Molecular identification of Achyranthis Bidentatae Radix by using DNA barcoding.

Achyranthis Bidentatae Radix has a long history in China as a commonly used herb that can be used to treat various diseases, including those related to the liver, muscles, bones, and kidneys. Recently, an increase in the number of adulterants has been reported, which affects the clinical safety of Achyranthis Bidentatae Radix. To identify adulterants of Achyranthis Bidentatae Radix, we collected samples from major regions and conducted an in-depth genetic comparison of the herb and its commonly used adulterants. We amplified and sequenced three genomic regions, internal transcribed spacer (ITS), psbA-trnH, and internal transcribed spacer 2 (ITS2), to confirm whether ITS2 is a suitable identifier for Achyranthis Bidentatae Radix. Results showed that the ITS2 sequence length of Achyranthis Bidentatae Radix was 199 bp, with no variation between samples. The inter-specific genetic distance of ITS2 between Achyranthis Bidentatae Radix and its adulterants was 0.390. Neighbor-joining trees showed that Achyranthis Bidentatae Radix and its adulterants are easily differentiated by monophyly. In conclusion, ITS2 regions accurately and effectively distinguished between Achyranthis Bidentatae Radix and its adulterants.

Genetics and molecular mapping of genes for high-temperature resistance to stripe rust in wheat cultivar Xiaoyan 54.

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the most widespread and destructive wheat diseases worldwide. Growing resistant cultivars is the preferred means of control of the disease. The winter wheat cultivar Xiaoyan 54 has high-temperature resistance to stripe rust. To identify genes for stripe rust resistance, Xiaoyan 54 was crossed with Mingxian 169, a winter wheat genotype susceptible to all Chinese races of the pathogen. Seedlings and adult plants of the parents and F(1), F(2), F(3) and F(4) progeny were tested with Chinese race CYR32 under controlled greenhouse conditions and in the field. Xiaoyan 54 has two recessive resistance genes, designated as Yrxy1 and Yrxy2, conferring high-temperature resistance. Simple sequence repeat (SSR) primers were used to identify molecular markers flanking Yrxy2 using 181 plants from one segregating F(3) line. A total of nine markers, two of which flanked the locus at genetic distances of 4.0 and 6.4 cM on the long arm of chromosome 2A were identified. Resistance gene analog polymorphism (RGAP) and SSR techniques were used to identify molecular markers linked to Yrxy1. A linkage group of nine RGAP and two SSR markers was constructed for Yrxy1 using 177 plants of another segregating F(3) line. Two RGAP markers were closely linked to the locus with genetic distances of 2.3 and 3.5 cM. Amplification of a set of nulli-tetrasomic Chinese Spring lines with RGAP markers M8 and M9 and the two SSR markers located Yrxy1 on the short arm of chromosome 7A. The SSR markers Xbarc49 and Xwmc422 were 15.8 and 26.1 cM, respectively, from the gene. The closely linked molecular markers should be useful for incorporating the resistance genes into commercial cultivars and combining them with other genes for stripe rust resistance.

The value of PCT, IL-6, and CRP in the early diagnosis and evaluation of COVID-19.

OBJECTIVE: The aim of the present study was to assess the value of inflammatory factors procalcitonin (PCT), interleukin 6 (IL-6), and C-reactive protein (CRP) in the early diagnosis and evaluation of novel coronavirus pneumonia (COVID-19). MATERIALS AND METHODS: The data of 140 patients with pneumonia in our hospital, including 70 who had COVID-19 and 70 who had community-acquired pneumonia (CAP), were statistically analyzed. The levels of PCT, IL-6, and CRP were measured and statistically analyzed to determine the differences between the two groups. The differences in the COVID-19 group were analyzed after subgrouping into the ordinary type, severe type, and critical type. RESULTS: The PCT and CRP levels in the COVID-19 group were statistically lower than those in the CAP group (p < 0.05), but IL-6 was not statistically different between the two groups (p > 0.05). Statistically significant differences existed in IL-6 and CRP when comparing the COVID-19 subgroups of the critical type, severe type, and ordinary type (p < 0.05). However, there was no clinical meaning in the evaluation of the difference in PCT levels among the three subgroups with COVID-19. CONCLUSIONS: PCT and CRP could be used as indicators in the differentiation between COVID-19 and CAP, but IL-6 was of little significance in the differentiation. The higher the IL-6 and CRP, the more severe the condition of COVID-19 might be.

