scholarly journals Combating antimicrobial resistance using antimicrobial combination therapy and β–lactamase inhibitors

2018 ◽  
Vol 12 (02.1) ◽  
pp. 14S
Author(s):  
Bassam El-Hafi ◽  
Sari Shawki Rasheed ◽  
Noor A Salloum ◽  
Antoine Abou Fayad ◽  
George F Araj ◽  
...  
Keyword(s):  
Gene Expression ◽  
Antimicrobial Resistance ◽  
Combination Therapy ◽  
Bacterial Infections ◽  
Antimicrobial Agents ◽  
Treatment Approaches ◽  
Expression Levels ◽  
Gene Expression Levels

Introduction: The range of antimicrobial agents used to treat bacterial infections is becoming limited with the constant increase in antimicrobial resistance (AMR). Several genetic factors underlie AMR, including β-lactamase-encoding genes such as blaCTXM-15 that confers resistance to third-generation cephalosporins, and blaOXA-48, blaNDM-1, and blaKPC-2 that confer resistance to carbapenems. Remaining treatment approaches for such resistant infections include antimicrobial combination therapy and the use of β-lactamase inhibitors. This study assesses the molecular effects of such treatment approaches on antimicrobial resistant Enterobacteriaceae clinical isolates in vitro and in vivo. Methodology: Nine clinical Enterobacteriaceae isolates were included in the study. One harboring blaCTXM-15, one harboring blaOXA-48, one harboring blaKPC-2, two harboring blaNDM-1 and blaCTXM-15, and four harboring blaOXA-48 and blaCTXM-15. Minimal inhibitory concentrations were determined for carbapenems with β-lactamase inhibitors: avibactam, Ca-EDTA, and relebactam. Synergism between antibiotic combinations was determined by double disc diffusion when using colistin with several antibiotics. In vitro and in vivo gene expression levels were done on these combinations with and without inhibitors. Results: The use of meropenem, imipenem, and ertapenem with the selected β-lactamase inhibitors restored isolate susceptibility in 100%, 87.5%, and 25% of the cases, respectively. Antimicrobial synergism was mostly detected between colistin and meropenem, fosfomycin, or tigecycline. Survival studies revealed the survival of most mice receiving antimicrobial combination therapy with inhibitors as compared to the controls. Overall gene expression levels of resistance genes were variable depending on treatment. Conclusions: The threat of antibiotic resistant bacterial infections remains viable; however, different approaches to therapy are available.

scholarly journals The Synergistic Effect of Azoles and Fluoxetine against Resistant Candida albicans Strains Is Attributed to Attenuating Fungal Virulence

2016 ◽  
Vol 60 (10) ◽  
pp. 6179-6188 ◽  
Author(s):  
Wenrui Gu ◽  
Dongmei Guo ◽  
Liuping Zhang ◽  
Dongmei Xu ◽  
Shujuan Sun
Keyword(s):  
Gene Expression ◽  
Candida Albicans ◽  
Synergistic Effects ◽  
Mechanism Study ◽  
Content Type ◽  
Expression Levels ◽  
Time Kill ◽  
Gene Expression Levels

ABSTRACTThis study evaluated the synergistic effects of the selective serotonin reuptake inhibitor, fluoxetine, in combination with azoles againstCandida albicansbothin vitroandin vivoand explored the underlying mechanism. MICs, sessile MICs, and time-kill curves were determined for resistantC. albicans.Galleria mellonellawas used as a nonvertebrate model for determining the efficacy of the drug combinations againstC. albicansin vivo. For the mechanism study, gene expression levels of theSAPgene family were determined by reverse transcription (RT)-PCR, and extracellular phospholipase activities were detectedin vitroby the egg yolk agar method. The combinations resulted in synergistic activity againstC. albicansstrains, but the same effect was not found for the non-albicans Candidastrains. For the biofilms formed over 4, 8, and 12 h, synergism was seen for the combination of fluconazole and fluoxetine. In addition, the time-kill curves confirmed the synergism dynamically. The results of theG. mellonellastudies agreed with thein vitroanalysis. In the mechanism study, we observed that fluconazole plus fluoxetine caused downregulation of the gene expression levels ofSAP1toSAP4and weakened the extracellular phospholipase activities of resistantC. albicans. The combinations of azoles and fluoxetine showed synergistic effects against resistantC. albicansmay diminish the virulence properties ofC. albicans.

