Long noncoding RNA PVT1 predicts poor prognosis and promotes the progression of colorectal cancer through the miR-24-3p/NRP1 axis in zebrafish xenografts.

Long noncoding RNAs (lncRNAs) play important roles in the progression of human cancer. It is reported that lncRNA plasmacytoma variant translocation 1 (PVT1) is involved in colorectal cancer (CRC), however, the underlying mechanism remains to be explored deeply, especially by in vivo models. In the present study, bioinformatics analysis showed that the expression level of PVT1 was upregulated in CRC tissues and highly associated with poor prognosis of CRC patients. In cultured CRC cells, knockdown of PVT1 inhibited cell proliferation and migration of CRC cells, while overexpression of PVT1 promoted the progression of CRC cells. In zebrafish xenografts, the silencing of PVT1 also suppressed the growth and metastasis of CRC cells. For mechanism studies, the binding relationships among PVT1, miR-24-3p, and Neuropilin 1 (NRP1) were predicted by starBase firstly. The luciferase reporter assays verified that PVT1 and NRP1 could bind with miR-24-3p directly. Further studies showed miR-24-3p negatively regulated the progression of CRC cells, the inhibition of miR-24-3p counteracted the repression effects of CRC progression when knocking down PVT1. In addition, the expression of NRP1 was regulated by PVT1, and NRP1 overexpression could partially rescue the inhibition effects of CRC progression when knocking down PVT1 in vitro and in vivo. Our study reveals that PVT1 promotes the proliferation and metastasis of CRC via regulating the miR-24-3p/NRP1 axis, which provides a prognosis biomarker and a potential therapeutic target for CRC patients.

Simultaneous ground-state cooling of multiple degenerate mechanical modes through the cross-Kerr effect.

Simultaneous ground-state cooling of multiple degenerate mechanical modes is a difficult issue in optomechanical systems, owing to the existence of the dark mode effect. Here we propose a universal and scalable method to break the dark mode effect of two degenerate mechanical modes by introducing cross-Kerr (CK) nonlinearity. At most, four stable steady states can be achieved in our scheme in the presence of the CK effect, unlike the bistable behavior of the standard optomechanical system. Under a constant input laser power, the effective detuning and mechanical resonant frequency can be modulated by the CK nonlinearity, resulting in an optimal CK coupling strength for cooling. Similarly, there will be an optimal input laser power for cooling when the CK coupling strength stays fixed. Our scheme can be extended to break the dark mode effect of multiple degenerate mechanical modes by introducing more than one CK effect. To fulfill the requirement of the simultaneous ground-state cooling of N multiple degenerate mechanical modes, N - 1 CK effects with different strengths are needed. Our proposal provides new, to the best of our knowledge. insights into dark mode control and might pave the way to manipulating multiple quantum states in a macroscopic system.

Heterodyne detection of backscattering for whispering-gallery-mode sensors.

Whispering-gallery-mode (WGM) microcavities have shown significant applications in nanoparticle sensing for environmental monitoring and biological analysis. However, the enhancement of detection resolution often calls for active cavities or elaborate structural designs, leading to an increase of fabrication complexity and cost. Herein, heterodyne amplification is implemented in WGM microsensors based on backscattering detection mechanism. By interfering with an exotic reference laser, the reflecting light backscattered by perturbation targets can be strongly enlarged, yielding an easy-to-resolve and consequently sensitive microsensor. The dependence of detection laser frequency has also been characterized with the assistance of optothermal dynamics. We show that exploiting heterodyne interferometry boosts the detection of weak signals in microresonator systems and provides a fertile ground for optical microsensor development.

Tuning the Quantum Properties of ZnO Devices by Modulating Bulk Length and Doping.

The quantum transport properties of ZnO devices with five different bulk configurations are investigated with numerical methods. The calculation results reveal that the transport property at a higher energy range can be tuned by changing the length of central scattering. By substituting some Zn atoms with Cu atoms, it is found that the doped Cu atoms have an obvious effect on the quantum properties at the entire energy range investigated, and could result in different transmission. The properties of ZnO devices are also influenced by the doping positions of Cu atoms. The tuning mechanism relies on the shifting of carrier distributions in the scattering center of the device.

Phase-controlled dual-wavelength resonance in a self-coupling whispering-gallery-mode microcavity.