Desalination of brackish groundwater by direct contact membrane distillation.

The direct contact membrane distillation (DCMD) applied for desalination of brackish groundwater with self-made polyvinylidene fluoride (PVDF) membranes was presented in the paper. The PVDF membrane exhibited high rejection of non-volatile inorganic salt solutes and a maximum permeate flux 24.5 kg m(-2) h(-1) was obtained with feed temperature at 70 degrees C. The DCMD experimental results indicated that the feed concentration had no significant influence on the permeate flux and the rejection of solute. When natural groundwater was used directly as the feed, the precipitation of CaCO(3) would be formed and clog the hollow fibre inlets with gradual concentration of the feed, which resulted in a rapid decline of the module efficiency. The negative influence of scaling could be eliminated by acidification of the feed. Finally, a 250 h DCMD continuous desalination experiment of acidified groundwater with the concentration factor at constant 4.0 was carried out. The permeate flux kept stable and the permeate conductivity was less than 7.0 microS cm(-1) during this process. Furthermore, there was no deposit observed on the membrane surface. All of these demonstrated that DCMD could be efficiently used for production of high-quality potable water from brackish groundwater with water recovery as high as 75%.

Fluoride removal from brackish groundwater by direct contact membrane distillation.

The direct contact membrane distillation (DCMD) applied for fluoride removal from brackish groundwater is presented. The self-prepared polyvinylidene fluoride membrane exhibited high rejection of inorganic salt solutes and a maximum permeate flux 35.6 kgm(-2) h(-1) was obtained. The feed concentration had no marked impact on the permeate flux and the rejection of fluoride. The precipitation of CaCO3 would clog the hollow fiber inlets and foul the membrane surface with the increase of concentration factor when natural groundwater was used directly as the feed, which resulted in a rapid decline of the module efficiency. This phenomenon was diminished by acidification of the feed. The experimental results showed that the permeate flux and the quality of obtained distillate kept stable before concentration factor reached 5.0 with the acidified groundwater as feed. The membrane module efficiency began to decline gradually when the feed continued to be concentrated, which can be mainly attributed to the formation of CaF2 deposits on the membrane surface. Finally, a 300 h continuous fluoride removal experiment on acidified groundwater was carried out with concentration factor at 4.0, the permeate flux kept stable and the permeate fluoride was not detected.

Induction of new ADAM related proteins from treated human Chang-liver cells.

Chang-liver cells is a cell line generated from human liver tissue, which is often used in scientific research. ADAMs are a family of proteins that consist of multi-domains, possess multi-functions and play a central role in normal or abnormal physiological conditions, such as regeneration and tumorigenesis. To investigate the expression and functional alteration of the ADAMs or ADAM related proteins in Chang-liver cells, this cell line was treated with heat stress, modified Hanks solution containing ATP or other buffers. Our results showed that the treatment with Hanks solution containing ATP induces Chang-liver cells to express new ADAM related proteins. To analyze these new ADAM related proteins, a cDNA expression library was constructed for the treated Chang-liver cells. A series of positive clones were obtained through immunoscreening with an ADAMs common antibody. A new ADAM related protein possessing alkaline protease activity was confirmed in these clones.

The Cardioprotective Effects of Late-Phase Remote Preconditioning of Trauma Depends on Neurogenic Pathways and the Activation of PKC and NF-κB (But Not iNOS) in Mice.