scholarly journals A Review of the Combination Therapy of Low Frequency Ultrasound with Antibiotics

2017 ◽  
Vol 2017 ◽  
pp. 1-14 ◽  
Author(s):  
Yun Cai ◽  
Jin Wang ◽  
Xu Liu ◽  
Rui Wang ◽  
Lei Xia
Keyword(s):  
Combination Therapy ◽  
Bacterial Infections ◽  
Bacterial Resistance ◽  
Low Frequency ◽  
Bactericidal Effect ◽  
Low Frequency Ultrasound ◽  
Biofilm Bacteria ◽  
Frequency Ultrasound

Single antimicrobial therapy has been unable to resist the global spread of bacterial resistance. Literatures of availablein vitroandin vivostudies were reviewed and the results showed that low frequency ultrasound (LFU) has a promising synergistic bactericidal effect with antibiotics against both planktonic and biofilm bacteria. It also can facilitate the release of antibiotics from medical implants. As a noninvasive and targeted therapy, LFU has great potential in treating bacterial infections. However, more in-depth and detailed studies are still needed before LFU is officially applied as a combination therapy in the field of anti-infective treatment.

Effects of the HDAC inhibitor scriptaid on the in vitro development of bovine embryos and on imprinting gene expression levels

2018 ◽  
Vol 110 ◽  
pp. 79-85 ◽  
Author(s):  
R. Laguna-Barraza ◽  
M.J. Sánchez-Calabuig ◽  
A. Gutiérrez-Adán ◽  
D. Rizos ◽  
S. Pérez-Cerezales
Keyword(s):  
Gene Expression ◽  
Hdac Inhibitor ◽  
Bovine Embryos ◽  
In Vitro Development ◽  
Expression Levels ◽  
Imprinting Gene ◽  
Vitro Development ◽  
Gene Expression Levels

scholarly journals In vivo measurements reveal a single 5’ intron is sufficient to increase protein expression level in C. elegans

2018 ◽  
Author(s):  
Matthew M. Crane ◽  
Bryan Sands ◽  
Christian Battaglia ◽  
Brock Johnson ◽  
Soo Yun ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Expression Level ◽  
Protein Activity ◽  
Coding Sequence ◽  
Expression Levels ◽  
C Elegans ◽  
Hsp 90 ◽  
Gene Expression Levels

AbstractIntrons can increase gene expression levels using a variety of mechanisms collectively referred to as Intron Mediated Enhancement (IME). To date, the magnitude of IME has been quantified in human cell culture and plant models by comparing intronless reporter gene expression levels to those of intron-bearing reporter genes in vitro (mRNA, Western Blots, protein activity), using genome editing technologies that lacked full control of locus and copy number. Here, for the first time, we quantified IME in vivo, in terms of protein expression levels, using fluorescent reporter proteins expressed from a single, defined locus in Caenorhabditis elegans. To quantify the magnitude of IME, we developed a microfluidic chip-based workflow to mount and image individual animals, including software for operation and image processing. We used this workflow to systematically test the effects of position, number and sequence of introns on two different proteins, mCherry and mEGFP, driven by two different promoters, vit-2 and hsp-90. We found the three canonical synthetic introns commonly used in C. elegans transgenes increased mCherry protein concentration by approximately 50%. The naturally-occurring introns found in hsp-90 also increased mCherry expression level by about 50%. Furthermore, and consistent with prior results examining mRNA levels, protein activity or phenotypic rescue, we found that a single, natural or synthetic, 5’ intron was sufficient for the full IME effect while a 3’ intron was not. IME was also affected by protein coding sequence (50% for mCherry and 80% for mEGFP) but not strongly affected by promoter 46% for hsp-90 and 54% for the stronger vit-2. Our results show that IME of protein expression in C. elegans is affected by intron position and contextual coding sequence surrounding the introns, but not greatly by promoter strength. Our combined controlled transgenesis and microfluidic screening approach should facilitate screens for factors affecting IME and other intron-dependent processes.