We report a novel, to the best of our knowledge, way to achieve phase-controlled dual-wavelength resonance based on whispering-gallery-mode (WGM) microcavities experimentally. With the help of a feedback waveguide, not only two optical pathways but also a unidirectional coupling between counter-propagating waves are formed, which is the requirement of all-optical analogues of electromagnetically induced transparency and Autler-Townes splitting. By adjusting the accumulating phase introduced from the fiber waveguide, we observe the signal lineshape changes from symmetric to asymmetric, i.e., the resonant transmission and extinction ratio of two splitting modes can be controlled, which brings a new degree of freedom to the WGM resonator system. These results may boost the development of quantum state control and pave the way for reconfiguring devices such as narrow-band filters.

The efficacy and safety of one-stage endoscopic treatment for ascending acute cholangitis caused by choledocholithiasis with severe comorbidities.

BACKGROUND: Emergency endoscopic retrograde cholangiopancreatography (ERCP) for ascending acute cholangitis in patients with severe comorbidities is challenging. Here, we evaluated the efficacy and safety of one-stage ERCP in such patients by performing a retrospective study. METHODS: We included all patients with ascending acute cholangitis and undergoing ERCP between January 2017 and March 2019. In total, we recruited 212 patients: 74 and 138 with and without severe comorbidities, respectively. We collected and analyzed data related to basal characteristics, ERCP, and clinical outcomes. RESULTS: Elderly age (76.20 ± 9.99 years vs. 66.52 ± 8.16 years, P = 0.000), higher levels of leukocyte count (15.86 ± 2.47 × 109/ml vs. 13.49 ± 1.65 × 109/ml, P = 0.000), and serum bilirubin (3.11 ± 1.29 mg/dl vs. 1.94 ± 0.90 mg/dl, P = 0.000) were present in patients with severe comorbidities. A significantly higher proportion of these patients were severe cases (32.4% vs. 6.5%, P = 0.000), American Society of Anesthesiologists (ASA) stage V status (37.8% vs. 10.1%, P = 0.000) and had undergone general anesthesia (56.8% vs. 18.8%, P = 0.000). Successful biliary cannulation and complete stone clearance in one session were achieved in 207 and 202 patients, respectively. Mean length of hospital stay was 8.02 ± 2.71 days. Forty-three patients required ICU stay with the mean length of 3.26 ± 3.51 days. In-hospital mortality occurred in seven patients; all these patients had severe comorbidities. ERCP details, including urgent and early ERCP, biliary cannulation, complete stone clearance in one session, stent insertion, and complications were not significantly different between the two groups. Patients with severe comorbidities had a longer in-hospital stay (9.39 ± 3.15 days vs. 7.29 ± 2.11 days, P = 0.000), a higher proportion of ICU admission (45.9% vs. 6.5%, P = 0.000), and a longer ICU stay length (4.88 ± 4.37 days vs. 1.44 ± 0.52 days, P = 0.000). Our data also revealed that early diagnosis is an important predictor associated with clinical outcomes. CONCLUSIONS: One-stage ERCP is safe and effective for ascending acute cholangitis caused by choledocholithiasis. Early diagnosis is a significant predictor of clinical outcomes.

Shuxuening injection, derived from Ginkgo biloba leaf, induced pseudo-allergic reactions through hyperactivation of mTOR.

Context: Shuxuening injection (SXNI), derived from the leaf of Ginkgo biloba L. (Ginkgoaceae), is widely used to treat cardio-cerebral vascular system related disease due to the efficacy of dilating the blood vessels and improving the function of microcirculation. Nevertheless, SXNI induces immediate hypersensitivity reactions in clinics and the molecular mechanisms are unknown.Objective: The present study investigates the molecular mechanism of SXNI mediated hypersensitivity reactions.Materials and methods: Naive male ICR mice (n = 10) were administered (i.v.) with negative control combined with Evans blue (EB) (CTL-EB), SXNI (14 or 70 mg/kg) combined with EB (SXNI/1-EB or SXNI/4-EB), vascular leakage was evaluated, ears and lungs were collected for histopathological analysis. In vitro, TSC1 was knockdown in human umbilical vein endothelial cells (HUVECs). HUVECs were incubated with SXNI, and the alterations of endothelial cell permeability were observed. Rapamycin (mTOR inbibitor) was used to investigate SXNI-induced hypersensitivity reactions both in mice and HUVECs.Results: SXNI (70 mg/kg) induced vascular leakage in mice. Slight oedema and microvascular dilation in the ears, and broaden of alveolar septal and monocyte infiltration in the lungs were observed in SXNI (70 mg/kg) treated mice. mTOR inhibitor alleviates SXNI mediated vascular endothelial hyperpermeability both in vitro and in vivo.Discussion and conclusions: SXNI stimulates pseudo-allergic reactions through hyperactivation of mTOR signalling pathway. Our work provides the new molecular mechanism of drug related pseudo-allergic reactions, and a potential drug to prevent and treat SXNI mediated hypersensitivity reactions.

