Revolutionizing medicine: Molecularly imprinted polymers as precision tools in cancer diagnosis and antibiotic detection.

Molecularly imprinted polymers (MIPs) are pivotal in medicine, mimicking biological receptors with enhanced specificity and affinity. Comprising templates, functional monomers, and cross-linkers, MIPs form stable three-dimensional polymer networks. Synthetic templates like glycan and aptamers improve efficiency, guiding the molecular imprinting process. Cross-linking determines MIPs' morphology and mechanical stability, with printable hydrogels offering biocompatibility and customizable properties, mimicking native extracellular matrix (ECM) microenvironments. Their versatility finds applications in tissue engineering, soft robotics, regenerative medicine, and wastewater treatment. In cancer research, MIPs excel in both detection and therapy. MIP-based detection systems exhibit superior sensitivity and selectivity for cancer biomarkers. They target nucleic acids, proteins, and exosomes, providing stability, sensitivity, and adaptability. In therapy, MIPs offer solutions to challenges like multidrug resistance, excelling in drug delivery, photodynamic therapy, photothermal therapy, and biological activity regulation. In microbiology, MIPs serve as adsorbents in solid-phase extraction (SPE), efficiently separating and enriching antibiotics during sample preparation. They contribute to bacterial identification, selectively capturing specific strains or species. MIPs aid in detecting antibiotic residues using fluorescent nanostructures and developing sensors for sulfadiazine detection in food samples. In summary, MIPs play a pivotal role in advancing medical technologies with enhanced sensitivity, selectivity, and versatility. Applications range from biomarker detection to innovative cancer therapies, making MIPs indispensable for the accurate determination and monitoring of diverse biological and environmental samples.

Ameliorative effect of Stevia rebaudiana Bertoni on sperm parameters, in vitro fertilization, and early embryo development in a streptozotocin-induced mouse model of diabetes.

Diabetes mellitus (DM) is a common metabolic disease characterized by high blood sugar levels. It is well known that men with diabetes frequently experience reproductive disorders and sexual dysfunction. In fact, sperm quality has a significant effect on fertilization success and embryo development. The current study aimed to investigate the effect of Stevia rebaudiana hydroalcoholic extract on serum testosterone levels, sperm parameters, in vitro fertilization (IVF) success, and in vitro embryonic developmental potential to reach the blastocyst stage in a streptozotocin (STZ)-induced mouse model of diabetes. In this research, 30 male mice were distributed randomly into control, diabetic (streptozotocin 150 mg/kg) and diabetic + Stevia (400 mg/kg) groups. The results revealed a decrease in body and testis weight and elevated blood fasting blood sugar (FBS) levels in the diabetic group, compared with the control. However, Stevia treatment significantly increased body and testis weight, while serum FBS levels were decreased compared with the diabetic group. In addition, Stevia significantly increased blood testosterone levels compared with the diabetic group. Moreover, sperm parameters were improved considerably by Stevia treatment compared with the diabetic group. Furthermore, Stevia administration significantly promoted IVF success rate and in vitro development of fertilized oocytes compared with the diabetic group. In summary, our data indicated that Stevia enhanced sperm parameters, IVF success, and in vitro embryonic developmental competency in diabetic mice, probably because of its antioxidant effects. Therefore, Stevia could ameliorate sperm parameters that, in turn, increase fertilization outcomes in experimental-induced diabetes.

Effects of cigarette smoke condensate on reproduction in mice in vivo.

Smoking has dangerous and sometimes irreversible effects on various body tissues, including the reproductive system. We conducted this research to determine the in vivo effects of cigarette smoke condensate (CSC) on reproduction in mice. In this experimental in vivo study, 32 male and female NMRI mice were divided into four groups. The mice were injected with CSC (CSC-1R3F) for 28 days. The mice were mated 1 day after the last injection and observed daily for 1 week for the presence of a vaginal plug to track mating. We evaluated mating success rate, and sperm and oocyte quality, pregnancy outcome, childbearing status, and in vitro fertilization (IVF). The results showed a decrease in successful mating in female mice that received the CSC injections. CSC significantly influenced the number of offspring born to males. When the CSC was injected into male mice, there was a significant increase in the number of offspring compared with the group in which only the females received CSC injections. According to the results, there was a negative effect of CSC on morphological parameters in male and female mice. Also, successful IVF after exposure to CSC was significantly decreased in the female mice treated group. The results indicated that CSC significantly affected the number of offspring and fecundity success in females.

