Designing a Multiplex PCR for Rapid and Accurate Detection of Metallobetalactamases Resistant Genes from Acinetobacter baumaniiIsolates in Tehran City, Iran.

Background & Objective: Acinetobacter baumannii strains harboring Meallobetalactamases (MBL) pose a significant threat in the context of nosocomial infections. The present investigation was undertaken with the objective of devising a Multiplex PCR methodology for the concurrent detection of MBL genes within A. baumannii strains prevalent in Tehran City, Iran. Methods: Between October 2020 and February 2021, 100 strains of A. baumannii were procured from burn specimens of hospitalized patients at Motahhari Hospital in Tehran. The identification of A. baumannii strains involved conventional biochemical techniques, coupled with confirmation of the presence of the bla OXA-51 gene. Antibiotic susceptibility was assessed using the Kirby-Bauer disc diffusion test. MBL-producing strains were characterized through a phenotypic approach employing the combined disk test, alongside Multiplex PCR for the simultaneous identification of bla VIM, bla IMP, bla GIM, and bla NDM genes. Statistical analyses were conducted using the chi-square test, with SPSS version 20.0 employed for data processing. Results: Among 100 strains examined, 96.1% exhibited positivity for MBL, as determined by the combined disk test. The study revealed a predominance of extensively drug-resistant (XDR) strains, with colistin demonstrating the highest level of sensitivity. The genotypic assay unveiled that Multiplex PCR identified bla VIM, bla NDM, and bla IMP in 20 strains, bla VIM and bla NDM in 30 strains, and exclusively the bla NDM gene in 45 strains. Notably, the Multiplex PCR technique exhibited the capacity to concurrently detect MBL genes (bla VIM, bla IMP, bla GIM, bla NDM) in 2 strains. Conclusion: The current investigation underscores prevalence of the bla NDM gene within clinical strains of A. baumannii. Furthermore, Multiplex PCR emerges as a robust and highly sensitive technique for rapid discernment of the MBL genes within in A. baumannii strains.

Studying the effect of gene fusion of A and C types capsular synthesizing enzymes and anticancer sequence on inducing the expression of apoptotic BCL-2, BAX, and Caspase-3 genes by Real-time RT-PCR method.

Background: Today, uterine cancer is one of the most important causes of death in the world and is one of the major problems in human health. There have been numerous reports of the effect of Streptococcus agalactiae peptide and capsular products against cancer cell lines. Objective: This study aimed to research recombinant peptide CPSA-CPSC-L-ACAN and investigate its apoptotic effect against the HeLa cell line by Real-Time-RT PCR. Design: In this study confirmation of the recombinant fusion peptide was performed by Western blotting. The effect of cytotoxicity of different concentrations of recombinant fusion peptide against the HeLa cell line was investigated by the MTT technique. The expression of apoptotic genes including BAX, BCL-2, and Caspase-3 in comparison with the GAPDH reference gene before and after exposure to recombinant fusion peptide was measured by Real-Time RT-PCR. Results: Recombinant fusion peptide at a concentration of 63 µg/ml destroyed 50% of the HeLa cell line in 24 h and cell treatment with this concentration increased gene expression of Caspase-3 genes by 16 times, bax by 6 times and decreased the expression of bcl-2 by 0.176 times. Conclusions: The results showed that treatment of the HeLa cell line with recombinant fusion peptide induced an apoptotic effect. The recombinant fusion peptide could probably help the medical community as a prophylactic or therapeutic treatment for cervical cancer.

Anti-biofilm and bystander effects of antimicrobial photo-sonodynamic therapy against polymicrobial periopathogenic biofilms formed on coated orthodontic mini-screws with zinc oxide nanoparticles.

