Detection of Microsatellite Instability by High-Resolution Melting Analysis in Colorectal Cancer

Background: Colorectal cancer is the third most common cancer worldwide. MSI is a molecular marker of a deficient MMR system and happens in almost 15% of CRCs. Because of a wide frequency of MSI⁺ CRC in Iran compared to other parts of the world, the importance of screening for this type of cancer is highlighted. Methods: The most common MSI detection technique is a fluorescent PCR-based method in which fragments are analyzed by CE. This technique is very time-consuming, difficult, and expensive. We sought to develop and evaluate a proper method with high accuracy, specificity, and sensitivity to screen the MSI⁺ CRC. A HRM analysis procedure is relying on the analysis of the melting curve attributes. Low cost, feasibility, high specificity, and sensitivity are outstanding attributes of HRM analysis. Results: Five mononucleotide microsatellite markers, including BAT-25, BAT-26, NR-21, NR-24, and NR-27, in 25 archival CRC tumor tissue samples were compared with normal tissue adjacent using HRM method. The specificity and sensitivity of BAT-25 with HRM method were 100% compared to CE, while other markers had lower sensitivity. However, when all the markers were considered together, the sensitivity and specificity became 100%. The number of MSI⁺ samples was 56%, which shows a higher ratio than previous Iranian studies. The highest MSI was related to BAT-26 (52%). Conclusion: The HRM method is much simpler and more cost-effective than current MSI techniques, and its sensitivity and accuracy are comparable. Therefore, it can serve as an alternative method in cases where CE is unavailable. Key Words: Biomarkers, Microsatellite instability, Colorectal cancer     

Comparative Assessment of Neutrophil Gelatinase-Associated Lipocalin (NGAL) and Cystatin C as Early Biomarkers for Early Detection of Renal Failure in Patients with Hypertension

Background:

Hypertension is one the most common causes of chronic kidney disease (CKD). One of the major concerns in hypertensive patients is early detection of renal disorders. In the past, serum creatinine (Scr) concentration was used as a marker of kidney function, but it proffers a late reflection of reduced glomerular filtration rate. Cystatin C and neutrophil gelatinase-associated lipocalin (NGAL) have been recently proven to be useful for quantification of CKD. Therefore, we compared the diagnostic value of NGAL with cystatin C and creatinine to evaluate kidney function in hypertensive patients.

Methods:

In this study, 42 hypertensive patients and 30 healthy volunteers were recruited. Serum cystatin C (Scys C) and plasma NGAL were measured using ELISA method. Creatinine, urea, hemoglobin, fibrinogen, and C-reactive protein were measured according to the routine methods. Estimated glomerular filtration rate (eGFR) was considered as the gold standard method (cut-off value of < 78 ml/min/1.73 m².

Results:

In the patient group, plasma NGAL, cystatin C, and creatinine were all significantly correlated with eGFR, and plasma NGAL correlated best with eGFR. Receiver-operating characteristics analysis indicated that plasma NGAL was a better indicator than creatinine and cystatin C for predicting a GFR < 78 ml/min/1.73 m². The sensitivity and specificity for NGAL were 96% and 100%, for cystatin C were 92% and 60% and for creatinine were 76% and 47%, respectively.

Conclusion:

Plasma NGAL demonstrated a higher diagnostic value to detect kidney impairment in the early stages of CKD as compared to Scys C and Scr in hypertensive patients.Key Words: Neutrophil gelatinase-associated lipocalin (NGAL), Cystatin C, Creatinine, Hypertension     

Effects of ammonia on growth and molting of Litopenaeus vannamei postlarvae reared under two salinity levels

ABSTRACT

Litopenaeus vannamei postlarvae were exposed to 0, 6, 13, and 19 mg/L total ammonia nitrogen (TAN) treatments. After 45 days, shrimp weight and length were lowest under TAN concentrations of 13 and 19 mg/L (P ≤ 0.05). Maximum weight gain was observed in control and 6 mg/L treatments. Mortality was highest (80.55 ± 4.80%) under 19 mg/L reared in 35 ppt salinity. Average intermolt periods of PLs exposed to 0, 6, 13, and 19 mg/L TAN were 11.5 ± 0.7, 10.8 ± 1.3, 9.4 ± 1.0, and 8.7 ± 0.6 days under 35 ppt and 11.1 ± 0.5, 10.7 ± 0.6, 10.1 ± 0.5, and 9.5 ± 0.2 days under 45 ppt salinity. Although TAN increased postlarvae molting frequency, its negative effects on the shrimp growth and survival of PLs was directly linked to its concentration and exposure duration. Higher salinity reduces the effects of ammonia and increases the survival.     

