The Effect of Curcumin on Aβ, Akt, and GSK3β in Brain and Retina of APP/PS1 Mice and Blood of Alzheimer’s Patients With Early Stage Disease

BackgroundCurcumin has multifunctional pharmacological properties, including anti-oxidant, anti-inflammatory, and anti-diabetic properties. We investigated whether curcumin can improve pathological changes associated with Alzheimer's disease, including amyloid β (Aβ), protein kinase B (PKB, also termed Akt), and glycogen synthase kinase 3β (GSK3β) levels and expression. MethodsAlzheimer’s transgenic APPSWE/PS1ΔE9 mice and wild type mice were treated with curcumin by intragastric administration for 2 weeks at 2 and 5 months of age. Aβ plaques and contents in the brain and retina were measured by immunohistochemistry and enzyme-linked immune sorbent assay, respectively, while the expression of Akt and GSK3β was tested by RNA isolation and quantitative real-time poly merase chain reaction. Bloods of patients with AD and age-matched health controls were used to determine the contents of Aβ, Akt and GSK3β. ResultsCurcumin treatment decreased Aβ accumulation in the early stages of AD at 5 months (p < 0.001). It also improved AD-associated pathological changes, including upregulation of Akt (p < 0.01) and downregulation of GSK3β (p < 0.01). In addition to AD-associated changes, the proinflammatory cytokine IL-1β was significantly decreased by curcumin treatment (p < 0.05). ConclusionsCurcumin can suppress Aβ accumulation during the early stages of AD, upregulate the expression of Akt, downregulate the expression of GSK3β, and inhibit the proinflammatory cytokine IL-1β. Curcumin can be used to improve pathological features of AD in the early stages of disease

Characterization of DDX4 Gene Expression in Human Cases with Non-Obstructive Azoospermia and in Sterile and Fertile Mice

Background: In mammals, spermatogenesis is the main process for male fertility that is initiated by spermatogonial stem cells (SSCs) proliferation. SSCs are unipotent progenitor cells accountable for transferring the genetic information to the following generation by differentiating to haploid cells during spermato-and spermiogenesis. DEAD-box helicase 4 (DDX4) is a specific germ cell marker and its expression pattern is localized to, spermatocytes, and spermatids. The expression in the SSCs on the basement membrane of the seminiferous tubules is low. Methods: Immunohistochemistry (IHC) and Fluidigm reverse transcriptase-poly-merase chain reaction (RT-PCR) were used to analyze the expression of DDX4 in testis tissue of fertile and sterile mice and human cases with non-obstructive azoospermia. Results: Our immunohistochemical findings of fertile and busulfan-treated mice showed expression of DDX4 in the basal and luminal compartment of seminiferous tubules of fertile mice whereas no expression was detected in busulfan-treated mice. The immunohistochemical analysis of two human cases with different levels of non-obstructive azoospermia revealed more luminal DDX4 positive cells. Conclusion: Our findings indicate that DDX4 might be a valuable germ cell marker for analyzing the pathology of germ cell tumors and infertility as global urological problems.

Multiplex Polymerase chain reaction to diagnose bloodstream infections in patients after cardiothoracic surgery

