[Preparation of interleukin-1ß-targeted nanobodies and their effects on apoptosis in hypoxic cardiomyocytes of mice].

Objective: To prepare interleukin-1ß-targeted nanoantibodies and observe their effects on apoptosis in hypoxic cardiomyocyte of mice. Methods: Using DNA recombination technology, the pET-16b and pHEN1 expression vectors were used to construct the prokaryotic expression plasmids of interleukin-1ß-targeted nanobodies (pET-16b-4G6M-VHH, pET-16b-5BVP-VHH, pET-16b-5MVZ-VHH, pHEN1-4G6M-VHH, pHEN1-5BVP-VHH and pHEN1-5MVZ-VHH, where VHH is a variable domain of heavy chain antibody, 4G6M-VHH, 5BVP-VHH, 5MVZ-VHH were three interleukin-1ß-targeted nanoantibodies respectively). The constructed plasmids were transferred into Escherichia coli Rosetta2 (DE3) for induction of expression and nickel column purification, respectively. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were employed to identify the expression product and purified product, and the enzyme-linked immune sorbent assay (ELISA) was performed to determine their affinity. The cardiomyocyte hypoxia model was used with the highest affinity IL-1ß-targeted nanobody (pHEN1-5MVZ-VHH), and cell survival and apoptosis rates were detected (the experiment was divided into normal control group, hypoxia model group, blank plasmid group and 12.5, 25.0, 50.0 µg/ml pHEN1-5MVZ-VHH treatment groups). Results: SDS-PAGE and Western blotting results showed that the anti-interleukin-1ß (IL-1ß) nanobodies with a relative molecular mass of about 15 000 were successfully obtained. Likewise, ELISA results found that the nanobodies expressed in pHEN1 vector group had higher affinity for IL-1ß antigen compared with pET-16b vector group (4G6M-VHH group: 3.20±0.03 vs 1.20±0.03, P<0.001; 5BVP-VHH group: 3.18±0.06 vs 1.21±0.02, P<0.001; 5MVZ-VHH group: 3.38±0.05 vs 1.62±0.04, P<0.001). Additionally, the results of cell survival assay and apoptosis assay detected that compared with the hypoxia model group, HL-1 cell activity was significantly increased in the 25.0 µg/ml and 50.0 µg/ml pHEN1-5MVZ-VHH treatment groups [(75.55±2.23)% vs (46.90±2.51)%, P<0.001; (74.36±1.96)% vs (46.90±2.51)%, P<0.001], and apoptosis rate was significantly reduced [(6.83±0.27)% vs (10.24±0.76)%, P<0.001; (6.68±0.38)% vs (10.24±0.76)%, P<0.001]. Conclusions: 4G6M-VHH, 5BVP-VHH, and 5MVZ-VHH are expressed by both pET-16b and pHEN1 expression vectors and the nanobodies produced by the pHEN1 vector display enhanced antigen affinity. Furthermore, in hypoxic cardiomyocytes, pHEN1-5MVZ-VHH treatment reduces cell apoptosis.

Gigantic jets between a thundercloud and the ionosphere.

Transient luminous events in the atmosphere, such as lighting-induced sprites and upwardly discharging blue jets, were discovered recently in the region between thunderclouds and the ionosphere. In the conventional picture, the main components of Earth's global electric circuit include thunderstorms, the conducting ionosphere, the downward fair-weather currents and the conducting Earth. Thunderstorms serve as one of the generators that drive current upward from cloud tops to the ionosphere, where the electric potential is hundreds of kilovolts higher than Earth's surface. It has not been clear, however, whether all the important components of the global circuit have even been identified. Here we report observations of five gigantic jets that establish a direct link between a thundercloud (altitude approximately 16 km) and the ionosphere at 90 km elevation. Extremely-low-frequency radio waves in four events were detected, while no cloud-to-ground lightning was observed to trigger these events. Our result indicates that the extremely-low-frequency waves were generated by negative cloud-to-ionosphere discharges, which would reduce the electrical potential between ionosphere and ground. Therefore, the conventional picture of the global electric circuit needs to be modified to include the contributions of gigantic jets and possibly sprites.

[Mechanism of cell autophagy for regulating skeletal muscle wasting of rats after severe burns].