Simultaneous selection for seed and forage production in cocksfoot (Dactylis glomerata): application of drought tolerance and susceptibility indices

An experiment was conducted to estimate drought tolerance of some cocksfoot genotypes based on seed and forage yield and related indices (including tolerance index, mean productivity, geometric mean productivity, reduction percentage index, stress tolerance index and stress susceptibility index) and to identify relationships between measured traits. Twenty‐five genotypes were evaluated in the field under control and drought‐stress environments over 2 years. The results revealed a high variation for seed yield and forage yield and the measured indices under both moisture conditions. No correlation was observed between seed yield and forage yield under the control treatment, but this correlation was positively significant under the water‐stress treatment. Drought‐tolerant and susceptible genotypes were identified based on seed and forage yield performance. Genotypes 2, 4 and 7 had high seed yield, while genotypes 7 and 10 had high forage yield under both stressed and non‐stressed conditions and were identified as the most tolerant genotypes in each category. The results also revealed that genotypes 1, 2, 4, 6, 7, 8 and 24 had relatively high seed and forage yield in both control and drought‐stressed conditions. These genotypes can be used for further studies to improve both seed and forage yields concurrently.     

Production of monoclonal antibody against recombinant bovine sex-determining region Y (SRY) and their preferential binding to Y chromosome-bearing sperm

Separation of X and Y chromosome-bearing sperm is an appropriate method for the selection of desired sex of offspring to increase the profit in livestock industries. The purpose of this study was the production of a monoclonal antibody against recombinant bovine sex-determining region Y protein for separation Y sperm. The hybridoma cells from splenocytes of immunized female's balb/C mice and Sp2/0 cells were made. The binding affinity of our monoclonal antibody (mAbSRY2) was compared with mouse monoclonal SRY-15. The Western blot method indicated that mAbSRY2 successfully detected the rbSRY protein. The specificity and sensitivity of mAbSRY2 is comparable to SRY-15 commercially ones. The SRY gene in 100% of bull semen contains the Y chromosome that had the strongest binding affinity to mAbSRY2 was synthesized. In other words, the binding affinity of semen contains the X sperms near the negative control. In general, this immunological method can help to separate X from Y sperms. However, the mAbSRY2 is bind to Y-bearing sexed sperm, but in the future; the sexed sperms need to apply in farms.     

Micropropagation of Prunus scoparia,a Suitable Rootstock for Almond under Drought Conditions

Prunus scoparia is a wild deciduous shrub, usually living on dry calcareous soils of the rocky mountains and has been used as a grafting rootstock for domesticated almonds to provide drought resistance. In the current study, micropropagation ability of P. scoparia was investigated using cytokinin and auxin. Uniform nodal shoot pieces (3–5 cm in length) of seedlings were used as explants. The explants were disinfected with 10% sodium hypochlorite solution. For adventitious shoot induction and proliferation, Murashige and Skoog (MS) media containing 7.00 g/l agar and 30.00 g/l sucrose containing five concentrations of benzyl adenine (BA) (0.00, 0.50, 1.00, 2.00, and 4.00 mg/1) and also containing six concentrations of Thidiazuron (TDZ) (0.00, 0.50, 1.00, 2.00, 5.00, and 7.00 mg/1) were compared. For rooting, in vitro shoots (2–3 cm) were transferred into ½ MS medium supplemented with 30 g/l sucrose, 7.50 g/l agar, and different concentrations of IBA (0.00, 0.25, 0.50, and 1.00 mg/l) and NAA (0.00, 0.25, 0.50, and 1.00 mg/l). Based on the results obtained for shoot proliferation, only 2.00 and 4.00 mg/l BA and 2.00 mg/l TDZ concentrations generated shoots, while other treatments did not show shoot proliferation. Among the three treatments that generated shoots, the best results for shoot number, leaf number, and leaf color quality were observed in media containing 2.00 mg/l TDZ. Based on the results obtained for rooting, the effect of IBA concentrations on the rooting percentage, root number, and root length was significant. Among IBA concentrations, only 0.50 mg/l IBA induced rooting, while there was no rooting in the media containing other IBA concentrations. None of the NAA concentrations showed rooting. In conclusion, MS culture medium supplemented with 2.00 mg/l TDZ and ½ MS culture medium supplemented with 0.50 mg/l IBA are suggested for in vitro shoot proliferation and rooting of P. scoparia, respectively. The results presented herein could be used for in vitro selection and micropropagation of P. scoparia.     