Extracellular vesicle isolation, purification and evaluation in cancer diagnosis.

Strategies for non-invasive biomarker discovery in early detection of cancer are an urgent need. Extracellular vesicles (EVs) have generated increasing attention from the scientific community and are under intensive investigations due to their unique biological profiles and their non-invasive nature. EVs are membrane-enclosed vesicles with variable sizes and function. Such vesicles are actively secreted from multiple cell types and are considered as key vehicles for inter-cellular communications and signalling. The stability and potential to easily cross biological barriers enable EVs for exerting durable effects on target cells. These along with easy access to such vesicles, the consistent secretion from tumour during all stages of tumorigenesis and their content providing a reservoir of molecules as well as mirroring the identity of the cell of origin are virtues that have made EVs appealing to be assessed in liquid biopsy approaches and for using as a promising resource of biomarkers in cancer diagnosis and therapy and monitoring targeted cancer therapy. Early detection of EVs will guide time-scheduled personalised therapy. Surveying reliable and sensitive methods for rapid isolation of EVs from biofluids, the purity of isolated vesicles and their molecular profiling and marker specification for clinical translation in patients with cancer are issues in the area and the hot topics of many recent studies. Here, the focus is over methods for EV isolation and stratification for digging more information about liquid biopsy-based diagnosis. Extending knowledge regarding EV-based strategies is a key to validate independent patient follow-up for cancer diagnosis at early stages and inspecting the efficacy of therapeutics.

Effects of embryonic stem cell-conditioned medium on the preimplantation development of mouse embryos.

The production of high-quality embryos in the laboratory and a successful pregnancy are closely related to the condition and contents of oocyte and embryo culture media. In this study, we investigated the effects of embryonic stem cell-conditioned medium (ESCCM) and embryonic stem cells growth medium (ESCGM) compared with potassium-enriched simplex optimized medium (KSOM) on preimplantation embryo development stages during natural or in vitro fertilization (IVF). Birth rate of pups was measured. To obtain mature oocytes, and 2-cell and 8-cell embryos, human chorionic gonadotropin (HCG) was injected 48 h after i.p. injection of 5 units of pregnant mare serum gonadotropin. Mature oocytes were obtained from non-mated female mice 14 h after HCG injection. To obtain 2-cell and 8-cell embryos, mated female mice, 1 day and 3 days, respectively, after HCG injection, were used. Mature oocytes were fertilized in HTF medium. Embryos obtained from natural or in vitro fertilization were cultured in experimental media, ESCCM and ESCGM, or KSOM as the control culture medium. Embryos that developed to the blastocyst stage were transferred to the uteri of pseudopregnant mice and effects of the experimental media on embryo viability were determined. ESCCM and ESCGM could not pass the embryo after the 2-cell stage, but they were suitable for the development of the embryo from the 8-cell stage to the blastocyst. It can be concluded that the embryo has various requirements at different stages of development.

Feeding role of mouse embryonic fibroblast cells is influenced by genetic background, cell passage and day of isolation.

Mouse embryonic fibroblast (MEF) cells are commonly used as feeder cells to maintain the pluripotent state of stem cells. MEFs produce growth factors and provide adhesion molecules and extracellular matrix (ECM) compounds for cellular binding. In the present study, we compared the expression levels of Fgf2, Bmp4, ActivinA, Lif and Tgfb1 genes at the mRNA level and the level of Fgf2 protein secretion and Lif cytokine secretion at passages one, three and five of MEFs isolated from 13.5-day-old and 15.5-day-old embryos of NMRI and C57BL/6 mice using real-time PCR and enzyme-linked immunosorbent assay. We observed differences in the expression levels of the studied genes and secretion of the two growth factors in the three passages of MEFs isolated from 13.5-day-old and 15.5-day-old embryos, respectively. These differences were also observed between the NMRI and C57BL/6 strains. The results of this study suggested that researchers should use mice embryos that have different genetic backgrounds and ages, in addition to different MEF passages, when producing MEFs based on the application and type of their study.

