Study on detection of mouse influenza serum by surface enhanced raman spectroscopy / 实用医学杂志

Objective A new method for serum detection based on surface-enhanced Raman spectroscopy was explored by comparing the Raman spectra between normal mice and influenza virus-infected mice. Methods The nano-silver sol was used as the active substrate.The Raman spectra of the normal group,the model group and the Tamiflu control group were detected by portable Raman spectroscopy,and the partial least squares discrimina-tion analysis(OPLS-DA)was performed by the orthogonal correction. The number of RNA replicas of influenza virus in lung tissue was measured by RT-PCR as a control method. Results At the 3rd or 5th days,the serum of the normal group,the model group and the Tamiflu control group showed a significant trend. By the ROC curve evaluation,the predictive ability of 3 groups of serum SERS models established by OPLS-DA was high,which could distinguish and differentiate 3 groups of serum. Conclusions The results of SERS and RT-PCR detections were consistent.The preliminary results show that SERS pattern can help to identify and diagnose whether the body is infected with influenza virus.

Development of a DNA-based microarray for detection of nine pathogens causing rash and fever illness / 军事医学

Objective To develop a chemiluminescence imaging DNA microarray method for simultaneous,fast and accurate detection of nine rash-and fever-causing pathogens,namely,Measles virus,Rubella virus,Enterovirus type 71, Varicella zoster virus,Dengue fever virus,Human small FDNA virus B19,Coxsackie virus type A16,A-βStreptococcus pyogenes (hemolytic streptococcus)and Salmonella typhi.Methods Primers and probes were designed based on the specific sequence in the conserved region of genomes of the nine pathogens.The nucleic acids of the nine pathogens were amplified and labelled by multiplex PCR method.The multiplex PCR amplification products were hybridized with specific probes of microarray that was scanned after washing and chemiluminescence coloration to identify the nine pathogens.After the optimization of the multiplex PCR system,hybridization and chemiluminescence imaging,the specificity,sensitivity and reproducibility of the chip were evaluated.The serial diluted nucleic acid of Enterovirus type 71 was detected using microarray and real-time PCR approach to compare the sensitivity of these two methods.Results Nine specific primers and eleven specific probes were selected.The microarray demonstrated high sensitivity and specificity.The minimum detection limit of plasmid DNA and in vitro transcribed RNAs was 3 ×103 copies per reaction.The detection sensitivity of this microarray was 10 percent of that by the real-time PCR method.The rate of sensitivity and specificity of clinical sample detection was 95% and 85.7% respectively,and the rate of accuracy was 93.2%.Conclusion A chemiluminescence imaging DNA microarray method for simultaneous,fast and accurate detection of nine pathogens that cause rash and fever illnesses is established successfully,which can serve as a new high throuthput screening method for clinical diagnosis and epidemiological investigation of rash and fever illnesses.

Protective effect of chalcone ketones compound L2H17 on mice infected with influenza virus / 军事医学

Objective To investigate the protective and therapeutic effect of chalcone ketones compound(code:L2H17,hereinafter referred to as L2)on mice infected with influenza A virus.Methods BALB/c mice were randomly divided into six groups:normal group,model group,positive drug-treated group,L2 treated groups (3 different concentrations).The mice were adapted for 72 hours,before a model was established by intranasal infection.Mice in each group were given medicine by i.g once daily for 6 days starting 24 h before virus challenge.Survival was observed daily for 14 days.The mortality,median survival time,rate of death protection and rate of prolonging life were determined to observe the therapeutic effect of chalcone(L2) against influenza virus infection.The whole lungs were taken under aseptic conditions on days 3 and 5 post-infection to calculat lung indexes and lung index inhibition.The left lung was fixed with 4% formaldehyde for pathological biopsy,the right lung was soaked in RNAstore to detect lung tissue viral load,and the double antibody sandwich ELISA method was used to detect the expression of inflammatory cytokines IL-6 and TNF-α in order to observe the therapeutic effect of L2 on viral pneumonia caused by influenza virus infection.Results Compared with the model group,the L2 80 mg/kg treatment group exhibited significant increases in median survival time(11 d),the rate of death protection (50%),and the rate of prolonging life(24.1%)but a moderate 50% decrease in mortality.In addition,the lung index decreased significantly both on d 3 and 5 after virus infection (P<0.05).The pathological results also improved significantly.The L2 80 mg/kg dose group had a significantly lower viral load of lung tissue on d 3 and 5 post-infection(P<0.05).Compared with model group,the expression of inflammatory factor IL-6 became lower to different degrees.Conclusion L2 has a protective effect on mice infected with influenza virus by reducing the degree of pathological changes of pneumonia caused by influenza viruses.