BACKGROUND: A superficial abdominal surgical incision elicits cardioprotection against cardiac ischemia-reperfusion (I/R) injury in mice. This process, called remote preconditioning of trauma (RPCT), has both an early and a late phase. Previous investigations have demonstrated that early RPCT reduces cardiac infarct size by 80% to 85%. We evaluated the cardioprotective and molecular mechanisms of late-phase RPCT in a murine I/R injury model. METHODS: Wild-type mice, bradykinin (BK) 2 receptor knockout mice, 3M transgenic mice (nuclear factor κB [NF-κb] repressor inhibitor of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha [IκBα((S32A, S36A, Y42F))]), and inducible nitric oxide synthase (iNOS) knockout mice were analyzed using a previously established I/R injury model. A noninvasive abdominal surgical incision was made 24 hours prior to I/R injury and the infarct size was determined at 24 hours post-I/R injury. RESULTS: The results indicated that a strong cardioprotective effect occurred during late-phase RPCT (58.42% ± 1.89% sham vs 29.41% ± 4.00% late RPCT, mean area of the infarct divided by the mean area of the risk region; P ≤ .05; n = 10). Furthermore, pharmacological intervention revealed the involvement of neurogenic signaling in the beneficial effects of late RPCT via sensory and sympathetic thoracic nerves. Pharmacological experiments in transgenic mice-implicated BK receptors, ß-adrenergic receptors, protein kinase C, and NF-κB but not iNOS signaling in the cardioprotective effects of late RPCT. CONCLUSION: Late RPCT significantly decreased myocardial infarct size via neurogenic transmission and various other signaling pathways. This protective mechanism differentiates late and early RPCT. This study describes a new cardiac I/R injury prevention method and refines the concept of RPCT.

Radiation therapy depletes extrachromosomally amplified drug resistance genes and oncogenes from tumor cells via micronuclear capture of episomes and double minute chromosomes.

PURPOSE: To determine if clinically relevant doses of ionizing radiation are capable of inducing extrachromosomal DNA loss in transformed human cell lines. MATERIALS AND METHODS: The multidrug-resistant (MDR) human epidermoid KB-C1 cell line and the human neuroendocrine colon carcinoma line COLO320, which contain extrachromosomally amplified MDR1 drug resistance genes and MYCC oncogenes, were irradiated with 2 Gy fractions up to a total dose of 28 Gy. To track the fate of extrachromosomally amplified genes, cells surviving radiation therapy and unirradiated control cells were analyzed by fluorescent in situ hybridization of chromosomes using MDR1 and MYCC-specific cosmid DNA probes. In addition, total DNA and protein isolated from irradiated and control cells was subjected to Southern and Western blotting procedures, respectively, to determine amplified gene copy number and protein expression levels. Dose-response assays to follow loss of function of the MDR1 gene from KB-C1 cells were also performed. RESULTS: A significant reduction in extrachromosomal DNA, amplified gene copy number, and expression was detected in surviving cells after relatively low doses of radiation. Entrapment of extrachromosomal DNA into micronuclei was a consistent feature of irradiated cells. CONCLUSIONS: Clinically relevant doses of radiation can deplete extrachromosomal DNA in viable human malignant cells and alter their phenotype. Depletion of extrachromosomally amplified genes from tumor cells occurs via entrapment in radiation-induced micronuclei.

The question of relative biological effectiveness and quality factor for auger emitters incorporated into proliferating mammalian cells.

The problem of determining RBE values for Auger emitters incorporated into proliferating mammalian cells is examined. In general, the reference radiation plays a key role in obtaining experimental RBE values. Using survival of cultured Chinese hamster V79 cells as the experimental model, new data are provided regarding selection of a reference radiation for internal Auger emitters. These data show that gamma rays delivered acutely (137Cs) are more than twice as lethal as gamma rays delivered chronically with an exponentially decreasing dose rate (99mTc). The results confirm that the reference radiation should be delivered chronically in a manner consistent with the extended exposure received by the cells in the case of incorporated radionuclides. Through a direct comparison of the radiotoxicity of Auger emitters and alpha emitters, the high RBE values reported for DNA-bound Auger emitters are confirmed. These studies reveal that the DNA binding compound [125I]iododeoxyuridine (125IdU) is about 1.6 times more effective in killing V79 cells than 5.3 MeV alpha particles from intracellularly localized 210Po-citrate. In addition, toxicity studies with the radiochemicals 125IdU and [125]-iododeoxycytidine (125IdC) establish the equivalence of the radiosensitivity of thymine and cytosine base sites in the DNA. In view of these results, and information already available, the question of establishing quality factors for Auger emitters is considered. Finally, a method for calculation of the dose equivalent for internal Auger emitters is advanced.

