Gene repression using synthetic small regulatory RNA in Methylorubrum extorquens.

AIM: Genetic tools are a prerequisite for engineering cell factories for synthetic biology and biotechnology. Methylorubrum extorquens is an important platform for a future one-carbon (C1) bioeconomy, but its application is currently limited by the availability of genetic tools. Small regulatory RNA (sRNA) is an important regulatory factor in bacteria and has been applied for gene repression in several strains. This study aimed to construct a synthetic sRNA system based on the MicC scaffold and the chaperone Hfq to control gene expression in M. extorquens. METHODS AND RESULTS: Initially, the exogenous lacZ gene was transposed into the M. extorquens chromosome as a reporter, and corresponding ß-galactosidase was measured to assess the knockdown efficiency of lacZ. A synthetic sRNA containing a 24-nt antisense RNA targeting lacZ and an Escherichia coli MicC scaffold were constructed, and different Hfqs from E. coli, M. extorquens AM1 and PA1 were further identified. The results showed that the expression of endogenous hfqs from the chromosome in M. extorquens strains was inadequate, and only when it was overexpressed via the plasmid did the colonies show a colour change and a corresponding decrease in ß-galactosidase expression. More specifically, M. extorquens strains with overexpressing their own Hfq showed the best gene repression efficiency. Furthermore, this E. coli MicC scaffold and AM1 Hfq system were combined to knock down crtI gene expression in AM1, leading to an 86% decrease in carotenoid production (0·09 mg g-1 ) compared to that (0·65 mg g-1 ) in the wild-type strain. CONCLUSION: A functional synthetic sRNA system combined with E. coli MicC and endogenous Hfq was constructed in M. extorquens strains, which was able to interfere with the target crtI gene and reduce carotenoid production. SIGNIFICANCE AND IMPACT OF THE STUDY: The synthetic sRNA system reported in this study provides a genetic tool for the manipulation of M. extorquens. The present findings might be helpful for achieving high-throughput gene knockdown expression.

[Determination of 1-methoxy-2-propanol in urine by headspace solid-phase microextraction coupled with gas chromatography].

Objective: To establish a method for the determination of 1-methoxy-2-propanol in urine using headspace solid phase micro-extraction coupled with gas chromatography. Methods: The 1-methoxy-2-propanol was enriched by headspace solid phase micro-extraction fiber coated with carbene/polydimethylsiloxane (CAR/PDMS) . Single factor rotation method was used to optimize the conditions of extraction temperature, salt amount, and extraction time. The separation was performed on DB-5 (30 m×0.32 mm×0.25 µm) capillary column and detected with flame ionization detector. The quantification was based on the standard curve. Results: The concentration of 1-methoxy-2-propanol in urine was linear in the range of 0.50-10.0 mg/L, and the linear correlation coefficient was 0.9993. The detection limit of the method was 0.14 mg/L, and the limit of quantification was 0.45 mg/L. The recovery was 85.8% to 104.7%, and the RSD of intra- and inter-batch precision were 3.25%-6.65% and 0.81%-3.96%, respectively. Conclusion: The method is high sensitivity and simple operation, and is suitable for the determination of 1-methoxy-2-propanol in urine of occupational exposure population.

Repeatability of vessel density measurement in human skin by OCT-based microangiography.

PURPOSE: To investigate the repeatability of vessel density measurement at human arm skin in healthy subjects with OCT-based microangiography (OMAG). METHODS: Four locations including volar wrist, volar forearm, shoulder, and volar upper arm were scanned using an optimized swept source OCT system, working at center wavelength of 1300 nm and A-line rate of 100 kHz. Three scans were acquired at each location at the same visit. Vascular images of papillary dermis, reticular dermis, and the whole dermis layer were generated with OMAG processing and automatic segmentation algorithms. The vessel density (VD) of each layer was calculated based on vascular images, and the repeatability of the VD at the same physiological location was thereafter assessed. RESULTS: Fifteen healthy volunteers were included. High repeatability of VD was found for wrist, forearm, shoulder, and upper arm (coefficient of variation (CV)=2.4, 2.7, 2.7, 2.0, and intraclass correlation coefficient (ICC)=0.906, 0.854, 0.943, 0.916 respectively). The VD measurements showed no significant difference between the four locations in any of the three layers, ie papillary layer (P=.1063), reticular layer (P=.3371), and whole dermis layer (P=.3233). CONCLUSION: Quantification of VD by using OCT/OMAG is repeatable when imaging skin tissue beds in healthy individuals.