Evaluation of the effect of procyanidin on drug permeability on different intestinal mucosa of rats with Ussing Chamber in vitro / 国际药学研究杂志

Objective To investigate the effect of procyanidin on permeability of rodamin 123(R123)and fluorescein sodium (FS)via different intestinal mucosa in rat. Methods The cumulative permeability and the apparent permeability cofficient(Papp)of R123 and FS via rat intestinal membranes at the procyanidin concentration of 20 mg/L was evaluated by the method of Ussing Cham?ber. The concentrations of R123 and FS in the receptor samples were determined by fluorospectrophotometry. Results The absorptive directed permeability of R123 across all membranes was increased by co-administration of procyanidins,whereas that of the secretive direct was decreased. Compared with control group,the secretive directed permeability of R123 was significantly decreased in colon (P<0.01). Conclusion Procyanidin could inhibit the secretion of R123 on different intestinal mucosa which might be related to the inhibition of P-gp function.

Dvelopment of a DNA-based microarray for detection of human adenovirus serotypes / 军事医学

Objective To develop a chemiluminescence imaging DNA microarray method for simultaneous,quick and accurate detection of serotypes of human adenovirus (HAdV ),namely,HAdV3,HAdV7,HAdV11,HAdV14 and HAdV55.Methods Based on the specific gene sequences in the conserved region of adenovirus from GenBank, oligonucleotide primers and probes were designed and synthesized to prepare the oligonucleotide microarray.The specific genomic sequences were amplified by multiplex PCR method.The multiplex PCR amplification products were hybridized with the specific probes of microarray that was scanned after washing and chemiluminescenceb before the result was analyzed.After optimization of the multiple PCR system,hybridization reactions and conditions of chemiluminescence,the specificity,sensitivity and reproducibility of the chip were evaluated.Results The microarray displayed high sensitivity, specificity and reproducibility.The minimum detection limit of plasmidg DNA was 3 ×103 copies/reaction.The microarray detection results of 38 clinical samples were approximately consistent with those using the direct sequencing method(37 /38).Conclusion A chemiluminescence imaging DNA microarray method for quick,sensitive and specific detection of five serotypes of HAdV is established,which can provide a new means for detecting serotypes of HAdV.

Effect of doxycycline on the proliferation of human small cell lung cancer H446 cells in vitro and its mechanisms / 国际药学研究杂志

Objective To investigate the effect of doxycycline on the proliferation of human small cell lung cancer H446 cells in vitro and its possible mechanisms. Methods H446 cells were treated with different concentrations of doxycycline for 24,48,72 or 96 h. Cell proliferation was measured by CCK-8 assay,IC50 was caculated by Bliss,and cell apoptosis was examined by TUNEL assay. The protein and mRNA expression of Bcl-2 and Bax in H446 cells were detected by Western blot and real time-PCR,respectively. Re-sults Doxycycline inhibited the proliferation of H446 cells in a concentration-time dependent manner. The half inhibitory concentra-tions(IC50)of doxycycline on cell proliferation in H446 cells were approximately 24.10 mg/L for 24 h treatment group,10.98 mg/L for 48 h treatment group,8.20 mg/L for 72 h treatment group and 8.03 mg/L for 96 h treatment group,respectively. Furthermore,doxycy-cline treatment could also induce the apoptosis of H446 cells in a concentration-dependent manner. Moreover,doxycycline treatment dramatically down-regulated the expression of Bcl-2 and up-regulated the expression of Bax in H446 cells both at the protein and at the mRNA levels. The differences were statistically significant(P<0.05). Conclusion Doxycycline can inhibit the proliferation of H446 cells by inducing apoptosis via down-regulating the expression of Bcl-2 and up-regulating the expression of Bax. Doxycycline has great potential as a chemotherapeutic agent for human small cell lung cancer.