Distribution of 111In-bleomycin complex in small cell lung cancer cells by autoradiography.

The distribution of 111In-bleomycin Complex (111In-BLMC) in small cell lung cancer (SCLC) cells was studied by autoradiography. SCLC cells were exposed to 111In-BLMC and 111Indium chloride (111InCl3) for 1 hour, 3 hours, and 4 hours; washed with fresh medium; and spread on slides. The slides were smeared with NTB2 (NTB3) emulsion by wet or dry-mount technique and exposed 3 to 15 days. 111In-BLMC was found to localize in the cell nucleus and nuclear membrane (78.3%); 111InCl3 located mainly in the cytoplasm (52.3%). This distribution of labeled BLM may explain the mechanism of killing SCLC cells by 111In-BLMC.

Enhanced killing of human small cell lung cancer by hyperthermia and indium-111-bleomycin complex.

Indium-111-bleomycin complex (111In-BLMC) is a radiopharmaceutical agent that produces tumor regression in mouse glioma in vivo and kills human small cell lung cancer (SCLC) cells in vitro. The interaction between hyperthermia and 111In-BLMC against human SCLC (N417) cells was studied for bleomycin (BLM) (15 micrograms/ml) or 111In-BLMC (40-50 microCi carried by 15 micrograms BLM/ml) for 5 min or 1.5, 2, or 4 hr at 37 degrees C or 43 degrees C exposures. Cell survival was determined by colony formation in soft agarose. There was a synergistic effect for 111In-BLMC and hyperthermia for cell killing. At 37 degrees C, the percent survival of N417 cells for BLM alone was 25.9%, and for 111In-BLMC it was 13.2%; at 43 degrees C, survival was 5.3% for BLM alone and 1.2% for 111In-BLMC by a 4 hr treatment. Effectiveness was greater when 111In-BLMC was combined with hyperthermia.

Chromosome aberrations of human small cell lung cancer induced by a new 111In-bleomycin complex.

A new 111Indium labeled bleomycin complex (111In-BLMC) was prepared and found to be effective for tumor imaging and therapy both in mouse glioma and human small cell lung cancer (SCLC) cells. Chromosome aberrations were studied in human SCLC cells to explore its mechanisms of killing cancer cells. SCLC cells (N417) were exposed to 111In-BLMC, BLM, or 111InCl3 (for control) for 1 hour, treated with colcemid, and chromosomal changes were analyzed. A dramatic increase in chromatic gaps, breaks, chromosome breaks, double minutes, rings, triradii, quadriradii, and chromosome stickiness were observed in the cells treated by 111In-BLMC compared to BLM or 111InCl3. These results indicated that 111In-BLMC has therapeutic potential for combination chemo-radiotherapy of cancer (e.g., by Auger electrons and local energy deposition).

Killing lung cancer cells at cell-cycle phase by a new indium-111-bleomycin complex.

The efficacy of killing small cell lung cancer (SCLC) cells at the G1, S, and G2-M phase of the cell-cycle by a new 111In-bleomycin complex (111In-BLMC) was investigated. SCLC cells (N417, H526, H209) were synchronized by double thymidine block and assessed by DNA content with flow cytometry, and the period for the maximal accumulation of cells in S, G1, or G2-M phase was determined. Cells in different cell cycle phases were exposed to 0.9% NaCl, BLM, or 111In-BLMC for 1 hour and observed for colony formation. The survival of H526 cells treated with 111In-BLMC was 71% (for enriched S phase), 46% (G1), and 31% (G2-M). For N417 cells, it was 25% (S), 20% (G1), and 8% (G2-M) for 111In-BLMC and 18% (S), 33% (G1), and 10% (G2-M) for BLM. These results indicated that SCLC cells in G2-M were most sensitive and those in S phase were least sensitive to 111In-BLMC; cells in G1 phase were the least sensitive to BLM.