[Different methods in predicting mortality of pediatric intensive care units sepsis in Southwest China].

Objective: To investigate the value of systemic inflammatory response syndrome (SIRS), pediatric sequential organ failure assessment (pSOFA) and pediatric critical illness score (PCIS) in predicting mortality of pediatric sepsis in pediatric intensive care units (PICU) from Southwest China. Methods: This was a prospective multicenter observational study. A total of 447 children with sepsis admitted to 12 PICU in Southwest China from April 2022 to March 2023 were enrolled. Based on the prognosis, the patients were divided into survival group and non-survival group. The physiological parameters of SIRS, pSOFA and PCIS were recorded and scored within 24 h after PICU admission. The general clinical data and some laboratory results were recorded. The area under the curve (AUC) of the receiver operating characteristic curve was used to compare the predictive value of SIRS, pSOFA and PCIS in mortality of pediatric sepsis. Results: Amongst 447 children with sepsis, 260 patients were male and 187 patients were female, aged 2.5 (0.8, 7.0) years, 405 patients were in the survival group and 42 patients were in the non-survival group. 418 patients (93.5%) met the criteria of SIRS, and 440 patients (98.4%) met the criteria of pSOFA≥2. There was no significant difference in the number of items meeting the SIRS criteria between the survival group and the non-survival group (3(2, 4) vs. 3(3, 4) points, Z=1.30, P=0.192). The pSOFA score of the non-survival group was significantly higher than that of the survival group (9(6, 12) vs. 4(3, 7) points, Z=6.56, P<0.001), and the PCIS score was significantly lower than that of the survival group (72(68, 81) vs. 82(76, 88) points, Z=5.90, P<0.001). The predictive value of pSOFA (AUC=0.82) and PCIS (AUC=0.78) for sepsis mortality was significantly higher than that of SIRS (AUC=0.56) (Z=6.59, 4.23, both P<0.001). There was no significant difference between pSOFA and PCIS (Z=1.35, P=0.176). Platelet count, procalcitonin, lactic acid, albumin, creatinine, total bilirubin, activated partial thromboplastin time, prothrombin time and international normalized ratio were all able to predict mortality of sepsis to a certain degree (AUC=0.64, 0.68, 0.80, 0.64, 0.68, 0.60, 0.77, 0.75, 0.76, all P<0.05). Conclusion: Compared with SIRS, both pSOFA and PCIS had better predictive value in the mortality of pediatric sepsis in PICU.

Acupuncture combined with exercise training at different time points on nerve repair of cerebral ischemia-reperfusion injury in rats and its effects on the expressions of Nestin, bFGF and EGF.

OBJECTIVE: The aim of the study was to observe the neuroreparative effect of electroacupuncture in rats with cerebral ischemia-reperfusion injury, and to explore the difference in the therapeutic effect of acupuncture on different acupoint groups after cerebral ischemia-reperfusion. MATERIALS AND METHODS: Experimental rats were randomly divided into: sham operation group, model group, electroacupuncture group, rehabilitation group, and Diankang group (electroacupuncture + rehabilitation training). There were 24 rats in each group, and the focal cerebral ischemia-reperfusion model was established by Zea-Longa suture method. After modeling, it took 4 hours to electroacupuncture at Baihui and Dazhui points, which was used to observe the changes of nerve function in rats with signs of keel nerve function defect. Protein expression was detected by immunohistochemistry. RESULTS: Compared with the model group, the EA 3d, 7d, 10d groups and the rehabilitation group had no significant difference in promoting the expression of Nestin (p>0.05). There was a significant difference (p<0.01). After cerebral ischemia-reperfusion injury, the expression of bFGF and EGF on the ischemic side was stronger. The peak of bFGF expression appeared earlier, and the peak of EGF expression appeared later. The expression of bFGF and EGF in cerebral ischemic cortex at different time points of ischemia in electroacupuncture group, rehabilitation group and Diankang group was increased, and the response was enhanced. The effect of Diankang group on the upregulation of bFGF and EGF was more significant (p<0.01, p<0.05). CONCLUSIONS: Under the influence of different effects, Diankang is superior to simple treatment in improving ischemic neurological dysfunction. This may be related to the fact that Diankang can promote the proliferation of neural stem cells and the expression of neurotrophic factors on the ischemic side of the rat brain.