Comparison of urapidil and nicardipine in the treatment of chronic renal failure patients with hypertensive emergencies / 药学实践杂志

Objective To study the efficacies and adverse reactions of urapidil and nicardipine in the treatment of the chronic renal failure patients with hypertensive emergencies .Methods 59 chronic renal failure patients with hypertensive emer-gencies were randomly divided into nicardipine treatment group and urapidil treatment group .The patients in the nicardipine group were given a 1 mg nicardipine intravenous injection ,and 30-100 μg/min intravenous transfusion was given continuously . The patients in the urapidil group were given a 12 .5 mg urapidil intravenous injection ,and 150-500μg/min intravenous trans-fusion was given continuously .The dosage were changed according to the patients′blood pressure in both of the groups .The patients′blood pressure ,heart rate and adverse reactions were recorded .Results The patients′blood pressure in both of the groups were significantly lower after treatment (P<0 .05) .The SBP in nicardipine treatment group was significant lower than SBP in urapidil treatment group in the first hour after treatment (P<0 .05) .There was no significant difference in SBP be-tween the two groups 2 hours after treatment (P>0 .05) .There was no significant difference in DBP between the two groups after treatment (P>0 .05) .In the nicardipine group ,the heart rate rose after the treatment ,the difference was statistically significant (P<0 .05) .While in the urapidil group ,the heart rate went down after the treatment ,and the difference was statis-tically significant (P<0 .05) .There was no significant difference in adverse reactions between the two groups (P>0 .05) . Conclusion Both of nicardipine and urapidil were effective in the treatment of chronic renal failure patients with emergency hy-pertensive .Nicardipine was more effective in reducing the SBP in the first stage of treatment .

Serum fingerprint detection based on surface-enhanced Raman scattering / 军事医学

Objective To optimize the experiment conditions of surface-enhanced Raman spectroscopy detection of serum fingerprint spectra.Methods Normal human serum was used as the sample and Ag nanoparticles as the active substrate.The enhanced signals of different optimized experiments were obtained , including serum dose(2.5 to 500 μl), incubation time(10 to 30 minutes) temperature(4℃,room temperature and 37℃),and different treatment(extraction and protein removal).Results and Conclusion Serum doses should not exceed 50μl.The ratio should range from 1∶1 to 5∶1, the incubation time is from 10 to 30 minutes, and the incubation temperature from 4℃ to 37℃.The signals of samples directly mixed with an active substrate are stronger than those of samples which are extracted or protein removed .

Alkanethiol modified magnetic silver flower nanoparticles applied to detection of chloramphenicol / 军事医学

Objective To prepare novel alkanethiol modified magnetic silver flower nanoparticles as SERS substrate to chloramphenicol for Raman detection and to determine their enhancement effect.Methods An alkanethiol was chosen as a surface modifier of the substrate and was self-assembled onto the magnetic silver flower nanoparticle surface.The chloram-phenicol molecules were enriched to the surface of the substrate by hydrophobic interaction and the effect for detection of chloramphenicol SERS signal was enhanced.Results It was found that the 1-hexanethiol-modified SERS substrate was able to lead to stronger enhancement than 1-dodecanethiol and octadecanethiol.Fe3 O4@SiO2-Ag-C6 was used to detect the chloramphenicol (10 -3 -10 -10 mol/L) and chloramphenicol in milk (10 -3 -10 -9 mol/L) by surface-enhanced Raman spectroscopy.The detection limits were 0.1 nmol/L (32 ppt) and 1 nmol/L (323 ppt) respectively.Conclusion Alkanethiol modified magnetic silver flower nanoparticles are a highly active SERS substrate, which can be used for detection of low concentrations of analytical substances.

Progress in research of immunoassay based on SERS labeling technique / 军事医学

A new medical research technology that combines surface enhanced Raman spectroscopy (SERS)with labeling immune technique is emerging with the development of SERS.This paper is intended to describe the principles, research progress and existing problems relating to SERS labeling immunoassay technology.We also summarize the research techniques for improving the sensitivity of SERS labeling immunoassay and the methods to eliminate nonspecific adsorption in SERS labeling immunoassay.Furthermore,the future development of SERS labeling immunoassay technology is discussed.