BACKGROUND: The present study evaluated the anti-biofilm and bystander effects of antimicrobial photo-sonodynamic therapy (aPSDT) on the polymicrobial periopathogenic biofilms formed on mini-screws coated with zinc oxide nanoparticles (ZnONPs). MATERIALS AND METHODS: Thirty orthodontic identical mini-screws were divided into 6 groups (n = 5) as follows: 1. negative control: uncoated mini-screw + phosphate-buffered saline (PBS), 2. positive control: uncoated mini-screw + 0.2% CHX, 3. coating control: coated mini-screw + PBS, 4. antimicrobial photodynamic therapy (aPDT): coated mini-screw+light emitting diode (LED), 5. Antimicrobial sonodynamic therapy (aSDT): coated mini-screw+ultrasound waves, and 6. aPSDT: coated mini-screw+LED+ultrasound waves. Electrostatic spray-assisted vapor deposition was employed to coat ZnONPs on titanium mini-screws. The biofilm inhibition test was used to assess the anti-biofilm efficacy against polymicrobial periopathogenic biofilms including Porphyromonas gingivitis, Prevotella intermedia, and Aggregatibacter actinomycetemcomitans, and the results were shown as the percent reduction of Log10 colony-forming unit (CFU)/mL. Following each treatment, the gene expression levels of TNF-α, IL-1ß, and IL-6 were evaluated on human gingival fibroblast (HGF) cells via quantitative real-time polymerase chain reaction (qRT-PCR) to reveal the bystander effects of aPSDT on HGF cells. RESULTS: A significant reduction in log10 CFU/mL of periopathogens was observed in groups treated with aPDT, aSDT, aPSDT, and 0.2% CHX up to 6.81, 6.63, 5.02, and 4.83 log, respectively, when compared with control groups (P<0.05). 0.2% CHX and aPSDT groups demonstrated significantly higher capacity in eliminating the periopathogen biofilm compared with other groups (P<0.05). The qRT-PCR showed that the expression level of inflammatory cytokines was significantly down regulated in aPDT, aSDT, and aPSDT groups (P<0.05). CONCLUSION: It was found that the ZnONPs-mediated aPSDT could significantly reduce periopathogen biofilm as well as the expression level of inflammatory cytokines.

Designing a novel fusion protein from Streptococcus agalactiae with apoptosis induction effects on cervical cancer cells.

Cervical cancer remains life-threatening cancer in women around the world. Due to the limitations of conventional treatment approaches, there is an urgent need to develop novel and more efficient strategies against cervical cancer. Therefore, the researchers attend to the alternative anti-cancer compounds like bacterial products. Rib and α are known as surface proteins of Streptococcus agalactiae with immunologic effects. In the present study, we designed a new anti-cancer fusion protein (Rib-α) originating from S. agalactiae with in silico methods, and then, the recombinant gene was cloned in the pET-22 (+) expression vector. The recombinant protein was expressed in E. coli BL21. To purify the expressed protein, we applied the Ni-NTA column. The molecular mechanism by which Rib-α is cytotoxic to cancer cells has been discussed based on MTT, flow cytometry, and real-time PCR methods. The engineered fusion protein suppressed the proliferation of the cancer cells at 180 µg/ml. Cytotoxic assessment and morphological changes, augmentation of apoptotic-related genes, upregulation of caspase-3 mRNA, and flow cytometric analysis confirmed that apoptosis might be the principal mechanism of cell death. According to our findings, Rib-α fusion protein motivated the intrinsic apoptosis pathway. Therefore, it can be an exciting candidate to discover a new class of antineoplastic agents.

Streptococcal bacterial components in cancer therapy.

The incidence rate of cancer is steadily increasing all around the world, and there is an urgent need to develop novel and more effective treatment strategies. Recently, bacterial therapy has been investigated as a new approach to target cancer, and is becoming a serious option. Streptococcus strains are among the most common and well-studied virulent bacteria that cause a variety of human infections. Everyone has experienced a sore throat during their lifetime, or has been asymptomatically colonized by streptococci. The ability of Streptococcus bacteria to fight cancer was discovered more than 100 years ago, and over the years has undergone clinical trials, but the mechanism is not yet completely understood. Recently, several animal models and human clinical trials have been reported. Streptococcal strains can have an intrinsic anti-tumor activity, or can activate the host immune system to fight the tumor. Bacteria can selectively accumulate and proliferate in the hypoxic regions of solid tumors. Moreover, the bacteria can be genetically engineered to secrete toxins or enzymes that can specifically attack the tumors.

Bacteriocins: Recent Advances in its Application as an Antimicrobial Alternative.