Effect of cigarette smoke condensate on mouse embryo development and expression of pluripotency and apoptotic genes in vitro.

The aim of the present study was to investigate the effect of cigarette smoke condensate (CSC) on in vitro development of mouse embryos. In total 3000 NMRI mice 2PN embryos were divided into six groups (n = 500). The test group was exposed to 20, 40, 80, 160 or 320 µg/ml of CSC. In the control group, CSC was not added to the culture medium during the development of 2PN embryos. The effects of 20 and 80 µg/ml of CSC on genes involved in pluripotency and apoptosis, and also, the aryl hydrocarbon receptor gene was assessed in the blastocysts. Our results showed that CSC had an adverse effect on the viability of mouse embryos at the concentrations of 80, 160 and 320 µg/ml compared with the control group (P < 0.05). In contrast, it had positive effects on the viability of mouse embryos at the concentrations of 20 and 40 µg/ml compared with the control group (P < 0.05). The 20 and 80 µg/ml concentrations of CSC increased the expression of pluripotency, apoptotic, and aryl hydrocarbon receptor genes in the blastocyst embryo stage compared with the control group (P < 0.05). It can be concluded that concentrations higher than 40 µg/ml of CSC have an adverse effect on mouse embryo development in the preimplantation stages. Also, 20 and 80 µg/ml concentrations of CSC have a significant effect on the expression of pluripotency, apoptotic, and the aryl hydrocarbon receptor genes in the blastocyst embryo stage compared with the control group.

The effect of the "segment" of spinal cord injury on the activity of the nucleotide-binding domain-like receptor protein 3 inflammasome and response to hormonal therapy.

Spinal cord injury (SCI) is a common devastating condition that causes neuronal loss and dysfunction. Neuroinflammation takes cardinal roles in the pathogenesis of SCI, and nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome is a mediator of inflammatory reactions occurring in SCI patients. The present study was designed to survey possible relation between thoracic segments whereby injury occurs with the activity of NLRP3 inflammasome complex, and to find the influence of hormonal therapy on the outcomes. Adult male Wistar rats underwent contusion SCI model at three different thoracic segments T1, T6 and T12, then receiving subcutaneous injection of either 10 mg/kg melatonin or 25 µg/kg 17-ß estradiol (E2) every 12 hours until 72 hours post-SCI. Inflammasome activity was assessed before and at the end of hormonal therapy. SCI rats showed decreased locomotor activity and myelination, and increased activity of the NLRP3, apoptosis-associated speck-like protein (ASC) and caspase-1 at gene and protein levels. Release of interleukins (ILs) 18 and 1ß was also augmented after SCI (P < 0.0.5). Hormonal therapy was most effective for targeting mRNA activity at T6 segment. Treatment with either melatonin or E2 caused a decrease in the protein activity of NLRP3 inflammasome at all segments (P < 0.0.5), except for T6 that NLRP3 protein had no response to melatonin. IL-1ß showed decreased activity in response to hormonal therapy at all segments, whilst IL-18 protein had no change at T1 segment. It is understood that although no alteration in the activity of NLRP3 was found for SCI at different segments, the response to hormonal therapy was influenced by segment. SIGNIFICANCE OF THE STUDY: From our results, the NLRP3 inflammasome activity is not influenced by segment, but there are differences in the effect of hormonal therapy on inflammasome activity at different segments in response to melatonin or E2. These findings also provide the beneficial effects of melatonin or E2 on inflammation caused by spinal cord injury in different thoracic segments. Finally, these data can have therapeutic importance for hormone therapy of spinal cord injury.