Objective: To investigate the mechanism of cell autophagy for regulating skeletal muscle wasting of rats after severe burns. Methods: Seventy-two Sprague-Dawley rats were collected and divided into sham injury group, simple burn group, burn+ phosphate buffer solution (PBS) group, and burn+ 3-methyladenine (3-MA) group according to the random number table, with 18 rats in each group. Rats in simple burn group, burn+ PBS group, and burn+ 3-MA group were inflicted with 30% total body surface area full-thickness scald (hereinafter referred to as burns). Rats in sham injury group were sham injured. Immediately after burns and fluid resuscitation, rats in burn+ PBS group were intraperitoneally injected with 1 mL PBS, and rats in burn+ 3-MA group were intraperitoneally injected with 1 mL 3-MA (125 g/L). On post injury day 3 and 7, the weights of anterior tibial muscle of right hind limbs and body of rats were measured to calculate percentage of anterior tibial muscle of right hind limbs weight. Protein expressions of microtubule related protein 1 light chain 3A (LC3A) and Beclin-1 of anterior tibial muscle were observed by immunofluorescence method and detected by Western blotting, and ratio of microtubule related protein 1 LC3A-Ⅱ to LC3A-Ⅰ was calculated. Data were processed with analysis of variance of factorial design, one-way analysis of variance, t-test and Bonferroni correction. Results: On post injury day 3 and 7, percentages of anterior tibial muscle of right hind limbs weight of rats in simple burn group were (0.148±0.009)% and (0.134±0.018)%, respectively, which were significantly lower than those in sham injury group [(0.203±0.009)%, (0.181±0.015)%, t=10.585, 4.913, P<0.01]. Percentages of anterior tibial muscle of right hind limbs weight of rats in burn+ 3-MA group were (0.187±0.004)% and (0.192±0.009)%, respectively, which were obviously higher than those in burn+ PBS group [(0.162±0.005)%, (0.167±0.005)%, t=9.564, 5.948, P<0.01]. On post injury day 3 and 7, protein expressions of Beclin-1 and microtubule related protein 1 LC3A of anterior tibial muscle of rats in simple burn group were significantly higher than those in sham injury group, while protein expressions of Beclin-1 and microtubule related protein 1 LC3A of anterior tibial muscle of rats in burn+ 3-MA group were significantly lower than those in burn+ PBS group. Ratios of microtubule related protein 1 LC3A-Ⅱ to LC3A-Ⅰ of anterior tibial muscle of rats in simple burn group were significantly higher than those in sham injury group (t=3.461, 3.353, P<0.05), while ratios of microtubule related protein 1 LC3A-Ⅱ to LC3A-Ⅰ of anterior tibial muscle of rats in burn+ 3-MA group were significantly lower than those in burn+ PBS group (t=3.129, 3.977, P<0.05). Conclusions: Cell autophagy induced by severe burns is involved in the process of skeletal muscle wasting of rats, and inhibition of cell autophagy may contribute to the remission of skeletal muscle wasting of rats induced by burns.

Operator-less processing of myocardial perfusion SPECT studies.

UNLABELLED: We have developed a completely automated algorithm to generate reoriented tomographic images from projections in myocardial perfusion SPECT. METHODS: The algorithm consists of three software modules. The first module determines reconstruction limits for the projection dataset using two-dimensional feature extraction techniques. The second module reconstructs the projection images into transaxial images using standard filtered backprojection. The third module reorients the transaxial images into short-axis images. RESULTS: The algorithm was validated on 350 rest 201Tl and 350 stress 99mTc-sestamibi studies acquired on a single-detector (178 studies), a 90 degrees dual-detector (230 studies) or a triple-detector camera (292 studies). The complete processing sequence was successful in 93.6% of the studies (166/178 + 216/230 + 273/292). As for the individual modules, myocardial boundaries were correctly determined in 96.3% of the studies (171/178 + 222/230 + 281/292), while reorientation was successful in 97.2% of the studies (166/171 + 216/222 + 273/281). No significant difference in success rates for 201Tl versus 99mTc-sestamibi images was found. CONCLUSION: Our automated approach to myocardial perfusion SPECT processing is highly successful, intrinsically reproducible and can produce time and cost savings while improving accuracy in a clinical or research environment.

Automatic quantification of ejection fraction from gated myocardial perfusion SPECT.