Due to the emergence and development of antibiotic resistance in the treatment of bacterial infections, efforts to discover new antimicrobial agents have increased. One of these antimicrobial agents is a compound produced by a large number of bacteria called bacteriocin. Bacteriocins are small ribosomal polypeptides that can exert their antibacterial effects against bacteria close to their producer strain or even non-closely-relatedstrains. Adequate knowledge of the structure and functional mechanisms of bacteriocins and their spectrum of activity, as well as knowledge of the mechanisms of possible resistance to these compounds, will lead to further development of their use as an alternative to antibiotics. Furthermore, most bacteria that live in the gastrointestinal tract (GIT) have the ability to produce bacteriocins, which spread throughout the GIT. Despite antimicrobial studies in vitro, our knowledge of bacteriocins in the GIT and the migration of these bacteriocins from the epithelial barrier is low. Hence, in this study, we reviewed general information about bacteriocins, such as classification, mechanism of action and resistance, emphasizing their presence, stability, and spectrum of activity in the GIT.

The chemical composition and antibacterial activity of a methanolic extract of Satureja khuzistanica

Abstract In the present study, the metabolite profiling of methanolic extract from aerial parts of Satureja khuzistanica Jamzad, as an endemic medicinal plant from Iran, was evaluated using HPLC-PDA-ESI. Then, the main compound from the extract was isolated and purified by using extensive chromatographic techniques. In addition, the structure of the isolated compounds was elucidated using 1D, 2D NMR, and MS spectrometry, upon which 22 compounds were identified. The antibacterial activity of diosmetin 7-rutinoside (6) and linarin (13) in combination with carvacrol as a major compound of the essential oil was tested against Pseudomonas aeruginosa and Staphylococcus aureus through disc diffusion and minimum inhibitory concentration methods. The results indicated that the linarin, when mixed with carvacrol as the main compounds in the essential oil of the plant, has a satisfactory activity against both Pseudomonas aeruginosa and Staphylococcus aureus with MIC values of 0.16 and 0.18 µg/mL, respectively. Further, the fractional inhibitory concentration (FIC) index indicated that this compound had synergism with carvacrol.

Evaluation of cold atmospheric-pressure plasma against burn wound infections and gene silencing.

BACKGROUND AND OBJECTIVES: Non-thermal atmospheric-pressure plasma or cold plasma is defined as an ionized gas. This study aimed to investigate the effect of cold plasma on Pseudomonas aeruginosa strains. Also, the expression level of the alp virulence gene before and after treatment with cold plasma was compared with the Housekeeping gene gyrA. MATERIALS AND METHODS: P. aeruginosa isolates recovered from hospitalized burn patients at Shahid Motahari Burns Hospital, Tehran, Iran. The Kirby Bauer disk diffusion method was used to determine the antimicrobial susceptibility test. Then, the antibacterial effect of atmospheric non-thermal plasma was evaluated on P. aeruginosa in as in vitro and in vivo studies at different times on Muller Hinton agar and in mouse model (treated by plasma every day/ 90 sec). The histopathological study was evaluated by Hematoxylin-Eosin staining. Data were analyzed using SPSS software by the Chi-square test and Pvalues less than 0.05 considered as statistically significant. RESULTS: Results indicated that non-thermal atmospheric plasma inhibited the growth of P. aeruginosa. The non-thermal helium plasma accelerates wound healing for 6 days. Results showed that cold plasma decreased virulence gene expression alp after treatment. Therefore, cold plasma can be suggested as a complementary therapeutic protocol to reduce bacterial infection and accelerate wound healing and reduce the expression of virulence genes of pathogens. CONCLUSION: Cold plasma showed pathogen inhibitory properties of P. aeruginosa and virulence alkaline protease and wound healing properties in animal models, so this inexpensive and suitable method can be presented to the medical community to disinfect burn wounds and improve wound healing.

Anticancer Effect of Enterocin A-Colicin E1 Fusion Peptide on the Gastric Cancer Cell.

Cancer is one of the most causes of death all over the world, although improvements in its treatment and recognition. Due to the limitations of common anticancer methods, including surgery, chemotherapy, and radiotherapy, attention has been drawn to other anti-cancer compounds, especially natural peptides such as bacteriocins. In this study, we used a combination of two bacteriocins, colicin E1 and enterocin A, against AGS gastric cancer cell lines. In order to evaluate anticancer properties of fusion peptide, we applied MTT assay, real-time PCR, and flow cytometry tests. This is the first report to show the cell growth inhibitory activity of the enterocin A in combination with colicin E1 against AGS human cancer cells. The results of this study showed that this fusion peptide at a concentration of 60.4 µg/mL and 24 h was able to kill half of the tested cancer cells, and treatment of the cells with this concentration increased the expression of bax and caspase 3 genes and reduced the expression of bacl-2 in 24 h. Flow cytometry analysis of annexin V-FITC/propidium iodide results also showed that our peptide was able to induce apoptosis in treated cells compared with control. Taken together, enterocin A-colicin E1 (ent A-col E1) can be considered as a good candidate for anticancer therapies.