Genome-wide transcriptional analysis of Aristolochia manshuriensis induced gastric carcinoma.

Context: Aristolochia manshuriensis Kom (Aristolochiaceae) (AMK) is known for toxicity and mutagenicity.Objective: The tumorigenic role of AMK has yet to be understood.Materials and methods: AMK extracts were extracted from root crude drug. SD (Sprague Dawley) rats underwent gavage with AMK (0.92 g/kg) every other day for 10 (AMK-10) or 20 (AMK-20) weeks. Stomach samples were gathered for histopathological evaluation, microarray and mRNA analysis.Results: The gastric weight to body weight ratio (GW/BW) is 1.7 in the AMK-10 cohort, and 1.8 in AMK-20 cohort compared to control (CTL) cohort. Liver function was damaged in AMK-10 and AMK-20 rats compared to CTL rats. There were no significant changes of CRE (creatinine) in AMK-10 and AMK-20 rats. Histopathological analysis revealed that rats developed dysplasia in the forestomach in AMK-10 rats, and became gastric carcinoma in AMK-20 rats. Genes including Mapk13, Nme1, Gsta4, Gstm1, Jun, Mgst2, Ggt6, Gpx2, Gpx8, Calml3, Rasgrp2, Cd44, Gsr, Dgkb, Rras, and Amt were found to be critical in AMK-10 and AMK-20 rats. Pik3cb, Plcb3, Tp53, Hras, Myc, Src, Akt1, Gnai3, and Fgfr3 worked in AMK-10 rats, and PDE2a and PDE3a played a pivotal role in AMK-20 rats.Discussion and conclusions: AMK induced benign or malignant gastric tumours depends on the period of AMK administration. Genes including Mapk13, Nme1, Gsta4, Gstm1, Jun, Mgst2, Ggt6, Gpx2, Gpx8, Calml3, Rasgrp2, Cd44, Gsr, Dgkb, Rras, Amt, Pik3cb, Plcb3, Tp53, Hras, Myc, Src, Akt1, Gnai3, Fgfr3, PDE2a, and PDE3a were found to be critical in aristolochic acid-induced gastric tumour process.

The Involvement of the RhoA/ROCK Signaling Pathway in Hypersensitivity Reactions Induced by Paclitaxel Injection.

A high incidence of hypersensitivity reactions (HSRs) largely limits the use of paclitaxel injection. Currently, these reactions are considered to be mediated by histamine release and complement activation. However, the evidence is insufficient and the molecular mechanism involved in paclitaxel injection-induced HSRs is still incompletely understood. In this study, a mice model mimicking vascular hyperpermeability was applied. The vascular leakage induced merely by excipients (polyoxyl 35 castor oil) was equivalent to the reactions evoked by paclitaxel injection under the same conditions. Treatment with paclitaxel injection could cause rapid histamine release. The vascular exudation was dramatically inhibited by pretreatment with a histamine antagonist. No significant change in paclitaxel injection-induced HSRs was observed in complement-deficient and complement-depleted mice. The RhoA/ROCK signaling pathway was activated by paclitaxel injection. Moreover, the ROCK inhibitor showed a protective effect on vascular leakage in the ears and on inflammation in the lungs. In conclusion, this study provided a suitable mice model for investigating the HSRs characterized by vascular hyperpermeability and confirmed the main sensitization of excipients in paclitaxel injection. Histamine release and RhoA/ROCK pathway activation, rather than complement activation, played an important role in paclitaxel injection-induced HSRs. Furthermore, the ROCK inhibitor may provide a potential preventive approach for paclitaxel injection side effects.