Application of surface-enhanced raman spectroscopy in human serum / 军事医学

Recently surface-enhanced Raman spectroscopy (SERS) has been widely used in physics, chemistry and bio-medical science.Due to its high sensitivity and specificity,SERS is often used to detect changes in serum components in humans.Various biomolecules in human serum, such as proteins, lipids and nucleic acids, have their own distinctive raman spectroscopy so that different raman shift, band intensity and width reflect different metabolic abnormalities of cells at the molecular level in human serum.In this paper we described the general situation of SERS and summarized the latest research progress in a variety of diseases of human serum.Prospects of developmenls are also outlined.

Pharmacokinetics of capsaicin via different intestinal segments of rats / 国际药学研究杂志

Objective To validate the correlation between in vitro and in vivo absorption of capsaicin, and study the distribution of capsaicin in tissue after oral administration. Methods In situ closed loop method was used to measure the absorption of capsaicin from different intestine segments of rats. Concentrations of capsaicin in rat plasma were examined by LC-MS/MS and pharmacokinetic parameters were calculated by the software WinNonlin. Results The AUC0-240min and Cmax of capsaicin absorbed from colon were higher than those of ileum and jejunum. However, there was no statistic difference. Conclusion In the primary study, we realized that permeability of capsaicin across the colonic mucosa is remarkably higher than that across jejunal or ileac mucosa in mucosal-to-serosal direction. However, there′s no statistical difference for the absorption of capsaicin across different intestinal regions by in situ assay. These results suggest the correlation between in vitro and in situ method is worth further study.

Preparation of a new surface-enhanced Raman scattering substrate and its applica-tion to the detection of E.coli / 军事医学

Objective To develop a new type of Raman-enhanced substrate for rapid detection of E.coli based on label-free surface-enhanced Raman scattering( SERS) technology.Methods Stober’ s improved method was used to prepare 360 nm silica ( SiO2 ) nanospheres.Prepared gold core-silver shell nanoparticles( Au@Ag) of different size were attached to 360 nm SiO2 to fabricate the nanocomposites ( SiO2-Au@Ag ) that were characterized by transmission electron microscopy (TEM) and UV-visucl light adsorption spectra (UV-Vis).PATP was detected to select SiO2-Au@Ag with optimal SERS effect.This optimal SiO2-Au@Ag was used to obtain the sensitivity of PATP and E.coli detection after a simple mixed culti-vation.Results TEM images showed that Au@Ag aggregated with the size of Au@Ag attached to 360 nm SiO2 .UV-Vis spectra indicated that the maximum absorption of Au@Ag and SiO2-Au@Ag had a red shift with the invrease of Au@Ag size.The experiment results suggested that detection sensitivity of PATP by SiO2-100 nm Au@Ag 10 -10 mol/L, while the lowest detectable E.coli concentration was 105 CFU/ml.Conclusion The 360 nm SiO2 binding with 100nm Au@Ag exhibits great potential for SERS applications.

DNA microarray for simultaneous screening and detection of seven rickettsia / 军事医学

Objective To develop a chemiluminescence ( CL ) imaging DNA microarray method for simultaneous detection of seven rickettsiae.Methods Primers and probes were designed based on the specific sequence of seven rickettsia genomes.The probes were immobilized on the aldehyde modified glass surface to prepare DNA microarray for rickettsiae.The nucleic acids of the selected rickettsiae were amplified and labelled by multiplex PCR method, and then hybridized with microarray that was scanned after washing and chemiluminescence coloration, before the results were analyzed.Facilitated by the optimization of the multiplex PCR system, hybridization, and chemiluminescence imagination, we evaluated the specificity,sensitivity and reproducibility of the chip.The serial diluted nucleic acid of Rickettsia mooseri was detected using microarray and real-time PCR approach to compare the sensitivity of these two methods.Double-blind simulated samples were prepared to further evaluate the accuracy of the detection methods.Results One universal primer, four specific primers, one universal probe, and nine specific probes were selected.This DNA microarray demonstrated high specificity and sensitivity.The detection sensitivity of plasmid DNA and double-blind simulated sample DNA was 1.5 ×102 -3 ×103 copies per reaction and 103 -104 copies/μl.The detection results of real-time PCR method was consistent with the microarray, and the microarray possessed 10 fold lower detection sensitivities than the real-time PCR method.The coincidence rate of double-blind simulated sample detection was 100%.Conclusion A chemiluminescence imaging DNA microarray method for simultaneous detection of seven rickettsiae is established successfully,which can serve as a new high throuthput detection method for clinical diagnosis and epidemiological investigation of rickettsiae.