De novo generation of permanent neovascularized soft tissue appendages by platelet-derived growth factor.

Treatment of wounds with pharmacologic doses of the BB homodimeric form of recombinant PDGF (rPDGF-BB) induces the recruitment, activation, and proliferation of mesenchymal cells, resulting in the deposition of provisional, and subsequently collagen-containing extracellular matrix. In preliminary experiments with an in vitro growth chamber model in the rat consisting of a silicone shell containing a dissected femoral vascular bundle, we found that rPDGF-BB incorporated into a rapidly dissolving collagen type I film induces the generation of a marked, but transient amount of de novo tissue around the femoral vascular bundle. In the present studies, the new tissue generated around the femoral vascular bundle was wrapped with a full thickness syngeneic skin graft to determine if functional support of the graft would lead to sustained maintenance of the underlying generated tissue and create an epithelialized soft tissue appendage. The tissue generated after a single application of rPDGF-BB was skin grafted on the 10th day, exteriorized 20 d later, and observed for an additional month. This led to the formation of soft tissue appendages which demonstrated marked neovascularization, fibroblast migration and proliferation, and increased glycosaminoglycan, fibronectin, and collagen fibril deposition, now leading to preservation of the newly generated tissue. In contrast, minimal new tissue was generated in control-treated vascular bundles or bundles treated with inactive PDGF-BB, and grafting with skin failed to sustain the underlying tissue. Thus, rPDGF-BB coupled with skin grafting induced the formation of functional large soft tissue appendages which are potentially useful clinically to fill tissue defects or to serve as a cell delivery system for transfected genes.

The effect of energy restriction on fatty acid profiles of longissimus dorsi and tissue adipose depots in sheep.

Sheep production systems in northwest China depend mostly on natural grasslands. Seasonal growth and maturity fluctuations can cause periodical restrictions in food quality and quantity. These fluctuations, in turn, result in variability in fat deposition and fatty acid profiles in different fat depots. Consequently, the study objective was to compare fat deposition, intramuscular fat (IMF) percentage and fatty acid profiles of the longissimus dorsi (LD), kidney fat (KF), tail fat (TF), and subcutaneous fat (SF) in lambs under ME restrictions similar to seasonal changes observed in the natural grasslands of northwest China. Nineteen male Dorper × Small Tailed Han lambs were assigned to 2 treatments, a control (CON) fed at 1.0 MJ / W × d and restricted (RES) by restricting ME sequentially every 30 d (0.56 MJ / W × d, 0.84 / W × d, 1.0 MJ / W × d, 0.84 MJ / W × d, 0.56 MJ / W × d, 0.28 MJ / W × d). All lambs were harvested at the end of the 180 d experimental period. Compared to CON fed lambs, restricting ME resulted in lesser IMF, fat deposition indexes ( < 0.05) except testicular and heart fat and greater ( < 0.05) SFA in LD, KF, and TF depots. The RES fed lambs had greater ( < 0.05) -3 PUFA, eicosatrienoic acid (C20:3n3), eicosapentaenoic acid (C20:5n3, EPA), and trans-linolelaidic acid (C18:2n6t) in LD muscle. The conjugated linoleic acids (CLA) content was greater in the SF depots of the CON fed lambs compared to the RES fed lambs. Fatty acid ratios (unsaturated fatty acid; USFA:SFA, MUFA:SFA, PUFA:SFA), and percentage USFA in RES fed lambs were lesser in muscle and adipose tissue compared to CON fed lambs ( < 0.05), except SF depots. In RES fed lambs, EFA were less ( < 0.05) in LD and KF depots and the ratios of functional fatty acids were lesser in LD and some adipose tissues ( < 0.05), including lesser n-6:n-3 in KF and SF ( < 0.05) depots, lesser USFA, SFA, MUFA, SFA in LD, KF, and TF ( < 0.05) depots, and lesser PUFA and SFA in LD and TF ( < 0.05) depots. Results from this research demonstrate that sequential energy restriction, as might be experience during seasonal forage quality and quantity changes in natural grasslands, result in lesser intramuscular fat with associated lesser quality, as well as, changes in fatty acid composition in different fat depots, which has implications for both meat quality and animal physiological functions.

Tissue generation with growth factors.