scholarly journals Impact of metallothionein-knockdown on cisplatin resistance in malignant pleural mesothelioma

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Sabrina Borchert ◽  
Pia-Maria Suckrau ◽  
Robert F. H. Walter ◽  
Michael Wessolly ◽  
Elena Mairinger ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Cell Line ◽  
Cell Lines ◽  
Cisplatin Resistance ◽  
Resistance Mechanism ◽  
Pleural Mesothelioma ◽  
Expression Levels ◽  
Gene Expression Levels

Abstract Malignant pleural mesothelioma (MPM) is a rare, but aggressive tumor with dismal prognosis. Platinum-based chemotherapy is regularly used as part of multimodality therapy. The expression of metallothioneins (MT) has been identified as a reason for cisplatin resistance, which often leads to early therapy failure or relapse. Thus, knockdown of MT expression may improve response to cisplatin treatment. The MT gene- and protein expression of the MPM-cell lines MSTO-211H, NCI-H2052 and NCI-H2452 and the human fibroblast cell line MRC-5, as well as their sensitivity to cisplatin treatment have been evaluated. Knockdown of MT1A, 1B and 2A expression was induced by RNA interference. MT expression was measured using quantitative real-time PCR. An in vitro Assay based on enzyme activity was used to detect cell viability, necrosis and apoptosis before and after incubation with cisplatin. MT2A gene expression could be detected in all MPM cell lines, showing the highest expression in NCI-H2452 and NCI-H2052, whereas gene expression levels of MT1A and MT1B were low or absent. The immunohistochemically protein expression of MT-I/II reflect MT2A gene expression levels. Especially for MSTO-211H cell presenting low initial MT2A levels, a strong induction of MT2A expression could be observed during cisplatin treatment, indicating a cell line-specific and platin-dependent adaption mechanism. Additionally, a MT2A-dependent cellular evasion of apoptosis during cisplatin could be observed, leading to three different MT based phenotypes. MSTO-211H cells showed lower apoptosis rates at an increased expression level of MT2A after cisplatin treatment (from sixfold to fourfold). NCI-H2052 cells showed no changes in MT2A expression, while apoptosis rate is the highest (8–12-fold). NCI-H2452 cells showed neither changes in alteration rate of MT2A expression nor changes in apoptosis rates, indicating an MT2A-independent resistance mechanism. Knockdown of MT2A expression levels resulted in significantly induced apoptotic rates during cisplatin treatment with strongest induction of apoptosis in each of the MPM cell lines, but in different markedness. A therapeutic meaningful effect of MT2A knockdown and subsequent cisplatin treatment could be observed in MSTO-211H cells. The present study showed MT2A to be part of the underlying mechanism of cisplatin resistance in MPM. Especially in MSTO-211H cells we could demonstrate major effects by knockdown of MT2A expression, verifying our hypothesis of an MT driven resistance mechanism. We could prove the inhibition of MT2A as a powerful tool to boost response rates to cisplatin-based therapy in vitro. These data carry the potential to enhance the clinical outcome and management of MPM in the future.

scholarly journals Identification of reference genes for RT-qPCR data normalisation in aging studies

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Lourdes González-Bermúdez ◽  
Teresa Anglada ◽  
Anna Genescà ◽  
Marta Martín ◽  
Mariona Terradas
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Quantitative Polymerase Chain Reaction ◽  
Expression Levels ◽  
Qpcr Data ◽  
Age Related ◽  
Specific Subset ◽  
Aging Studies