Effect of cyanocobalamin on oocyte maturation, in vitro fertilization, and embryo development in mice.

The aim of this study was to investigate the effect of cyanocobalamin supplementation on in vitro maturation (IVM), in vitro fertilization (IVF), and subsequent embryonic development competence to the blastocyst stage, and in vitro development of mouse 2-cell embryos. Cumulus cells were prepared from mouse cumulus-oocyte complexes (COCs) and incubated for 24 h in an in vitro culture (IVC) medium that contained different concentrations of cyanocobalamin (100, 200, 300 or 500 pM). We collected 2-cell embryos from superovulated NMRI mice and cultured them in the same concentrations of cyanocobalamin (100, 200, 300 or 500 pM). After 42 h of IVM, we observed significantly increased oocyte maturation in the 200 pM cyanocobalamin-treated group compared with the control group (P < 0.0001). Mature oocytes cultured in 200 pM cyanocobalamin were fertilized and cultured in IVC medium with cyanocobalamin (100, 200, 300 or 500 pM) during early embryogenesis. The matured oocytes that were cultured in 200 pM cyanocobalamin had significantly higher 2-cell development rates compared with the control oocytes (P < 0.01). Embryos obtained from in vitro mature oocytes and in vivo fertilized oocytes that were cultured in 200 pM cyanocobalamin had significantly greater frequencies of development to the blastocyst stage and a significant reduction in 2-cell blocked and degenerated embryos compared with the control embryos (P < 0.0001). Embryos derived from oocytes fertilized in vivo with 200 pM cyanocobalamin had a higher percentage of blastocyst embryos compared with those derived from matured oocytes cultured in vitro (P < 0.0001). These finding demonstrated that the effects of cyanocobalamin on oocyte maturation, fertilization, and embryo development in mice depend on the concentration used in IVC medium.

CRISPR-Cas9-mediated knockout of the Prkdc in mouse embryonic stem cells leads to the modulation of the expression of pluripotency genes.

Prkdc encodes for the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) playing a key role in nonhomologous end joining pathway during DNA double-strand break repair and also influencing the homologous recombination (HR) repair system by phosphorylation of proteins involved in HR. In addition, Prkdc has other critical functions in biological processes, such as transcriptional regulation, telomere stability, apoptosis, and metabolism. DNA-PKcs upregulates during in vitro differentiation of mouse embryonic stem cells (mESCs). To address the potential role of Prkdc in mESCs pluripotency and in vitro differentiation into ectoderm, mesoderm, and endoderm germ layers under normal physiological conditions, a bi-allelic Prkdc-knockout cell line was generated in the present study by employing CRISPR/Cas9 system, and subsequently, its potential role in stemness and development was studied. The results of the study showed that the expression of pluripotency-associated genes, including Nanog and Sox-2 were overexpressed in the bi-allelic Prkdc-knockout cell line. Also, bi-allelic Prkdc-knockout cell line was shown to have typical mESCs cell morphology, cell cycle distribution, and alkaline phosphatase activity. Furthermore, the results of the study revealed that the expression of several germ layer markers is modulated in Prkdc-knockout lines. In conclusion, the findings of our study demonstrated the role of Prkdc during differentiation and development of ESCs.

The expressions of NLRP1, NLRP3, and AIM2 inflammasome complexes in the contusive spinal cord injury rat model and their responses to hormonal therapy.