UNLABELLED: We have developed a completely automatic algorithm to quantitatively measure left ventricular ejection fraction (LVEF) from gated 99mTc-sestamibi myocardial perfusion SPECT images. METHODS: The algorithm operates in the three-dimensional space and uses gated short-axis image volumes. It segments the left ventricle (LV), estimates and displays endocardial and epicardial surfaces for all gating intervals in the cardiac cycle, calculates the relative left ventricular cavity volumes and derives the global EF from the end-diastolic and end-systolic volume, all without operator interaction. The algorithm for measuring LVEF was tested in 65 clinical patients undergoing 16-interval and 8-interval rest-gated SPECT and validated against first-pass radionuclide ventriculography. RESULTS: Automatic segmentation and contouring of the LV was successful in 65/65 (100%) of the studies. Agreement between EFs measured from 8-interval gated SPECT and EFs calculated from first-pass data was high (y = 2.44 + 1.03x, r = 0.909, p < 0.001, s.e.e. = 6.87). Agreement between EF values measured from 16-interval and 8-interval gated SPECT was excellent (y = -2.7 + 0.97x, r = 0.988, p < 0.001, s.e.e. = 2.65), the latter being on average lower by 3.71 percentage points. CONCLUSION: Our automatic method is rapid and highly agrees with conventional radionuclide measurements of EF, thus providing clinically useful additional information to complement myocardial perfusion studies.

Automatic reorientation of three-dimensional, transaxial myocardial perfusion SPECT images.

UNLABELLED: We developed a completely automatic technique to reorient transaxial images into short-axis (oblique) myocardial perfusion SPECT images. METHODS: The algorithm starts by isolating (segmenting) the left ventricle (LV) myocardium using a combination of iterative clusterification and rule-based location/size/shape criteria. The three-dimensional, mid-myocardial LV surface is initially estimated as the locus of the trilinearly interpolated maxima for the count profiles originating from the center of mass of the segmented LV. The final mid-myocardial surface is obtained by iteratively applying this process, incorporating additional constraints of shape and texture and using the nonsegmented, nonthresholded transaxial image to obtain information on hypoperfused areas of the myocardium. It is then fitted to an ellipsoid, of which the major axis is assumed to represent the long axis of the LV, and the three-dimensional image volume is resliced perpendicularly to it. RESULTS: The algorithm was retrospectively applied to 400 dual-isotope studies (200 rest 201TI, 200 stress 99mTc-sestamibi) from 200 consecutive patients. Segmentation was successful in 394/400 (98.5%) of the patients. The reproducibility of computer-based reorientation was perfect and significantly better than either intraobserver or interobserver reproducibility. CONCLUSION: Automatic reorientation offers the potential for consistently faster and more accurate image processing and analysis and is an important step towards totally operator-less management of myocardial perfusion SPECT data.

Comparison of four neural net learning methods for dynamic system identification.

Four types of neural net learning rules are discussed for dynamic system identification. It is shown that the feedforward network (FFN) pattern learning rule is a first-order approximation of the FFN-batch learning rule. As a result, pattern learning is valid for nonlinear activation networks provided the learning rate is small. For recurrent types of networks (RecNs), RecN-pattern learning is different from RecN-batch learning. However, the difference can be controlled by using small learning rates. While RecN-batch learning is strict in a mathematical sense, RecN-pattern learning is simple to implement and can be implemented in a real-time manner. Simulation results agree very well with the theorems derived. It is shown by simulation that for system identification problems, recurrent networks are less sensitive to noise.

Ambient and personal dose assessment of a container inspection site using a mobile X-ray system.

Ambient monitor and phantom studies of absorbed and effective doses by TLDs were carried out in a non-intrusive inspection station for containers, Terminal I, of Taichung harbor, Taiwan. The doses from the X-ray scan in the control room and driver waiting room, located outside of the radiation control area, were quite small and could not be distinguished from the natural background radiation. The doses in the driver cab and the inspector cab of the X-ray scan car were also within background radiation levels. The protection wall, a 40-cm thick concrete barrier, can effectively attenuate the intensity of the primary X-ray scan. The possible effective dose of a person in the container or trailer is about 3.15 ± 0.23 µSv/scan and 2.31 ± 0.38 µSv/scan. This dose is below the annual background dose. If someone was to be scanned by the X-ray, the effective dose would be at an acceptable level.

Roentgenologic manifestations of pulmonary metastases in choriocarcinoma and invasive mole.