Abstract Aging is associated with changes in gene expression levels that affect cellular functions and predispose to age-related diseases. The use of candidate genes whose expression remains stable during aging is required to correctly address the age-associated variations in expression levels. Reverse transcription quantitative-polymerase chain reaction (RT-qPCR) has become a powerful approach for sensitive gene expression analysis. Reliable RT-qPCR assays rely on the normalisation of the results to stable reference genes. Taken these data together, here we evaluated the expression stability of eight frequently used reference genes in three aging models: oncogene-induced senescence (OIS), in vitro and in vivo aging. Using NormFinder and geNorm algorithms, we identified that the most stable reference gene pairs were PUM1 and TBP in OIS, GUSB and PUM1 for in vitro aging and GUSB and OAZ1 for in vivo aging. To validate these candidates, we used them to normalise the expression data of CDKN1A, APOD and TFRC genes, whose expression is known to be affected during OIS, in vitro and in vivo aging. This study demonstrates that accurate normalisation of RT-qPCR data is crucial in aging research and provides a specific subset of stable reference genes for future aging studies.

51 AGGREGATION REDUCES THE VARIATION OF GENE EXPRESSION LEVELS IN BOVINE CLONES

2006 ◽  
Vol 18 (2) ◽  
pp. 134
Author(s):  
S. Kurosaka ◽  
N. A. Leu ◽  
K. J. McLaughlin
Keyword(s):  
Gene Expression ◽  
Coefficient Of Variation ◽  
Blastocyst Stage ◽  
Average Level ◽  
Cdna Libraries ◽  
Cell Stage ◽  
Glucose Transporter 1 ◽  
Expression Levels ◽  
Gene Expression Levels

Mammalian somatic cell clones frequently exhibit abnormal gene expression that presumably results from errors in reprogramming of the transplanted genome. In the mouse, aggregation of 4-cell stage clones with each other improves reprogramming with respect to Oct-4 expression in blastocysts and an increase in term development (Boiani et al. 2003 EMBO J. 22, 5304-5312). To determine if clone-clone aggregation has a similar beneficial effect in the bovine, we aggregated 8-16 cell bovine clones with each other and profiled gene expression levels in bovine clones and clone-clone aggregates at the blastocyst stage. Clone embryos were produced from fibroblasts and cultured in vitro in SOF supplemented with fetal bovine serum at 39�C in an atmosphere of 5% CO2, 5% O2, and 90% N2. For aggregation of embryos, we first removed the zonae pepellucidae by treatment with 0.5% pronase at the 8-16 cell stage and then placed two zona-free embryos per well into deep microwells produced on the bottom of a culture dish by pressing a heated darning needle onto the surface. Seven to 10 microwells in close proximity were covered by a culture 50-�L drop of culture medium, and embryos were cultured until Day 7. Real-time RT-PCR analysis for Oct-4, DNA methyltransferase 1 (Dnmt1), Dnmt3, glucose transporter 1 (Glut1), Glut3, and Poly(A) polymerase (PolyA) was performed on reusable Dynabead Oligo (dT)25-cDNA libraries synthesized from individual blastocysts at Day 7. In vitro-fertilized embryos were used as controls. To compare the variation of gene expression in each embryo within the group, the coefficient of variation (COV; standard deviation/mean) was calculated. Although spatial distribution of Oct-4 transcript is normal in bovine blastocyst stage clones (Kurosaka et al. 2004 Reprod. Fertil. Dev. 16, 147), we detected disturbances in the level of Oct-4 expression in clones: 44.4% (8 of 18) of clones expressed Oct-4 within a range of 0.5- and 1.5-fold of the average level of expression in IVF embryos, compared to 81.8% (9 of 11) of IVF embryos. Only 22.2% (4 of 18) of clones expressed all genes examined within a range of 0.5- and 2.0-fold of the average level of IVF embryos, versus 45.5% (5 of 11) of IVF embryos. Clone-clone aggregation did not increase the proportion of clones with normal expression levels but did reduce the coefficient of variation of gene expression levels between individual clones for the genes Oct-4, Dnmt1, Dnmt3a and PolyA, but not for Glut1 and Glut3. Interestingly, bovine clone-clone aggregates (n = 25) had less variation between individual embryos compared to IVF aggregates (n = 11) for all genes except Glut1 and Glut3, although variation of single clones was larger than that of single IVF embryos. Analysis of Oct-4 and �-Actin transcripts in mouse clone blastocysts indicated a similar decrease in gene expression variation subsequent to aggregation of mouse clones. These results demonstrate that bovine pre-implantation stage clones exhibit a high degree of variation in gene expression levels and suggest that aggregation of clones is beneficial in reducing the variation in expression of some genes.