Bacteriocins: New Potential Therapeutic Candidates in Cancer Therapy.

Cancer is one of the most important disorders which is associated with high mortality and high costs of treatment for patients. Despite several efforts, finding, designing and developing, new therapeutic platforms in the treatment of cancer patients are still required. Utilization of microorganisms, particularly bacteria has emerged as new therapeutic approaches in the treatment of various cancers. Increasing data indicated that bacteria could be used in the production of a wide range of anti-cancer agents, including bacteriocins, antibiotics, peptides, enzymes, and toxins. Among these anti-cancer agents, bacteriocins have attractive properties, which make them powerful anti-cancer drugs. Multiple lines evidence indicated that several bacteriocins (i.e., colcins, nisins, pediocins, pyocins, and bovocins) via activation/inhibition different cellular and molecular signaling pathways are able to suppress tumor growth in various stages. Hence, identification and using various bacteriocins could lead to improve and introduce them to clinical practices. Here, we summarized various bacteriocins which could be employed as anti-cancer agents in the treatment of many cancers.

Reduced Efflux Pumps Expression of Pseudomonas Aeruginosa with Satureja Khuzistanica Essential Oil.

BACKGROUND: Efflux pumps such as MexEF-OprN and mexXY-OprM play an important role in the resistance of Pseudomonas Aeruginosa (P. aeruginosa) to antibiotics. The present study aimed to assess the reduced expression of efflux pump genes of P. aeruginosa with Satureja Khuzistanica essential oil (SKEO). METHODS: The present cross-sectional study was conducted in 2016 at the Microbiology Laboratory of Baqiyatallah University of Medical Sciences, Tehran, Iran. The disk diffusion method was used for susceptibility testing of gentamicin and norfloxacin. Minimum inhibitory concentration (MIC) was determined for gentamicin and norfloxacin. The antibacterial efficacy of SKEO was defined by determining the MIC values using the microdilution method. In vitro, the synergistic interaction of SKEO combined with gentamicin or norfloxacin was examined via checkerboard assay and defined as a fractional inhibitory concentration index. The reverse transcription-polymerase chain reaction technique was used to measure changes in the expression of the efflux pump genes. The data were analyzed using SPSS software version 16.0, and P<0.05 was considered statistically significant. RESULTS: The MIC values of SKEO were in the range of 6 to 12 µg/mL. In the presence of sub-inhibitory concentrations (1.16 to 2 MIC) of SKEO, synergistic effects were revealed using the checkerboard method. The effect of norfloxacin and gentamicin increased up to 8-fold. The expression of mexY and mexE was reduced after treatment with SKEO. CONCLUSION: SKEO reduced the expression of efflux pumps and the MIC values of norfloxacin and gentamicin in vitro.

Evaluation of antibacterial activity of enterocin A-colicin E1 fusion peptide.

OBJECTIVES: Bacterial resistance to most common antibiotics is a harbinger of the requirement to find novel anti-infective, antimicrobials agents, and increase innovative strategies to struggle them. Numerous bacteria produce small peptides with antimicrobial activities called bacteriocin. This study aimed to investigate the antibacterial properties of the fusion protein of Enterocin A and Colicin E1 modified against pathogens. MATERIALS AND METHODS: Analysis of recombinant bacteriocin Enterocin A and Colicin E1 (ent A-col E1) was performed to assay the stability and antibacterial activity of this fusion protein. The pET-22b vector was employed to express the coding sequence of the ent A-col E1 peptide in Escherichia coli BL21 (DE3). Minimum inhibitory concentration (MIC), disk diffusion, and time-kill tests were performed to evaluate the antibacterial activity of the ent A-col E1 against Pseudomonas aeruginosa (ATCC 9027), Escherichia coli (ATCC 10536), Enterococcus faecalis (ATCC 29212), and Staphylococcus aureus (ATCC 33591). RESULTS: The suggested recombinant peptide had good antibacterial activity against both Gram-negative and Gram-positive pathogens. It has also good stability at various temperatures, pH levels, and salt concentrations. CONCLUSION: Because bacteriocins are harmless compounds, they can be recommended as therapeutic or preventive supplements to control pathogens. According to the obtained results, the ent A-col E1 peptide can serve as an efficient antibacterial compound to treat or prevent bacterial infections.