BACKGROUND: An in vivo experimental model was introduced to determine whether the mitogenic effect of recombinant platelet-derived growth factor (rPDGF) could be used to generate potentially useful tissue. METHODS: In Lewis rats, the extended femoral arteriovenous bundle was placed within silicone chambers containing collagen disks. The disks could deliver their content of rPDGF-BB (125 to 131 micrograms/disk) either as a rapid pulse or as a slow release. The time course of tissue generation was determined by harvesting the specimens at various postoperative days. The effect of continuous versus pulsed delivery was determined at 30 days. Analysis of the generated tissue was performed by use of histomorphometry. RESULTS: Pulsed delivery of rPDGF-BB induced the formation of a substantial amount of tissue that peaked at 10 to 15 days (145.9 +/- 13.8 vs 35.0 +/- 6.8 mm3, p < 0.0001); however, the generated tissue completely subsided by day 30. Sustained delivery of rPDGF-BB caused continuous growth of the tissue and was more effective than pulsed delivery. CONCLUSIONS: In an experimental model that approximates an in vivo tissue culture system, rPDGF-BB can induce a tenfold increase in tissue within the chamber. However, that tissue is labile and its survival necessitates continuous rPDGF-BB delivery. To become useful for reconstructive purposes, means to stabilize this new tissue growth are needed.

Progestin permeation through polymer membranes IV: Mechanism of steroid permeation and functional group contributions to diffusion through hydrogel films.

Hydrogel films were prepared from hydroxyethyl methacrylate, both with (Film II) and withouth (Film I) 5.25 mole% of ethylene glycol dimethacrylate. Permeation, diffusion, and partition coefficients for progesterone, testosterone, nandrolone, norethindrone, 17 alpha-hydroxyprogesterone, estradiol, and hydrocortisone were determined. A solute permeation model was proposed based on the separation of a domain (B) composed of "bulk-like" water and a doman (A) composed of polymer, interfacial water, and bound water present in the films. The separate contributions from the "pore" and "solution-diffusion" mechanisms to the total permeability were calculated from the model. Steroid permeabilities through Films I and II were analyzed in accordance with this model. Permeation of Film II occurred via the solution-diffusion mechanism. Permeation of Film I occurred predominately by the pore mechanism with a small contribution (approximate 20%) from the solution-diffusion mechanism. The latter contribution was dependent on the solubility of the solute within the A domains of the hydrogel film. Functional group contributions to permeation of Film II were ascribed to either steric or hydrogen bonding effects.

Progestin permeation through polymer membrane V: Progesterone release from monolithic hydrogel devices.

Progesterone release from monolithic devices prepared from various copolymers of poly(hydroxyethyl methacrylate) and poly(methoxyethoxyethyl methacrylate) or poly(methoxyethyl methacrylate) was examined. In general, plots of the fraction of drug released versus (time)1/2 were linear during the early stages of drug release. This behavior is similar to that found for drugs released from hydrophobic polymers such as polydimethyl siloxane. However, for some release curves using the hydrogels, a breakpoint appeared during the early stages of drug release. These breakpoints were due to the effects of water absorption by these polymers. From analyses of permeability coefficients, it was demonstrated that release rates also were dependent on the initial drug load and the equilibrium water content of the polymer. These conclusions were verified from cross-membrane diffusion studies on films depleted of their initial drug load. In conjunction with this work, the aqueous solubility of progesterone was determined by several methods; an average value of 38 micrograms/ml at 23 degrees was obtained.

[High-dose methotrexate with citrovorum factor rescue (HD-MTX-CFR) in the treatment of malignant solid tumors--clinical analysis of 62 patients].

From 1977 to 1982, 62 patients with various advanced malignant solid tumors were treated by HD-MTX-CFR therapy and totally 129 courses were given. Majority of the patients suffered from malignant lymphoma (10), osteogenic sarcoma (11), lung cancer (16), esophageal cancer (3), breast cancer (3) and malignant melanoma (4). All were confirmed by cytology or pathology except one primary liver cancer. There were clinically measurable lesions in 59 patients for evaluation of the treatment, and 3 osteogenic sarcoma patients without metastasis were given a postoperative adjuvant chemotherapy. 33 out of 62 had received chemotherapy and/or radiotherapy before. Dose of MTX ranged from 2 to 3 gm per course in most patients and dose of CF, from 9 to 12 mg every 6 hours for 3 days. 2 (3.4%) patients achieved complete remission (1 osteogenic sarcoma and 1 malignant lymphoma) and 8 (13.6%), partial remission (1 osteogenic sarcoma, 5 malignant lymphoma, 1 esophageal cancer and 1 breast cancer) with a total response rate of 15.9%. No response was observed in all 16 lung cancers. The main side effects of HD-MTX-CFR therapy were leukopenia, thrombocytopenia, elevation of SGPT, nausea, vomiting, mucositis, skin rash, fever and fatigue. All patients were followed more than 3 years. 4 patients are still alive (9, 9, 4 and 7 years, respectively), including 3 osteogenic sarcoma patients who received postoperative adjuvant chemotherapy and 1 mycosis fungoides.