179 EFFECT OF SERUM DURING CULTURE ON DAY 14 ELONGATED BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 191 ◽  
Author(s):  
J. Angulo ◽  
G. T. Gentry ◽  
R. A. Godke ◽  
K. R. Bondioli
Keyword(s):  
Gene Expression ◽  
Messenger Rna ◽  
Culture Media ◽  
Calf Serum ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Expression Levels ◽  
The Mean

It has been reported that the addition of serum to embryo culture media alters gene expression and triggers the development of large offspring syndrome. The objectives of this study were to determine gene expression levels in embryos cultured in the absence or presence of 5% calf serum and in vivo-derived (IVD) embryos and to determine the effects of serum on the length of elongated embryos. Abattoir-derived oocytes were obtained from a commercial provider and fertilized at 24 h of maturation with semen from a bull previously used for IVF. At 18 h post-insemination (hpi), embryos were denuded and groups of 15 presumptive zygotes were cultured in 30-μL drops of modified SOF medium with amino acids and 6 mg mL–1 of BSA (mSOFaa). At 72 hpi, cleavage rate was assessed and embryos were randomly allocated into 2 treatments: mSOFaa without and with 5% calf serum. Embryos were then cultured to 168 hpi and blastocyst rates were assessed and recorded. Blastocysts (n = 5 to 10) from each treatment were transferred into synchronized recipients, and Day 14 embryos were recovered 7 days post-transfer. Embryos were photographed, measured, and immediately stored at –80°C in a minimal volume of PBS + 0.1% polyvinyl alcohol. Messenger RNA was isolated using a Dynabeads mRNA Direct Kit™ (Invitrogen, Carlsbad, CA), and reverse transcription was performed using an iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., CA). Quantitative PCR was performed to determine the transcript abundance for COX6A, IFNT1a, PLAC8, IGF2R, and GAPDH for each sample. The GAPDH was used as a reference gene, and gene expression was calculated as a ratio of expression levels between each gene of interest and GAPDH. Expression levels for each gene were determined from standard curves generated by serial dilutions of PCR amplicons starting with 0.4 pg/reaction. Blastocyst development rates were higher in embryos cultured with serum compared with the nonserum treatment (14.9 and 7.4% respectively; chi-square, P < 0.001). Lengths of elongated embryos from the serum (3395.3 ± 414.7 μm) and nonserum (2784 ± 741.8 μm) culture treatments differed from the IVD (6297.7 ± 677.2 μm) treatment (mean ± SE; ANOVA, P < 0.0052). There were no differences in the mean expression levels for COX6A, IFNT1a, PLAC8, and IGF2R across treatment groups, but in the serum treatment, 3 out 11 overexpressed IFNT1a, 4 out of 11 overexpressed IGF2R, and 2 out of 11 overexpressed PLAC8, defined as being 2 standard deviations above the mean of the IVD treatment for each respective gene. In the in vitro-produced nonserum and IVD treatments, overexpression by this definition was not observed. Although mean expression levels were not affected by culture with serum under these conditions, very high expression of IFNT1a, IGF2R, and PLAC8 was observed in some embryos cultured with serum, but not in embryos cultured without serum or IVD embryos.