Killing of human lung cancer cells using a new [111In]bleomycin complex [111In]BLMC.

The ability of a [111In]bleomycin complex [( 111In]BLMC) to kill five cell lines of human lung cancer (small cell lung cancer) was investigated. Cells were exposed to either 0.9% NaCl, [111In]Cl3, BLM, [111In]BLMC, nonradioactive InCl3, or In-BLMC for 60 minutes, plated in soft agarose, and assessed for colony formation. [111In]BLMC (40-200 microCi carried by 15-25 micrograms BLM/ml) was more cytotoxic than BLM (15-25 micrograms BLM/ml) by a factor of 1.6-5.3 for five cell lines. The percent survival of N417 cells was 28.4 for [111In]BLMC (40 microCi/15 micrograms BLM/ml) and 54.3 for BLM (15 micrograms/ml); 1.9 for [111In]BLMC (200 microCi/25 micrograms BLM/ml), and 10.0 for BLM (25 micrograms/ml). 111InCl3 (200 microCi/ml) and nonradioactive InCl3 failed to inhibit colony formation. The new [111In]BLMC may be useful for therapy of some lung cancer patients.

Stability of 111In-bleomycin in vivo--properties compared with 57Co-bleomycin.

111Indium-bleomycin (111In-BLM) and 57Co-bleomycin (57Co-BLM) were prepared and their distributions were compared in the tissues, blood, and urine in tumor-bearing and in untreated mice and rats. Autoradiographs of electrophoresis gels showed that patterns for urine from untreated and tumor-bearing animals, collected 1-3 h or 48 h after injection of 111In-BLM were similar to those for in vitro mixtures of urine and 111In-BLM, but differed from the patterns obtained with 111InCl3 under in vivo or in vitro conditions. In rats bearing mammary adenocarcinoma, 48 h after administration of the radiopharmaceutical, the activity ratio of tumor to eleven different tissues was 1.2-4.6 times higher for injected 111In-BLM than for 111InCl3 (P less than or equal to 0.001 or P less than or equal to 0.05). Imaging with a gamma camera depicted tumors in mice more distinctly with 111In-BLM than with 111InCl3. These findings were interpreted as reflecting the stability of 111In-BLM in vivo. The tumor concentration (%dose/g) was higher for the viable area than for the necrotic area for 111In-BLM, but the reverse was true for 57Co-BLM.

Use of 111In-bleomycin for combining radiotherapy and chemotherapy on glioma-bearing mice.

Mice bearing transplanted glioma received 0.9% NaCl, 0.1 mg of BLM, or 200-250 microCi of 111In-BLM (0.1 mg BLM) daily for 5 days intraperitoneally. After therapy, tumor sizes were in the order NaCl greater than BLM greater than 111In-BLM. On the 11th day after the first injection, tumor size (mm3) in the 111In-BLM group was 1,220; in the BLM group, it was 2,310 (P less than .025). After intratumor injection of a total dose of 0.1 mg of BLM/gm tumor weight, or of 1 mCi/gm tumor weight of 111In-BLM (carried by 0.1 mg of BLM/gm tumor weight), the tumor size decreased in the 111In-BLM group more than in the BLM group. On the 5th day after the 2nd dose therapy, the tumor size in the 111In-BLM group was 2,020; in the BLM group it was 4,220 (P less than .05). Host weights for these two groups were similar. The necrotic area in the tumor was much greater in the 111In-BLM group than in the BLM group. These results suggest the use for radiotherapy and chemotherapy.

Distribution and stability of [111In]bleomycin and its fractions in tumor-bearing mice.