Gene chip technique in detection of vancomycin-resistant Enterococcus gene / 军事医学

Objective To develop a detection method based on the technology of gene chips which can quickly distinguish genes of Enterococcus faecalis, E.faecium and vancomycin resistance.Methods Based on the specific gene ( ddl) sequences of two types of Enterococcus from GenBank, oligonucleotide probes which could detect and distinguish special genes and drug resistance genes ( vanA,vanB) of Enterococcus were designed and compounded.Then,the probes were dotted to modified slide.The target DNA fragments of vancomycin-resistant Enterococcus ( VRE) were labeled with biotin by multiple PCR amplification, and then hybridized with oligonucleotide probes on slide.The results were analyzed by portable imager.The multiple PCR system, hybridization reaction and condition of the chemiluminescence method were optimized before the specificity, sensitivity and reproducibility of the chip were evaluated.Results One universal primer, four specific primers, one universal probe and four specific probes were selected.This gene chip was demonstrated of high specificity and repeatability.The detection sensitivity was 103 CFU/ml.The gene chip detection results of 10 clinical samples were basically consistent with the drug sensitivity test ( 8/10 ) .Conclusion A gene chip technique for the detection of VRE is established successfully.It is possible to distinguish the type of VRE and detect the genetic phenotypes of drug resistance by gene chip technique.

Procyanidin inhibits multidruy resistance 1 mRNA expression via regulating microRNA-27a and microRNA-451 / 国际药学研究杂志

Objective To investigate the effect of expression of microRNA-27a(miR-27a) and microRNA-451(miR-451) in A2780/T cells and its relativity to multidrug resistance (MDR)1 mRNA inhibition by procyanidin. Methods Stem-loop PCR method was performed to evaluate the expression of miR-27a and miR-451 in use of procyanidin (0-40μmol/L) in 0-48 h in A2780/T cells. Additionally, over-expressing or interfecting microRNAs by using mimics or inhibitor of miR-27a and miR-451, the expression of MDR1 mRNA was assessed by RT-PCR in cells exposing to procyanidin. Results The expression of miR-27a and miR-451 was significant inhibited by procyanidin in both time- and concentration-dependency. Over-expressed MDR1 mRNA associated with miR-27a or miR-451 mimics was blocked by procyanidin, whereas there was no effect on down-expressed MDR1 mRNA associated with miR-27a or miR-451 inhibitor by procyanidin. Conclusion Procyanidin inhibits MDR1 mRNA expression by inhibiting miR-27a and miR-451 expression in A2780/T cells.

Effect of capsaicin on intestinal permeation of P-glycoprotein substrate rhodamine 123 and fluorescein sodium in rats / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the role of capsaicin in regulating permeation of P-gp substrate rhodamine 123 (R123) across the jejunum, ileum and colon membranes of rats.</p><p><b>METHODS</b>The permeability of R123 or fluorescein sodium (CF) across the jejunum, ileum and colon membranes of male SD rats was evaluated using a Ussing chamber. The concentration of R123 or CF in the receptor was determined using fluorospectrophotometry to calculate the apparent permeability coefficient (Papp).</p><p><b>RESULTS</b>Compared with the blank control group, capsaicin increased the permeability of R123 across jejunal membranes in the mucosal-to-serosal (M-S) direction and decreased its permeability in the serosal-to-mucosal (S-M) direction, but produced no obvious effect on R123 transport across the ileum or colon membranes. Capsaicin caused a regional increase in the permeability of CF across the jejunal membranes compared with the control group, but CF transport across the ileum and colon membranes was not affected.</p><p><b>CONCLUSION</b>Capsaicin can affect the transport of R123 and CF across rat jejunal membranes, and this effect is shows an obvious intestine segment-related difference probably because of the different distribution of P-gp or tight junction in the intestines. This finding suggests that capsaicin is a weak P-gp inhibitor and an improver of mucous membrane channels.</p>