Opioid receptors of the central amygdala and morphine-induced antinociception

BACKGROUND: The amygdala is a forebrain region, which is known as a modulator of pain sensation. The amygdala, particularly the central nucleus, has high concentrations of enkephalins relative to dynorphins and has high concentrations of opioid receptors. We here studied the role of central nuclei of amygdala in morphine antinociception. METHODS: In this study, we used 130 male Wistar rats (200-250 g). Bilateral two guide cannula were inserted into central nuclei of amygdala. The drugs were administrated via intra central-amygdala and intraperitoneal. The antinociceptive effect was measured by formalin test. RESULTS: Bilateral microinjections of morphine (50 and 100 microg/rat) into the central nuclei of amygdala elicited powerful suppression of nociceptive behaviors in both phases of formalin test. The intraperitoneal administration of naloxone (1 and 2 mg/kg) decreased significantly the antinociception induced by the intra-amygdaloid injection of morphine. Our data also showed that microinjection of naloxone (50 and 100 microg/rat) into the central nuclei of amygdala could reduce the analgesic effects of systemic morphine (7 mg/kg). On the other hand, bilateral neurotoxic lesions of the central nuclei of amygdala attenuated the antinociception induced by subcutaneous or intra-amygdaloid injection of morphine. CONCLUSION: These findings suggest that morphine analgesia in the formalin test depends on ascending connections to the forebrain, probably the amygdala.     

Willingness to travel to avoid recreation conflicts in Danish forests

Conflicts among forest visitors have direct effects on the quality of a recreational experience. As the number of visitors to forests close to residential areas increases, as well as the number of different activities, so does the potential for perceived conflicts. According to the literature, expanding knowledge of conflict characteristics and their causes is important for recreation planners and managers who aim to reduce conflicts.In the present study, different forest user groups were identified and categorised according to their pursued activities, and for each group, causes of conflict were identified. Furthermore, a choice experiment was constructed to estimate the distance visitors are willing to travel to encounter few visitors as opposed to many visitors, and thereby potentially experience fewer conflicts. Comparing the marginal willingness to travel (WTT) of different user groups suggests that some groups have a WTT further than the average to reach a forest with ‘Few’ visitors. The average WTT to reach a forest area with ‘Few’ visitors. ‘Mountain bikers,’ ‘Peace and nature lovers’ and ‘Horse riders’ are willing to travel 4 km more than the average per visit to reach a less crowded forest. At the other end of the scale, we find that people who are doing physical exercise are willing to travel 2 km less than the average to reach a less crowded forest.     

Conductive 3D structure nanofibrous scaffolds for spinal cord regeneration

The complex nature of spinal cord injuries has provided much inspiration for the design of novel biomaterials and scaffolds which are capable of stimulating neural tissue repair strategies. Recently, conductive polymers have gained much attention for improving the nerve regeneration. In our previous study, a three-dimensional (3D) structure with reliable performance was achieved for electrospun scaffolds. The main purpose in the current study is formation of electrical excitable 3D scaffolds by appending polyaniline (PANI) to biocompatible polymers. In this paper, an attempt was made to develop conductive nanofibrous scaffolds, which can simultaneously present both electrical and topographical cues to cells. By using a proper 3D structure, two kinds of conductive scaffolds are compared with a non-conductive scaffold. The 3D nanofibrous core-sheath scaffolds, which are conductive, were prepared with nanorough sheath and aligned core. Two different sheath polymers, including poly(lactic-co-glycolic acid) PLGA and PLGA/PANI, with identical PCL/PANI cores were fabricated. Nanofibers of PCL and PLGA blends with PANI have fiber diameters of 234±60.8 nm and 770±166.6 nm, and conductivity of 3.17×10^-5 S/cm and 4.29×10^-5 S/cm, respectively. The cell proliferation evaluation of nerve cells on these two conductive scaffolds and previous non-conductive scaffolds (PLGA) indicate that the first conductive scaffold (PCL/ PANI-PLGA) could be more effective for nerve tissue regeneration. Locomotor scores of grafted animals by developed scaffolds showed significant performance of non-conductive 3D scaffolds. Moreover, the animal studies indicated the ability of two new types of conductive scaffolds as spinal cord regeneration candidates.     

Wide hybridization and introgression breeding in safflower: Effectiveness of different selection methods

Possibility of using interspecific hybridization to increase genetic diversity of drought tolerance in safflower (Carthamus tinctorius L.) and effectiveness of different selection methods is poorly understood. The main objectives of this study were to evaluate the effectiveness of (a) interspecific hybridization to expand genetic diversity in safflower for agronomic traits and drought tolerance in advance generations and (b) different selection methods (direct vs. indirect) for improving seed yield. Interspecific hybridizations were performed between species of C. tinctorius × C. palaestinus (TP), C. palaestinus × C. oxyacantha (PO) and C. tinctorius × C. oxyacantha (TO). Based on the field screening of genotypes in F3 generation, four groups of selected genotypes (derived from combination of direct and indirect selection under both drought and normal environments along with selection based on drought tolerance index (STI)) were evaluated in F4 and F5 generations. Indirect selection for seed yield was significantly more efficient than direct selection. Selection in stress environment resulted to more efficiency than selection in normal environment. No significant difference was observed between direct selection and selection based on STI. The result showed that TP was the best population to combine high seed yield and drought tolerance in safflower.