Spinal cord injury (SCI) is a devastating condition with a growing incidence in developing countries. The activity of inflammasome complexes initiates neuroinflammation, which is a key player in SCI pathogenesis. Here, NLRP1, NLRP3, and absent in melanoma 2 (AIM2) inflammasome complexes were assessed in the contusive (T6) SCI rats for their expression profiles and their response to hormonal therapy (10 mg/kg melatonin or 25 µg/kg 17ß-estradiol [E2] every 12 h until 72 h). Two phases was considered in this study: the dominant time of inflammasome activation, which was 72 h post-SCI and the response from each complex to hormonal therapy at this time. Gene and protein expressions of NLRP1, NLRP3, AIM2, ASC, and caspase-1 were evaluated by real-time PCR (for gene analysis), western blot, and immunohistochemistry (IHC), and biochemical presence of IL-18 and IL-1ß in spinal cord tissue homogenates was analyzed by enzyme-linked immunosorbent assay (ELISA). The whole inflammasome complexes showed high expressions in the SCI group, while after hormonal therapy, these alterations were counteracted, which were more conspicuous for the NLRP1 and NLRP3. Melatonin had no predilection over E2 for such effect. Finally, the expression profile of signaling related to the synthesis (TLR4/NF-κB) and activation (NADPH oxidase 2 [NOX2]/TXNIP) of inflammasome complexes was surveyed, and there were low activities for the two pathways in SCI rats that underwent hormone therapy. From the findings, it is concluded that both melatonin and E2 are efficient to target inflammasome activation in the SCI rats.

MiR-646 prevents proliferation and progression of human breast cancer cell lines by suppressing HDAC2 expression.

BACKGROUND: Breast cancer is a type of cancer with a high incidence and mortality rate worldwide. Change in epigenetic mechanisms enhances cancer cell progression. Histon deacetylase 2 (HDAC2) was found to act as a potential oncogene in different malignancies. For better understanding the mechanisms related to breast cancer development, we investigated the role of HDAC2 in breast cancer and the inhibitory effect of miR-646 on this oncogene. METHODS: A total of thirty cancerous tissues and 30 adjacent non-cancerous specimens and also three breast cancer cell lines were enrolled in the study. Quantitative reverse transcriptase PCR (qRT-PCR) was employed to detect the HDAC2 and miR-646 expression level in the studied samples. The biological roles of HDAC2 and miR-646 were investigated through manipulating the expression level of HDAC2 or miR-646 in breast cancer cells. Finally, we evaluated whether the HDAC2 is a direct target for miR-646. RESULTS: In this study, we found HDAC2 is significantly upregulated in cancerous specimens and cell lines compared to non-cancerous tissues and normal cell line. On the other hand, miR-646 expression was decreased in clinical specimens and breast cancer cells compared to non-cancerous samples. Knocking out of the HDAC2 and overexpression of miR-646 inhibited breast cancer cell growth but promoted cell death, while untreated groups showed inverse results. Furthermore, we showed that in the breast cancer cells, miR-646 regulates the progression and proliferation by suppressing HDAC2. CONCLUSION: Taken together, our study identified a miR-646/HDAC2 regulatory function in the breast cancer development and introduced a therapeutically target for breast cancer.

Effect of embryo cryopreservation on derivation efficiency, pluripotency, and differentiation capacity of mouse embryonic stem cells.

Mouse embryonic stem cells (mESCs) are pluripotent cells that have the capability for self-renewal. One of the most important factors that affect the efficiency of their isolation is the condition of the mouse embryos. The main objective of this study is to isolate mESCs from C57BL/6 frozen/thawed eight-cell mouse embryos using serum-free culture. We generated mESCs from blastocysts that developed from frozen/thawed embryos of C57BL/6 mice by the 3i + LIF medium. Assessments of the isolated mESC lines (MUKF-1, MUKF-2, and MUKF-3) included simple karyotype analysis; polymerase chain reaction of the testis-determining gene (Sry); determination of alkaline phosphatase (ALP) activity; expressions of pluripotent transcription factors Oct4, Rex1, Sox2, and Nanog by reverse transcription polymerase chain reaction; and immunocytochemistry assessment of OCT4 and SSEA-I expressions at the protein level. We evaluated the ability of these mESC lines to differentiate into three germ layers by embryoid body (EB) formation. The cell doubling time (DT) of isolated mESCs was determined. The 2-C57 cell line was served as control. Germline competence of the male mESC line (MUKF-3) was tested through chimeric mouse production. Three independent mESC lines (MUKF-1, MUKF-2, and MUKF-3) were established from five cryopreserved embryos. The MUKF-1 and MUKF-2 lines were female, whereas MUKF-3 was a male mESC line. Karyotype analysis showed that MUKF-3 had a diploid karyotype, whereas MUKF-1 and MUKF-2 had abnormal karyotypes. All three lines had ALP activity and expressed Oct4, Rex1, and Nanog. Immunocytochemistry assessment for OCT4 and SSEA-I was positive for all three lines. The DT differed in the three mESC lines. MUKF-1 and MUKF-3 could form EB and express developmental genes after spontaneous differentiation. These data demonstrated that probably cryopreservation affected the efficiency of derivation, karyotype, DT, expression of pluripotency, developmental genes, and differentiation capacity of the independent mESC lines.