Decreased Urine miR-199-3p may be a Potential Biomarker for Diabetic Nephropathy via Targeting Zinc Finger E-box-Binding Protein 1.

BACKGROUND: The current study aims to evaluate the expression pattern changes of miR-199-3p in diabetic nephropathy (DN) patients and explore whether it could be used as a potential biomarker. METHODS: Real time PCR analysis was performed to examine the level of miR-199-3p in normoalbuminuria group (diabetes mellitus [DM]), microalbuminuria group (DNE), macroalbuminuria group (DNC), and healthy controls. ROC analysis was carried out to determine whether urine miR-199-3p could be used as a potential biomarker. Dual luciferase reporter assay was used to identify the target gene of miR-199-3p. RESULTS: Here we showed novel data that urine miR-199-3p was significantly decreased in the diabetes patients compared to healthy controls. Meanwhile, urine miR-199-3p was lowest in DNC group, lower in DNE group, but was relatively higher in DM group. Additionally, urine miR-199-3p level positively correlated with UAE level in both DNE group and DNC group. ROC analysis showed that the urine miR-199-3p may be used to differentiate DNE and DNC subjects from healthy controls and DM group. Besides, miR-199-3p could significantly suppress the relative luciferase activity of pmirGLO-ZEB1, indicating ZEB1 was a target gene of miR-199-3p. CONCLUSIONS: In summary, for the first time, we showed novel data that decreased urinary miR-199-3p could screen DN patients from DM patients and healthy controls, which may be a non-invasive biomarker for DN patients.

[Mechanism of nephrotoxicity of rhubarb in rats].

The aim of this study was to investigate the renal toxicity of rhubarb and its mechanism. The SD rats were randomly divided into three groups: normal group and two rhubarb extract groups (16, 2 g·kg⁻¹). According to the dose conversion method between human and animal, rhubarb 16 g·kg⁻¹ and 2 g·kg⁻¹ were equivalent to 10 times and 1.25 times of human clinical dose respectively. Rhubarb extract was administered by a gastric gavage to rats once daily for 30 days. Serum urea nitrogen （BUN）, creatinine （CRE） and urine KIM-1, NGAL and renal morphology were analyzed. The expressions of OAT1, OAT3 and clusterin mRNA in kidney were measured. The results showed that the low dose of rhubarb had no obvious renal toxicity. The high dose group showed mild and moderate renal injury and a down-regulation of clusterin mRNA expression in the kidney tissue. The renal toxicity in male animals was heavier than that in female animals. There was no significant change in blood BUN and CRE in the high dose group. But urine NGAL level of the high dose group increased by 51.53% compared with normal group, of which male animals increased more significantly (P<0.05, compared with the normal group). The expressions of renal OAT1 and OAT3 mRNA in the low dose group were obviously higher than that in the normal group. The results indicated that the high dose of rhubarb could cause the renal toxicity. The dosage should be controlled reasonably in the clinical use. OAT1 and OAT3 mRNA related to anionic transport in kidney tissue played a compensatory protective role in rhubarb-induced renal injury. But the compensatory effect is relatively weak at the high dose level. In addition, routine renal function indicators BUN and CRE had limitation for monitoring the kidney toxicity of rhubarb. It is suggested that urine NGAL detection might be helpful for monitoring the renal toxicity of rhubarb.

The Association of CYP1A1 Gene With Cervical Cancer and Additional SNP-SNP Interaction in Chinese Women.

AIMS: The aim of this study was to investigate the association between CYP1A1 gene polymorphism and cervical cancer risk, and the impact of SNP-SNP interaction on cervical cancer risk in Chinese women. METHODS: A total of 728 females with a mean age of 60.1 ± 14.5 years old were selected, including 360 cervical cancer patients and 368 normal controls. Logistic regression was performed to investigate association between single-nucleotide polymorphisms (SNP) and cervical cancer risk. Generalized multifactor dimensionality reduction (GMDR) was used to analyze the SNP-SNP interaction. RESULTS: Logistic analysis showed a significant association between rs4646903 and increased cervical cancer risk. The carriers of homozygous mutant of rs4646903 polymorphism revealed increased cervical cancer risk than those with wild-type homozygotes, OR (95%CI) were 1.45 (1.20-1.95). There was a significant two-locus model (P = 0.0107) involving rs4646903 and rs1048943, indicating a potential SNP-SNP interaction between rs4646903 and rs1048943. Overall, the two-locus models had a cross-validation consistency of 10 of 10, and had the testing accuracy of 60.72%. Subjects with TC or CC of rs4646903 and AG or GG of rs1048943 genotype have the highest cervical cancer risk, compared to subjects with TT of rs4646903 and AA of rs1048943 genotype, OR (95%CI) was 2.03 (1.42-2.89). CONCLUSIONS: rs4646903 minor alleles and interaction between rs4646903 and rs1048943 were associated with increased cervical cancer risk.