O-152 Investigation of the relationship of sperm motility and Kisspeptin in subfertile men

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
A Kocaman ◽  
B Ayas
Keyword(s):  
Gene Expression ◽  
Intracellular Calcium ◽  
Sperm Motility ◽  
Laser Scanning ◽  
Calcium Release ◽  
Expression Levels ◽  
Significant Difference ◽  
Motility Parameters ◽  
Gene Expression Levels

Abstract Study question Does kisspeptin administration affect the motility parameters in sperm samples of subfertile cases? Summary answer Kisspeptin administration significantly increased gene expression levels related with sperm motility as well as intracellular calcium concentrations. What is known already Sperm motility problems are among the most important causes of male infertility. In recent years, a peptide named kisspeptin has been discovered that may have effects on sperm motility. Kisspeptin is known to trigger calcium release in hypothalamic neurons. In addition, kisspeptin administration increased sperm progressive motility in studies conducted on normozoospermic individuals. Furthermore, it is suggested that kisspeptin protein in seminal plasma is positively associated with semen quality. However, there is no evidence that how kisspeptin can affect sperm in men with infertility problems. Study design, size, duration This basic research study was an in vitro experimental approach involving the use of semen samples from an infertil cases between September to December in 2020. 40 men were included in both control and experimental groups. Participants/materials, setting, methods All analyses were performed on semen samples from 10 normozoospermic (NZ), 10 asthenozoospermic (AZ), 10 oligoasthenozoospermic (OAZ) and 10 oligoastenoteratozoospermic (OATZ) men, aging between (21-40) years. Basal serum and seminal kisspeptin levels were analyzed by ELISA. Sperm were divided into two groups. Kisspeptin-13 administered in vitro. KISS1, KISS1R, CATSPER1, AKAP4 gene expressions analyzed by qRT-PCR using 2−ΔΔCt algorithm. Intracellular calcium concentration was determined with floresence spectroflurometer and laser scanning confocal microscope. Main results and the role of chance The serum kisspeptin level of NZ was significantly higher than other groups (p &lt; 0.05). The semen kisspeptin level was significantly higher than OAZ and OATZ (p &lt; 0.05), but not in NZ (p &gt; 0.05). Also, KISS1 gene expression was higher in AZ compared to other groups (p &lt; 0.05). Biochemical and gene expression analysis of kisspeptin were consistent with each other. There was a significant increase in the expression of CATSPER1 gene in AZ compared to other groups (p &lt; 0.05). Also, AKAP4 gene expression was significantly higher in OATZ compared to other groups (p &lt; 0.05). No significant difference was documented for the expression of KISS1R (p &gt; 0.05). Intracellular calcium was significantly increased in AZ and NZ after kisspeptin administration. The intracellular calcium increase is consistent with increased CATSPER1 gene expression levels in AZ. Kisspeptin administration may have a significant effect on sperm motility parameters. Limitations, reasons for caution The biochemical and gene expression levels of KISS1 were consistent. However, gene expression was explored at the mRNA level for CATSPER1 and AKAP4. The protein expression analyses of these genes may confirm the results. Also, using kisspeptin antagonists may strength the results of intracellular calcium analysis. Wider implications of the findings Kisspeptin treatment for individuals diagnosed with asthenozoospermia may have therapeutic results. KISS1 quantitation may be a determining factor for the subfertility in routine semen analysis. Trial registration number OMU KAEK 2019/462

CALM2 gene expression levels in the cumulus cells as a marker for chromosomal abnormalities in the embryo in in vitro fertilization programs

2016 ◽  
Vol 10_2016 ◽  
pp. 64-72
Author(s):  
Safronova N.A. Safronova ◽  
Kalinina E.A. Kalinina ◽  
Donnikov A.E. Donnikov ◽  
Burmenskaya O.V. Burmenskaya ◽  
Makarova N.P. Makarova ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Fertilization ◽  
Chromosomal Abnormalities ◽  
Cumulus Cells ◽  
Vitro Fertilization ◽  
Expression Levels ◽  
Gene Expression Levels
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