The tissue distributions in glioma-bearing mice given injections of [111In]bleomycin (BLM) indicated that tumor concentrations and ratios of tumor to blood, muscle and brain for [111In]BLM-B2 and -A2 were higher than those for unfractionated [111In]BLM. Autoradiographs of electrophoretic gels of urine containing [111In]BLM or one of its fractions differed from those containing 111InCl3. [111In]BLM and its fractions (A2 and B2) were found to be stable in vivo. The fractions may be more useful in the clinic than [111In]BLM.

A new 111In-bleomycin complex for tumor imaging: preparation, stability, and distribution in glioma-bearing mice.

A new 111In-bleomycin complex (111In-BLMC) is here reported. Its radiochemical purity was 99% by thin-layer chromatography (TLC) (Rf 0.65) and in 5% agarose gel electrophoresis in 0.02 M NaHCO3 it migrated toward the anode. Autoradiographs of TLC and gel electrophoresis plates showed no change on storage for 3 weeks. Urine and plasma from untreated or glioma-bearing mice after injection of 111In-BLMC were analyzed by TLC and gel electrophoresis. Results indicated stability in vivo, nonbinding to transferrin, affinity to viable tumor, and excretion faster than 111In-BLM-B2, 111In-BLM, or 57Co-BLM. Tissue distributions 24 hr after injection of radiopharmaceutical showed activity ratios of tumor to blood, muscle, and brain of 13.1, 12.4, and 81.6, respectively, which were significantly higher than those for previously prepared 111In-BLM-B2 or 111In-BLM (except for brain, 0.05 less than P less than 0.1). The new 111In-BLM complex may be useful in clinical imaging and for combining radionuclide radiotherapy and chemotherapy.

A new 111In-bleomycin complex for combined radiotherapy and chemotherapy.

Six days after tumor transplantation three daily intraperitoneal doses of 0.9% NaCl, bleomycin (BLM), or a new 111In-bleomycin complex (BLMC, 15 microCi/g body weight) were administered to glioma-bearing mice. After therapy, tumors in mice treated with 111In-BLMC were smaller than those treated with BLM. Sixteen days after the first injection tumor size for 111In-BLMC-treated mice was 560 (240-1,030) mm3, 1,980 (1,400-3,290) mm3 for BLM (P less than 0.025), and 4,830 (2,580-9,180) mm3 for NaCl (0.1 less than P less than 0.2). Thirteen days after tumor transplantation glioma-bearing mice received single intratumor injection of 0.9% NaCl, BLM, or 111In-BLMC (1.5 mCi, carried by 0.5 mg BLM/g tumor weight). The average tumor size for 111In-BLMC was smaller than that for BLM by a factor of 2.5-3.7. Host weights for these two groups were similar, and morphologic abnormalities were not found in kidney or liver.

A new tumor imaging agent--111In-bleomycin complex. Comparison with 67Ga-citrate and 57Co-bleomycin in tumor-bearing animals.

We have found a new 111In-bleomycin complex (BLMC), which has high affinity to tumor, does not bind to transferrin and is stable in vivo. Distribution in animals bearing glioma, hepatoma, or mammary adenocarcinoma at 48 hours showed: the ratios of tumor to blood, brain, heart, lung, liver, pancreas, stomach, and femur were 1.4-22.4 times as high for 111In-BLMC as for 67Ga-citrate. In mammary adenocarcinoma, 111In-BLMC bound more to viable and 57Co-Bleomycin (BLM) more to necrotic tumor. In viable tumor, the concentration of 111In-BLMC was similar to that of 57Co-BLM. The ratios of tumor to stomach and pancreas were higher, to blood, brain, muscle, heart, and femur were lower for 111In-BLMC than those for 57Co-BLM. The ratios of tumor to lung, liver, spleen, skin, and kidney were similar for the two compounds. Tumors were imaged more distinctly with the new 111In-BLMC and 57Co-BLM than with 67Ga-citrate. 111In-BLMC is promising for tumor imaging.