Increasing maternal age of blastocyst affects on efficient derivation and behavior of mouse embryonic stem cells.

Mouse embryonic stem cells (mESCs) have the capability to undergo unlimited cell division and differentiate into derivatives of all three embryonic germ layers. These fundamental features enable mESCs to potentially be appropriate, efficient models for biological and medical research. Therefore, it is essential to produce high-performance mESCs. In the current study, we have produced mESCs from blastocysts that developed from fertilized oocytes of 2 (2-C57)-, 4 (4-C57)-, and 6 (6-C57)-month-old C57BL/6 mice. A comparison of isolated stem cells was done from the viewpoint of the efficiency of mESC derivation, self-renewal, and their differentiation capacity. All generated mESCs showed a similar expression of the molecular markers protein of pluripotency and AP activity. In the 3i medium, there was a significant decrease in undifferentiated marker genes expression in the 2-C57 cells compared with the other two groups ( P < 0.05) but developmental genes significantly increased in the 4-C57 and 6-C57 cells compared with the 2-C57 cells ( P < 0.05). The differentiation capacity into three germ layers through the embryoid body formation and percentage of cell lines with normal numbers of chromosomes reduced with increased maternal age. The highest DT and highest percentage of cells in the S phase belonged to 2-C57 cells. These data demonstrated that blastocysts which developed from fertilized oocytes of 2-, 4-, and 6-month-old C57BL/6 mice can generate pluripotent stem cells, and suggested that both the efficiency of mESC isolation and the behavior of these isolated mESCs including pluripotency, self-renewal, cell cycle, and DT changed with increasing maternal age.

Effects of cigarette smoke condensate on proliferation and pluripotency gene expression in mouse embryonic stem cells.

BACKGROUND: Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of blastocysts. They can be used as valuable experimental models to test the effects of drugs, chemicals, and environmental contaminants such as cigarette smoke condensate (CSC) on preimplantation embryo development. The aim of this study was to evaluate the effect of CSC on ESCs derived from mice with different genetic backgrounds and maternal ages. METHODS: The study groups consisted of mouse ESCs (mESCs) obtained from three sources: blastocysts developed from fertilized oocytes of two-month-old (2-C57) and six-month-old (6-C57) C57BL/6 inbred mice and those developed from fertilized oocytes of two-month-old (2-NMRI) NMRI outbred mice. The groups of mESCs were exposed to 0.04, 4, and 40 µg/mL CSC. After exposure, we measured cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and real-time polymerase chain reaction for changes in expressions of Oct4, Sox2, Nanog, Ahr, Bax, Bcl2, TFAM, and POLG. The cell doubling time (DT) of these populations was also determined. RESULTS: We observed that CSC changed proliferation and DT in the 2-C57 and 6-C57 cells. There was no change in 2-NMRI cells. Exposure to CSC caused changes in the gene expressions and induced apoptosis in all three cell lines. CONCLUSION: Based on the results of the study, it can be concluded that CSC has an effect on the viability, DT and gene expression patterns in mouse ESCs and its effects vary based on the genetic background and maternal age of isolated mouse ESCs.

Generation of Fam83h knockout mice by CRISPR/Cas9-mediated gene engineering.