Helicobacter pylori Recombinant CagA Regulates Th1/Th2 Balance in a BALB/c Murine Model.

Purpose: Helicobacter pylori is recognized as one of the prevalent causes of human gastricinfection. In the present study, the role of mixed immunization with H. pylori lipopolysaccharide(LPS) and recombinant cytotoxin-associated gene A (rCagA) as a stimulator of host immuneresponses was determined. Methods: BALB/c mice were immunized with different formulations by the systemic administrationat 14-day intervals. The effects of the formulations plus CpG adjuvants were assessed before andpost-immunization in separated studies. Moreover, the expression of Th1/Th2 cytokines wasquantified in sera of immunized mice using reverse transcription polymerase chain reaction (RTPCR)test and the protein levels confirmed with enzyme linked immunosorbent assay (ELISA).Finally, the specific antibody levels in sera were studied by ELISA and the tendency of cellularresponse was examined by IgG1/IgG2a ratio. Results: Data of Western blotting verified the presence of constructed protein. Analysisof lymphocyte proliferation showed that CpG-conjugated rCagA increases lymphocytesproliferation compared to the control group. Also, it was shown that formulations containing LPSand rCagA promote a Th1 response indicated by interferon-gamma expression and induced Th1/Th2 balance. Additionally, the specific IgG1, total IgG and IgG2a levels elevated in response toall treatments. Ultimately, the IgG2a/IgG1 ratio in the mice immunized with rCagA-containingformulations increased. Conclusion: These results indicated that rCagA protein carried with CpG adjuvant not onlymaintained its antigenicity throughout the experiment but also induced robust Th1-biasedimmune responses. Therefore, it holds promise for the production of an efficient vaccine against H. pylori infection.

Effect of Satureja khuzistanica essential oil (SKEO) extract on expression of lasA and lasB genes in Pseudomonas aeruginosa.

BACKGROUND AND OBJECTIVES: Expressions of lasA and lasB genes of Pseudomonas aeruginosa are associated with bacterium pathogenicity. The present study was aimed to assess the effect of Satureja khuzistanica essential oil (SKEO) extract on expression of lasA and lasB genes in P. aeruginosa. MATERIALS AND METHODS: Pseudomonas aeruginosa isolates were cultured in Mueller Hinton broth containing sub-inhibitory concentrations of SKEO and total RNA extracted using Trizol method. cDNA was synthesized using random Hexamer primer and finally the expression of lasA and lasB genes carried out by real-time PCR. RESULTS: The MICs of SKEO extract for PA9, PA10, PA11, PA13, PA41 and PA42 isolates were 8, 8, 8, 9, 7 and 12 µg/ml, respectively. Statistical analysis for 6 isolates revealed that the reduction in expression of lasA and lasB genes under SKEO treatment was significant (P<0.05). CONCLUSION: The insignificantly increasing of lasB gene expression may lead to low virulent strains, for probably reason that the strain's exotoxin A are destroyed in the high amount of protease. In conclusion, using of SKEO in burned patients infected with P. aeruginosa may be effective; however, it is better to assess the spectrum activity of SKEO, pharmacokinetics, potency and its toxicity in human cells.

Protection of BALB/c mice against pathogenic Brucella abortus and Brucella melitensis by vaccination with recombinant Omp16.