From 1949 to 1975, a total of 3,915 chest films were taken for 429 cases of choriocarcinoma and 441 cases of invasive mole. The incidences of pulmonary metastases were 85.1% and 65.0%, respectively. The various forms of pulmonary metastases were studied and correlated with clinical symptoms and pathologic changes. In order to follow up the progression or regression of the various forms, serial chest films were taken at intervals of 10 to 14 days in 27 cases. In eight cases postmortem pulmonary arteriograms were obtained on the autopsied lung specimens for the study of vascular changes of metastatic lesions. Pathologic examinations and pulmonary arteriography were also done on the surgically resected lung specimens. As a result of this study, a relative comprehensive knowledge about the nature and development of the various forms of metastatic shadows has been deduced. It is rational to say that the various forms seen on the chest films represent only the various evolutionary changes of the same lesion.

A comparison of neural networks and partial least squares for deconvoluting fluorescence spectra.

This article compares backpropagation neural networks (BNN) with partial least squares (PLS) techniques in terms of their ability to deconvolute fluorescence spectra. Both actual experimental and simulated spectral data are studied for 2 binary systems. These systems consist of mixtures of tryptophan and tyrosine, and NADH and tryptophan over a total concentration range of 10(-7) to 10(-4) M. It is shown that BNN is superior to PLS for both systems.

Audible in utero sound caused by the ultrasonic radiation force from a real-time scanner.

While investigating in utero sound levels during vibro-acoustic stimulation on the maternal abdomen it was noticed that noise level increased when the real-time ultrasonic scanner beam was directed at the sensing hydrophone. The noise was recorded and later analysed for frequency content and waveform. It appeared related to the scanning and frame rate frequencies of the scanner used. Sounds may originate from radiation pressure produced when the ultrasound beam is absorbed by tissue or reflected from bone or the metal hydrophone. This implies that although ultrasound cannot be heard per se, any modulation of its intensity will produce vibrations in the maternal tissues or reflecting structures such as skull bone, and especially stapes, malleus and incus, that would be heard as sound by the fetus. The intensity of the sound produced varied with orientation of the transducer beam and this may itself produce a stimulation. Based on our recordings (Fig. 1), it was calculated (please see Appendix) that the fetus would hear a sound corresponding to 84dB noise pressure level in air.

Nuclear localization of the human mutY homologue hMYH.

The cDNA of the human mutY homologue (hMYH) was cloned from the total RNA of the tumor cell line SU-DHL-1 by reverse transcription-polymerase chain reaction (RT-PCR). Expression of hMYH in a plasmid can partially revert the mutator phenotype of the Escherichia coli mutY mutant MK609(DE3). The majority of the recombinant hMYH protein in E. coli was precipitated in the inclusion bodies. A minor fraction of the soluble recombinant protein was concentrated as the source of the protein in the activity assay. Recombinant hMYH displayed both glycosylase and AP lyase activity. Three independent rabbit antisera against an N-terminal peptide, HY90, a recombinant C-terminal fragment, and the full-length hMYH recombinant protein were prepared and affinity-purified, and these antisera recognized the 59 kDa endogenous hMYH protein in HeLa cells. Immunofluorescent staining experiments with these three antisera showed a consistent nuclear distribution of hMYH, excluding the nucleoli. This nuclear staining pattern was abolished if the antisera were incubated with specific peptide/protein competitors, whereas the staining pattern was unaffected if the antisera were incubated with nonspecific peptide competitors. Consistent with the immunofluorescent staining results, a flag-tagged transfected hMYH also showed a nuclear staining pattern excluding the nucleoli. These results suggest that hMYH is indeed a functional homologue of E. coli MutY and is localized in the nuclei of mammalian cells.

Genomic aberrations of human papillomavirus recovered from cervical cancers.

Human papillomaviruses (HPV) contribute to the development of malignancies of the uterine cervix and the viral E6 and E7 oncogenes are invariably retained and expressed in cervical cancer tissues. Minor, but not major, structural aberrations have been found quite frequently in viral DNA recovered from cervical cancer tissues. We examined the presence of the DNA sequence of HPV type 18 in 33 cervical cancer tissues by polymerase chain reaction. HPV type 18 DNA sequences was found in 24 of these 33 cervical cancer tissue specimens, and at least 21 of these 24 specimens did not appear to retain all the region and open reading frames examined. Twelve of these 24 tissues seemed to harbor only the E6 and/or E7 genes. These results can be construed to suggest that the absence of viral genes other than E6 and E7 is quite frequent in HPV recovered from cervical cancer tissues and that the E6 and E7 genes are important in the carcinogenesis of cervical carcinoma. It is possible that the E6 and/or E7 alone may be sufficient to maintain the transformed phenotype of cervical cancer.