[COMVp regimen in the treatment of small cell lung cancer--report of 106 patients].

One hundred and six consecutive small cell lung cancer patients were treated by a combination regimen COMVp (CTX 1,000-1,400 mg IV D 1,8; VCR 1-2 mg IV D 1,8; MTX 20-40 mg IV or IM D 3,5,10,12; VP-16 100 mg IV drip D 3-7; three weeks as a cycle and 2-3 cycles as a course) in our hospital during 1983 to 1984. Among the 95 patients who were evaluated, 10 (10.5%) gave CR, 57 (60%) PR, 19 (20%) no change and 9 (9.5%) progression. The overall response rate was 70.5%. In this paper, factors influencing the response and side effects are also analysed. The data show that COMVp regimen is one of the good front line combination chemotherapy regimens currently available in the management of small cell lung cancer.

[Flow cytometry in determination of DNA content of lymphoid cells: study of methodology and its preliminary clinical application].

The methodology of flow cytometry (FCM) in the determination of DNA content of lymphoid cells was studied. We conclude that the optimal conditions for the Ortho Instruments L-30 Cytofluorograf flow cytometer are as follows: 70% ethanol should be used as fixative and propidium iodide as DNA fluorescent stain at a final concentration of 50 micrograms/ml. RNase and Triton-X-100 added to the mixture and the use of fresh chicken red blood cells (CRBC) as internal standard will ensure the reliability of the data. The interference of doublet cells is eliminated by selecting single cell area signals. We discovered that aneuploidy detection can be optimized by using the coefficient of variation ratio (CVR) together with the DNA ratio (DR). A special computer program was written to calculate the percentage of cells in different phases of the cell cycle, according to DNA histograms derived from FCM. Based on assessment of the DNA content of lymphoid cells of 33 normal subjects, the normal ranges of DR, CVR and S% were found to be 2.57-2.83, 1.02 and less than 9.6% respectively. The diagnosis of two cases of lymphoid cell malignancy is reported and the potential of this method for wide clinical application discussed.

[Clinical study on therapeutic effects of treatment according to syndrome differentiation of traditional Chinese medicine combined with captopril on severe viral myocarditis complicated heart failure].

OBJECTIVE: To observe the therapeutic effect and mechanism of the treatment according to Syndrome Differentiation of TCM combined with captopril (CAP) on severe viral myocarditis (SVM) complicated heart failure (CHF). METHODS: One hundred and nine patients of SVM complicated CHF were randomly divided into the treated group (n = 72) and the control group (n = 37), the former was treated with TCM combined with CAP, while the latter was treated with dexamethasone and interferon. The TCM prescriptions were made depending on types of diseases by Syndrome Differentiation, i.e. Heart-Spleen deficiency type, Qi-Yin deficiency type and Spleen-Kidney Yang deficiency type. The efficacy of treatment was evaluated by the criteria including NYHA classification, myocardial enzymology, electrocardiogram, cardiac function and motorial toleration measured before and after treatment. RESULTS: The therapeutic effect of the treated group according to NYHA classification was obviously better than that of the control group; the creatine phosphokinase isoenzyme (CPK-MB), aspartate transaminase (AST), lactate dehydrogenase (LDH) content lowered in both groups, but more significantly lowered in the treated group than in the control group (P < 0.05, P < 0.01); the improvement of S-T segment of ECG in the treated group was better than that in the control (P < 0.01); also some parameters of heart function and motorial toleration were bettered in the treated group more significantly (P < 0.01). CONCLUSION: TCM treatment according to Syndrome Differentiation combined with CAP in treating SVM complicated CHF could elevate the clinical efficacy.