[Review on requirements of drug allergy or pseudoallergic reactions in pre-clinical evaluation].

Drug allergy and pseudoallergic reactions are main adverse drug reactions. Allergy is mainly induced by the immunogenicity of drug, drug metabolic products or drug additive. Pseudoallergic reactions may result from the irritation or activation of inflammatory material release. Pre-clinical evaluation of drug allergy and pseudoallergic reactions is included in immunotoxicity evaluation. Now there is no in vivo or in vitro method that could predict all kinds of allergy or pseudoallergic reactions due to the different mechanisms. In the past few years, FDA, SFDA OECD, ICH and WHO have published several guidelines on per-clinical immunotoxicity evaluation, however, no agreement has been reached on allergy and pseudoallergic reactions evaluation. This article reviews the requirements of allergy and pseudoallergic reactions in pre-clinical evaluation.

[Genotoxicity research thought and method on traditional Chinese medicine].

Genotoxicity research takes an important place in traditional Chinese medicine safety evaluation. Genotoxicity test on traditional Chinese medicine has been paid great attention since 1970s. Currently, the most developed genotoxicity test methods included: bacterial reverse mutation test and mouse lymphoma assay which are used to detect relevant genetic changes, micronucleus test and chromosomal analysis which are used to measure chromosomal aberration, and single cell electrophoresis assay which is used to test DNA damage. This article reviews research progress on genotoxicity of traditional Chinese medicine, evaluation methods of genotoxicity, the problems and solutions on genotoxicity evaluation of traditional Chinese medicine, and new technique used in genotoxicity test.

[Preclinical evaluation of pseudoallergic reactions on Chinese herbal injections: study on animal strain and gender difference].

Pseudoallergic reactions occured after the first administration of patients, and the pathogenic mechanisms of them were different from the allergic reactions which needed excitation after antigen sensitization. To provide a basis for evaluation, clinical use and drug development of pseudoallergic reactions, the models were established by two kinds of Chinese herbal injections (CHI) both on different strain or gender mice. With the use of ICR, Kunming, BALB/C, C57 mice, pseudoallergic tests of two CHI were conducted to compare the sensitivity of four strains mice, and compared the differences in male and female animals. Test substances contain 0.8% Evans blue (EB) were intravenously injected into different strain and gender mice. Scores of ear blue staining and quantitation of ear EB exudation were the parameters for pseudoallergic reaction. Results of strain difference indicated that both CHI A and B could cause severe pseudoallergic reactions indicated by obvious vascular hyperpermeability on ICR mice. The pseudoallergic reactions in ICR mice are more obvious under the the same dose of injection, which stated the sensibility of ICR mice. And the reactions of KM mice and BALB/C mice were slightly reduced which compared to ICR mice, even alomost nothing on C57 mice. Comparison results of gender difference showed that one CHI was not have significant difference in male and female animals, but male animals were more susceptible than females on another CHI. Therefore, ICR mice were preferable experimental strain on the model of pseudoallergic reactions induced by CHI A and B. Because of female animals were easily influenced by estrous cycle, the pseudoallergic reactions induced by CHI A and B select and use male mice befittingly.

[Study of screening pseudoallergenic substances of Shuanghuanglian injection].

In this study, chemistry, biology and pharmacology were combinated to screen pseudoallergenic substances of Shuang-huanglian injection (SHLI) so that to establish a scientific and systematic approach to screen pseudoallergenic substances of traditional Chinese medicine injections. The mouse pseudoallergic reaction models were used to screen the pseudoallergic reaction of SHLI's intermediate extract and the intermediate extract's component or ingredient. Among the three intermediates of Shuanghuanglian injection (extract of Scutellaria baicalensis, extract of Lonicera japonica, extract of Forsythia suspensa) , pseudoallergic action of Forsythia suspensa was the strongest, Forsythia suspesnsa's pseudoallergic reaction mainly associated with the composition with largerchemical polarity. Further it was found that forsythiaside A and arctiin which existed in the the composition with largerchemical polarity caused obvious pseudoallergic reactions. SHLI with removal forsythoside A with the technology of HPLC-MS displayed reduced pseudoallergic reaction and a significant improved safety. This study provided a scientific basis for SHLI process improvements and also offered idea and research foundation for screening pseudoallergenic substances injections in other TCM injections.