Family with sequence similarity 83 member H (FAM83H) protein-coding geneplay an essential role in the structural organization, calcification of developing enamel, and keratin cytoskeleton disassembly by recruiting Casein kinase 1 alpha (CSNK1A1) to keratin filaments. In this study, we have applied CRISPR Cas9 nickase (D10A) to knockout (KO) the Fam83h gene in NMRI outbred mice. We generated homozygous Fam83h KO mice ( Fam83h Ko/Ko ) through a premature termination codon, which was validated by Sanger sequencing in F0 generation. Next, we also bred the FAM83H KO for two generations. Reverse-transcription polymerase chain reaction and Western blot analysis approved the Fam83h KO mice. The Fam83h KO mice had evidence of normal morphology at the cervical loops, secretory and maturation stages, and mandibular molars. In comparison with the normal wild-type mice ( Fam83h W/W ), the F2 homozygous KO ( Fam83h Ko/Ko ) had sparse, scruffy coats with small body size and decreased general activity. Also, they had the natural reproductive ability and natural lifespan. In addition, delay in opening the eyes and dry eyes among infant mice were seen. The F1 heterozygous mice looked comparable to the normal wild-type mice ( Fam83h W/W ), which showed autosomal recessive inheritance of these phenotypes. The KO of FAM83H had controversial effects on the development of teeth and the formation of enamel. The phenotype defect in dental development and the enamel formation were seen in three mice among four generations. It can be concluded that null FAM83H in outbred mice not only showed the reported phenotypes in null inbred mouse but also showed normal lifespan and reproductive ability; dental deficiency in three homozygous mice; and the symptoms that were similar to the symptoms of dry eye syndrome and curly coat dog syndrome in all four evaluated KO generations.

Ratiometric enhanced fluorometric determination and imaging of intracellular microRNA-155 by using carbon dots, gold nanoparticles and rhodamine B for signal amplification.

An ultrasensitive and highly reliable ratiometric assay is described for the determination of microRNA-155. It works at the attomolar concentration level and has high selectivity which warrants its potential application in cancer biomarker tracking. The excellent performance of this method results from (a) the use of a hybrid conjugate prepared from Rhodamine B (RhB), carbon dots (CDs) and probe-microRNA, and (b) from the measurement of fluorescence resonance energy transfer (FRET) that is observed in the AuNP/target-microRNA system as a result of RNA hybridization. The dye RhB (emission peak at 580 nm) serves as an internal reference. The sensitivity of this assay is increased by about 30% because of the broad emissions of CDs (489 nm and 665 nm) through a sequential FRET phenomenon. RhB-CDs were covalently bio-conjugated to probe microRNA. In the presence of AuNPs, the fluorescence of the CDs is quenched, while in the presence of microRNA-155, the ratio of fluorescences at 489 and 665 nm (I489/I665) is enhanced again. A linear relationship exists between the ratio of fluorescence and the concentration of microRNA-155 in the range from 1 aM to 0.1 µM, and the detection limit is 0.3 aM. The assay was applied to quantitative studies of target microRNA-155 in multiple pathways associated with cancer progression in biological fluids include human serum samples and cancer cells. The nanoprobe also deliver clear signal to microRNA target in fixed and lived MDA-MB-231 cells. Graphical abstract A ratiometric FRET sensing method used for microRNA-155 detection at aM concentration level using CDs and AuNPs as donor-acceptor respectively and Rhodamine B as amplification reagent. The application of assay for imaging of microRNA-155 in fixed and live MDA-MB-231 cells is demonstrated.

A FRET immunosensor for sensitive detection of CA 15-3 tumor marker in human serum sample and breast cancer cells using antibody functionalized luminescent carbon-dots and AuNPs-dendrimer aptamer as donor-acceptor pair.