Effect of Radix euphorbiae pekinensis extract on bioavailability of paclitaxel after their oral co-administration / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the effect of Radix euphorbiae pekinensis extract on the permeability and bioavailability of paclitaxel co-administered orally.</p><p><b>METHODS</b>Based on Ussing Chamber and in vivo experiment, the permeability and bioavailability of paclitaxel were evaluated after oral co-administration with radix euphorbiae pekinensis in rats. The contents of paclitaxel in the permeates and the blood samples were determined using HPLC and LC-MS/MS method, respectively.</p><p><b>RESULTS</b>In Radix euphorbiae pekinensis co-administration group, the Papp of the mucosal-to-serosal (M-S) transport or serosal-to-mucosal transport (S-M) of paclitaxel in the jejunum or ileum segment differed significantly from those in verapamil co-administration group and blank control group (P<0.05), but the Papp of S-M transport in the colon showed no significant difference from that in the blank control group. In the blank group, the average absolute bioavailability (AB%) of orally administered paclitaxel was only 2.81%, compared to that of 7.63% in radix euphorbiae pekinensis group. The average AB% in verapamil group was about 1.5 times that of the blank group.</p><p><b>CONCLUSION</b>Co-administration of Radix euphorbiae pekinensis extract can increase the bioavailability of orally administered paclitaxel.</p>

Preparation and evaluation of flutide-loaded PLGA nanoparticles modified with cell-penetrating peptides / 军事医学

Objective To prepare and evaluate flutide-loaded PLGA nanoparticles modified with cell-penetrating peptide-TAT.Methods The sequence of TAT was synthesized with florenl methyoxycarbonyl amino acids .The purity and molecular weight of TAT were determined using RP-HPLC and MALDI-TOF-MS.PLGA was modified with the TAT peptide and then prepared into flutide-loaded nanoparticles ( TAT-PLGA NPs) with the double emulsion method .The physical and chemical properties were evaluated , including size distribution, Zeta potential, SEM of nanoparticles , loading ratio of drug content and release profiles of TAT-PLGA NPs in vitro.The cytotoxicity of TAT-PLGA NPs was evaluated by CCK-8 methods.Results The purity of synthesized TAT was 95.6%, and molecular weight was 1495.8.The mean diameter,Zeta potential, drug loading ratio of TAT-PLGA nanoparticals were (159.5 ±2.1) nm, -(1.87 ±0.6) mV, and (5.75 ±0.17)μg/mg, respectively.The nanoparticles observed by transmission electron microscopy (TEM) had a spherical shape and uniform size without aggregation .In vitro release test showed sustained release of flutide from TAT-PLGA nanoparticles .Cell proliferation assay revealed that the TAT-PLGA nanoparticles did not damage the cell growth in vitro and showed good compatibility.Conclusion TAT-PLGA nanoparticles are prepared successfully by double emulsion method,and have sustained-release effect and good compatibility in vitro.They have potential application prospect in prevention and treatment of influenza .

Pharmacokinetics of cantide, an antisense oligonucleotide, and its metabolites in rhesus monkeys / 药学学报

To study the pharmacokinetics of cantide, an antisense oligonucleotide, and its metabolites after iv gtt administration in rhesus monkeys, a dual solid phase extraction pretreatment method coupling with non-gel sieving capillary electrophoresis analysis method was used for determination of cantide and its metabolites in plasma and their pharmacokinetic parameters were calculated. The pharmacokinetic behavior of cantide and its metabolites (M1 and M2) after iv gtt administration (8, 16 and 24 mg kg(-1)) in rhesus monkeys were investigated. After iv gtt administration of cantide to rhesus monkeys, cantide in plasma was eliminated rapidly and the terminal elimination half-life (t1/2) was 57.91-77.97 min, the correlation coefficients (r) to the dose of Cmax AUC(o-inf) and AUC(0-t) of the prototype was 0.9918, 0.9568 and 0.9773, respectively. The metabolites of cantide reached the Cmax following cantide immediately and the Cmax of metabolites were lower than that of the prototype. The CL(S) of cantide and its metabolites (M1 and M2) were 1.60-2.19, 5.92-8.58 and 6.07-8.78 mL min(-1) kg(-1), respectively. So, it is concluded that the Cmax of cantide and its metabolites increased with the dose, which is the same as their AUC(0-inf) and AUC(0-t). The CL(S) of metabolites were higher than that of the prototype. The MRT and t1/2 of metabolites in the high dose group increased obviously.