[Thought and method of reproductive toxicity research in traditional Chinese medicine].

Reproductive toxicity research takes an important place in traditional Chinese medicine pre-clinical safety evaluation. Modern reproductive toxicity experiment includes drug-related miscarriage, fetal death, teratism, and adverse effects on fertility, genital system, embryonic development and fetus, which is different from contraindicated in pregnancy in traditional Chinese medicine theory. Now the three-phases reproductive toxicity study is the method mainly applied in traditional Chinese medicine reproductive toxicity evaluation. Besides that, alternative methods of whole embryos culture and embryonic stem cell test are also used in traditional Chinese medicine embryo toxicity evaluation. This article reviews research progress and pre-clinical evaluation on reproductive toxicity of traditional Chinese medicine.

[Experimental study on pseudoallergic reaction of Qingkailing injection].

Pseudoallergic reactions of Qingkailing injection (QKLI) was assessed by vascular hyperpermeability which were indicated by ear blue staining in ICR mice after single intravenous injection of QKLI mixed with Evans blue (EB) and skin blue spot formation in SD rats after intradermal injection of QKLI and intravenous injection of EB. In addition, QKLI-induced histamine, VEGF, TNF-alpha release was measured after ICR mice received the single dosing of QKLI iv. The mild vascular hyperpermeability characterized by ear blue staining could be observed in mice after intravenous injection of QKLI and EB. Intracutaneous injection of 50 micro L of test solution containing QKLI (25,50 microL) in rat back skin caused obvious local vascular hyperpermeability at the injection sites so as to result the larger diameters of blue spots than that in negative control group (P <0. 01). QKLI induced a significant increase of VEGF and a slight elevation of histamine in mice after intravenous administration, while TNF-alpha showed no change after QKLI iv. The results in this study indicated that both intravenous injection and intracutanous injection of QKLI could induce vascular hyperpemeability so as to cause pseudoallergic reaction in mice and rats. QKLI-induced pseudoallergic reaction may be associated with the release of histamine and VEGF.

A novel data fusion based intelligent identification approach for working cycle stages of hydraulic excavators.

Accurately identifying the stage of the excavator working cycle is the prerequisite to achieve the staged energy-saving control. However, current identification methods often overlook the influence of hydraulic system latency on identification results and depend on a single model, resulting in poor generalization performance of the identification approaches. Moreover, expert calibration system remains a necessary factor for improving identification accuracy. Aiming at these issues, a hybrid multi-scale feature extractor and a decision-level data fusion classifier approach (HMSFE-DFC) is proposed to identify the working cycle stages of excavator. The input signal employs mixed signals from the main pump pressure and the control current of the proportional solenoid valve to reduce the response delay caused by the single main pump pressure signal. A hybrid multi-scale feature extractor is constructed using a convolutional neural network temporal self-attention feature extraction mechanism and one-dimensional ResNet-50 architecture to extract multiscale features. To prevent overfitting, a decision-level data fusion classifier is used to fuse the decisions information of numerous classifiers. The accuracy of stage identification for 10 consecutive working cycles reaches 95.21%, which verifies its effectiveness.

Copper-Catalyzed Difunctionalization of Propargylic Carbonates through Tandem Nucleophilic Substitution/Boroprotonation.

Highly functionalized organic molecules are in high demand, but their preparation is challenging. Copper-catalyzed transformation of alkynyl- and allenyl-containing substrates has emerged as a powerful tool to achieve this objective. Herein, an efficient copper-catalyzed difunctionalization of propargylic carbonates through tandem nucleophilic substitution/boroprotonation has been developed, affording the formation of thiol-, selenium-, and boron-functionalized alkenes with high yield and stereoselectivity. Two distinct catalytic mechanisms involving a single reaction without any requirement of catalyst change were successfully demonstrated.