We proposed a FRET immunosensing for detection of CA15-3 tumor marker by highly biospecific interactions between CA 15-3 antigen and the corresponding antibody and aptamer. In this sandwich type immunoassay, CA15-3 antibody-functionalized carbon dots and AuNPs labeled PAMAM-Dendrimer/aptamer were used as donor/acceptor, respectively. When CA 15-3 Ag was added to homogenous immunoassay, the strong complex interaction between CA15-3 Ab-CA15-3 Ag- aptamer caused in more coming closer carbon dot and AuNPs and more decreasing fluorescence signal. The decreased fluorescence intensity was linear at three ranges including in concentration range 1.1 µUmL-1 to 16 µU mL-1 with regression of R2 = 0.9879, at the concentration range 16 µU mL-1 to 0.163 mU mL-1 with regression of R2 = 0.9944 and at the concentration range 0.163 mU mL-1 to 5.0 mU mL-1 with regression of R2 = 0.9805. The detection limit of the FRET immunoassay was 0.9 µU mL-1. This assay revealed good sensitivity and specificity with MDA-MB-231 breast cancer cells concentrations from 1000 to 40000 cells/mL with correlation coefficient of 0.9955 and detection limit of 300 cells/mL (3 cells in 10 µL of injected sample). In addition, this FRET immunosensing is applicable in diluted human serum. The recovery values were in the range of 95.86-96.97% for CA 15-3 Ag in spiked serum sample with RSD lower than 7.3%. The proposed immunoassay could be a valid model for establishing other immunoassays for detection of different cancer tumor markers with relevant antigens and antibodies.

Ni-hemin metal-organic framework with highly efficient peroxidase catalytic activity: toward colorimetric cancer cell detection and targeted therapeutics.

BACKGROUND: Given the great benefits of artificial enzymes, a simple approach is proposed via assembling of Ni2+ with hemin for synthesis of Ni-hemin metal-organic-frameworks (Ni-hemin MOFs) mimic enzyme. The formation of the Ni-hemin MOFs was verified by scanning electron microscopy, Transmission electron microscopy, X-ray powder diffraction, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, Energy-dispersive X-ray spectroscopy and UV-vis absorption spectroscopy. This novel nanocomposite exhibited surprising peroxidase like activity monitored by catalytic oxidation of a typical peroxidase substrate, 3,3,5,5'-tetramethylbenzidine, in the presence of H2O2. By using folic acid conjugated MOF nanocomposite as a recognition element, we develop a colorimetric assay for the direct detection of cancer cells. RESULTS: The proposed sensor presented high sensitivity and selectivity for the detection of human breast cancer cells (MCF-7) and Human Caucasian gastric adenocarcinoma. By measuring UV-vis absorbance response, a wide detection range from 50 to 105 cells/mL with a detection limit as low as 10 cells/mLwas reached for MCF-7 cells. We further discuss therapeutics efficiency of Ni-hemin MOFs in the presence of H2O2 and ascorbic acid. Peroxidase-mimic Ni-hemin MOFs as reactive oxygen species which could damage MCF-7 cancer cells, however for normal cells (human embryonic kidney HEK 293 cells) killing effect was negligible. CONCLUSIONS: Based on these behaviors, the developed method offers a fast, easy and cheap assay for the interest in future diagnostic and treatment application.

Dimethyl sulphoxide and electrolyte-free medium improve exogenous DNA uptake in mouse sperm and subsequently gene expression in the embryo.

SummaryOne of the methods to generate transgenic animals is called sperm-mediated gene transfer (SMGT). Mature sperm cells can take up exogenous DNA molecules intrinsically and transfer them into the oocyte during fertilization. This study assessed the effect of dimethyl sulfoxide (DMSO) and electrolyte-free medium (EFM) on DNA uptake (EGFP-N1plasmid) in mouse sperm. Sperms cells cultured in human tubular fluid (HTF) without any treatment were considered as the control group. Sperms cells that were incubated in EFM and HTF with DNA/DMSO at 4°C were classified into EFM and HTF groups. Sperm motility and viability were assessed following treatment. In vitro fertilization (IVF) with sperm in all groups was performed. Fertilization, embryo development and GFP-positive blastocyst rates were analyzed and compared. The result showed that sperm motility and viability in EFM were better than those in the HTF group. The rate of development to reach the blastocyst stage and GFP-positive blastocysts was significantly higher in the EFM group compared with the HTF group (P<0.05). Our data demonstrate that sperm stored in the EFM group can improve the efficiency of SMGT for the generation of GFP-positive blastocysts.