OBJECTIVES: Prevention of the globally spread zoonotic infection, brucellosis which affects an extensive range of hosts is still challenging researchers. There are no approved vaccines for the prevention of human disease and those used for animal brucellosis have adverse properties, which limit their application. We investigated the immunological and protective effects of recombinant 16 kDa outer membrane protein of Brucella abortus (Omp16) which introduced a new candidate for brucellosis subunit vaccine. MATERIALS AND METHODS: Brucella Omp16 gene was cloned in pET-23a and expressed in Escherichia coli BL21 (DE3). Recombinant Omp16 (rOmp16) was purified using nickel resin and confirmed by Western blot analysis. BALB/c mice were immunized with rOmp16, afterward, specific serum antibodies and cytokine responses were evaluated. Protection of immunized mice against pathogenic B. abortus 544 and B. melitensis 16M was evaluated by the intraperitoneal bacterial challenge. RESULTS: Sequencing results of the recombinant plasmid vector along with Western blotting confirmed the cloning procedure. Recognition of rOmp16 by specific IgG from serum samples of infected cases suggests the stimulation of immune response to this protein. Significant total serum IgG along with remarkable IgG1 and IgG2a response to the protein was recorded. A significant increase in IFN-γ, and IL-4 levels were observed from splenocyte cultures of immunized mice which were stimulated with rOmp16 suggesting the development of T-lymphocyte mediated immunity against the recombinant antigen. CONCLUSION: The intraperitoneal challenge with B. abortus 544 and B. melitensis 16M confirmed that rOmp16 is able to elicit efficient protective immune responses in the animal host.

Prevalence of Legionella Species in Water Resources of Iran: A Systematic Review and Meta-Analysis.

BACKGROUND: Legionella species are ubiquitous and naturally found in lakes, rivers, streams and hot springs, and other water resources. The present study aimed to investigate the prevalence of Legionella species in water resources of Iran by a systematic review and meta-analysis. METHODS: In search of papers relevant to the prevalence of Legionella in water resources of Iran, the scientific information database in both English and Persian languages was used. The search was limited to studies between the year 2000 and end of July 2016. Each cohort and cross-sectional study that reported the contamination of water with Legionella was included in the present study. For data analysis, comprehensive meta-analysis software with Cochran's Q and I2 tests were used. P values less than 0.05 were considered statistically significant. RESULTS: The prevalence of Legionella species in water resources of Iran was 27.3% (95% CI: 25.3-29.3). The prevalence of Legionella spp. in hospital water, dental settings water, and other water resources were 28.8% (95% CI: 26.4-31.2), 23.6% (95% CI: 16.1-33.2), and 29.6% (95% CI: 25.6-33.8), respectively. The most common Legionella species was L. pneumophila with a prevalence of 60.5% (95% CI: 53.3-67.2) and the prevalence of all other species was 52.5% (95% CI: 44.7-60.2). The highest prevalence was reported in Isfahan with 55.7% (95% CI: 48.0-63.0). CONCLUSION: Based on the results, the prevalence rate of Legionella species in water resources of Iran was high and the most common Legionella species was L. pneumophila.

Aqueous extract of saffron administration along with amygdala deep brain stimulation promoted alleviation of symptoms in post-traumatic stress disorder (PTSD) in rats.

OBJECTIVE: Post-traumatic stress disorder (PTSD) as one of the most devastating kinds of anxiety disorders, is the consequence of a traumatic event. Crocus sativus L., commonly known as saffron have been traditionally used for treatment of stress and anxiety. In this study, we evaluated the effects of peripheral administration of saffron, along with deep brain stimulation (DBS) in a post-traumatic stress disorder (PTSD) model caused by contextual fear conditioning (electrical foot shock chamber) in male Wistar rats. MATERIALS AND METHODS: rats (220-250 g) were divided into 7 groups (n=8) and underwent stereotactic surgery for implantation of the electrodes in the right-baso lateral of the amygdala (BLA). After 7 days, some animals received the foot shock, followed by another 7-day treatment (DBS treatment or combination treatment by saffron 5 mg/kg (i.p)) then freezing behavior as a predicted response in the absence of the foot shock (re-exposure time) and general anxiety were measured using elevated plus maze test. Serum corticosterone level and amygdala c-Fos protein expression were assessed using ELISA and Western blot analysis, respectively. RESULTS: DBS treatment and the combination therapy of saffron (5 mg/kg (I.P)) with DBS significantly (p<0.001) increased serum corticosterone levels. Also both treatments could significantly (p<0.001) reduce c-Fos protein expression and freezing behaviors time. However, DBS treatment had no effect on the general anxiety in rats with PTSD. On the other hand, combination therapy significantly (p<0.001) reduced anxiety behavior in rats with PTSD. CONCLUSION: These results might show the potential of this combination therapy for treatment of treatment-resistant PTSD patients.