relieves this repression, presumably through an intra- or intermolecular redox reaction mediated through the flavin bound to the N-terminal photolyase-like domain.

The tyrosine kinase c-Abl regulates p73 in apoptotic response to cisplatin-induced DNA damage.

Cancer chemotherapeutic agents such as cisplatin exert their cytotoxic effect by inducing DNA damage and activating programmed cell death (apoptosis). The tumour-suppressor protein p53 is an important activator of apoptosis. Although p53-deficient cancer cells are less responsive to chemotherapy, their resistance is not complete, which suggests that other apoptotic pathways may exist. A p53-related gene, p73, which encodes several proteins as a result of alternative splicing, can also induce apoptosis. Here we show that the amount of p73 protein in the cell is increased by cisplatin. This induction of p73 is not seen in cells unable to carry out mismatch repair and in which the nuclear enzyme c-Abl tyrosine kinase is not activated by cisplatin. The half-life of p73 is prolonged by cisplatin and by co-expression with c-Abl tyrosine kinase; the apoptosis-inducing function of p73 is also enhanced by the c-Abl kinase. Mouse embryo fibroblasts deficient in mismatch repair or in c-Abl do not upregulate p73 and are more resistant to killing by cisplatin. Our results indicate that c-Abl and p73 are components of a mismatch-repair-dependent apoptosis pathway which contributes to cisplatin-induced cytotoxicity.

Three-dimensional coupled fluid-structure simulation of pericardial bioprosthetic aortic valve function.

A computational, three-dimensional coupled fluid-structure dynamics model was developed for a generic pericardial aortic valve in a rigid aortic root graft with physiologic sinuses. Valve geometry was based on that of the natural valve. Blood flow was modeled as pulsatile, laminar, Newtonian, incompressible flow. The structural model accounted for material and geometric nonlinearities and also simulated leaflet coaptation. A body fitted grid was used to subdivide the flow domain into computational finite volume cells. Shell finite elements were used to discretize the leaflet volume. A finite volume computational fluid dynamics code and finite element structure dynamics code were used to solve the flow and structure equations, respectively. The fluid flow and structural equations were coupled using an implicit "influence coefficient" technique. Physiologic ventricular and aortic pressure waveforms were prescribed as the flow boundary conditions. The aortic flow field, valve structural configuration, and leaflet stresses were computed at 2 msec intervals. Model predictions on aortic flow and transient variation in valve orifice area were in close agreement with corresponding experimental in vitro data. These findings suggest that the computer model has potential for being a powerful design tool for bioprosthetic aortic valves.

Apoptosis of human glioma cells in vitro and in vivo induced by a neutralizing antibody against human basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) is mitogenic to neuroectoderm- and mesoderm-derived cells and is a potent angiogenic factor. Abundant amounts of this factor and its receptor are detected in human glioma tissues and cells, and bFGF in glioma is thought to be involved in autonomous cell growth as an autocrine growth factor. A neutralizing mouse monoclonal antibody (MAb) against bFGF, 3H3 MAb, has been shown to inhibit both in vitro and in vivo growth of human glioma cell lines. This study shows that the human glioma cell lines U-87MG and U-251MG, which express high levels of bFGF and its receptor, can be induced to undergo apoptosis when cultured with 3H3 MAb. It is also demonstrated that 3H3 MAb can cause apoptosis in the same glioma cells that were transplanted into nude mice. Furthermore, enforced overexpression of bcl-2 protein by gene transfection prevented 3H3 MAb-induced apoptosis of glioma cells. It is concluded that induction of apoptosis by the neutralizing antibody is a promising therapeutic strategy for glioma.

Preference of calcium-dependent interactions between calmodulin-like domains of calpain and calpastatin subdomains.

Calpastatin molecule contains four repeated inhibition domains, each having highly conserved internal regions A, B and C. The synthetic oligopeptides of regions A and C had no calpain inhibition activity while region B oligopeptide showed weak inhibition activity. Real-time biomolecular interaction analysis using a BIAcore instrument revealed that the bacterially expressed calmodulin-like domain of the calpain large subunit (L-CaMLD) and that of the small subunit (S-CaMLD) interacted, in a Ca(2+)-dependent fashion, preferentially with the immobilized synthetic oligopeptide of region A and that of region C, respectively. Calmodulin showed no specific binding to these oligopeptides. The tripartite structure of the calpastatin functional domain may confer the specific interactions with the protease domain and the two CaMLDs of calpain.