Background: Sepsis and other infectious complications are major causes of mortality and mor-bidity in patients after cardiac surgery. Whereas conventional blood culture (BC) suffers from low sensitivity as well as a reporting delay of approximately 48–72 h, real-time multiplex poly-merase chain reaction (PCR) based technologies like “SeptiFast” (SF) might offer a fast and reliable alternative for detection of bloodstream infections (BSI). The aim of this study was to compare the performance of SF with BC testing in patients suspected of having BSI after cardi-ac surgery. Methods: 279 blood samples from 168 individuals with suspected BSI were analyzed by SF and BC. After excluding results attributable to contaminants, a comparison between the two groups were carried out. Receiver operating characteristic (ROC) curves were generated to de-termine the accuracy of clinical and laboratory values for the prediction of positive SF results. Results: 14.7% (n = 41) of blood samples were positive using SF and 17.2% (n=49) using BC (n.s. [p>0.05]). In six samples SF detected more than one pathogen. Among the 47 microorgan-isms identified by SF, only 11 (23.4%) could be confirmed by BC. SF identified a higher num-ber of Gram-negative bacteria than BC did (28 vs. 12, χ2=7.97, p=0.005). The combination of BC and SF increased the number of detected microorganisms, including fungi, compared to BC alone (86 vs. 49, χ2=13.51, p<0.001). C-reactive protein (CRP) (21.7±11.41 vs. 16.0±16.9 mg/dl, p=0.009), procalcitonin (28.7±70.9 vs. 11.5±30.4 ng/dl, p=0.015), and interleukin 6 (IL 6) (932.3±1306.7 vs. 313.3±686.6 pg/ml, p=0.010) plasma concentrations were higher in patients with a positive SF result. Using ROC analysis, IL-6 (AUC 0.836) and CRP (AUC 0.804) showed the best predictive values for positive SF results. Conclusion: The SF test represent a valuable method for rapid etiologic diagnosis of BSI in patients after cardiothoracic surgery. In particular this method applies for individuals with sus-pected Gram-negative blood stream. Due to the low performance in detecting Gram-positive pathogens and the inability to determine antibiotic susceptibility, it should be used in addition to BC only.

Interaction Between ACE I/D and ACTN3 R557X Polymorphisms in Polish Competitive Swimmers

We hypothesized that the ACE ID / ACTN3 R577X genotype combination was associated with sprint and endurance performance. Therefore, the purpose of the present study was to determine the interaction between both ACE ID and ACTN3 R577X polymorphisms and sprint and endurance performance in swimmers. Genomic DNA was extracted from oral epithelial cells using GenElute Mammalian Genomic DNA Miniprep Kit (Sigma, Germany). All samples were genotyped using a real-time poly- merase chain reaction. The ACE I/D and the ACTN3 R577X genotype frequencies met Hardy-Weinberg expectations in both swimmers and controls. When the two swimmer groups, long distance swimmers (LDS) and short distance swimmers (SDS), were compared with control subjects in a single test, a significant association was found only for the ACE polymorphism, but not for ACTN3. Additionally, four ACE/ACTN3 combined genotypes (ID/RX, ID/XX, II/RX and II/XX) were statistically significant for the LDS versus Control comparison, but none for the SDS versus Control comparison. The ACE I/D and the ACTN3 R577X polymorphisms did not show any association with sprint swimming, taken individually or in combination. In spite of numerous previous reports of associations with athletic status or sprint performance in other sports, the ACTN3 R577X polymorphism, in contrast to ACE I/D, was not significantly associated with elite swimming status when considered individually. However, the combined analysis of the two loci suggests that the co-occurrence of the ACE I and ACTN3 X alleles may be beneficial to swimmers who compete in long distance races

Genetic Relationships Among Magnaporthe poae Isolates from Turfgrass Hosts and Relative Susceptibility of ‘Penncross’ and ‘Penn A-4’ Creeping Bentgrass

Isolates of Magnaporthe poae from turfgrass hosts were analyzed for mating type, genetic relatedness according to ITS sequences, reaction to a previously developed species-specific poly-merase chain reaction (PCR) assay, and virulence on two creeping bentgrass cultivars in growth chamber experiments. Analysis of internal transcribed spacer (ITS) sequences revealed three clades, designated A, B, and C. Clade A contained isolates of both mating types from creeping bentgrass, annual bluegrass, and Kentucky bluegrass. Clade B contained only mating type ‘A’ isolates from annual bluegrass, whereas Clade C contained only mating type ‘a’ isolates from creeping bentgrass. The M. poae PCR assay failed to positively identify several North Carolina isolates from annual bluegrass and creeping bentgrass. M. poae isolates from Kentucky blue-grass were most virulent toward creeping bentgrass in growth chamber experiments. Although isolates of M. poae are not host specific, certain ITS clades may have a limited host or geographical range. The improved creeping bentgrass cv. Penn A-4 was more susceptible to summer patch than cv. Penncross. Additional research is needed to develop methods for accurate diagnosis of summer patch and other patch diseases in creeping bentgrass and to determine how creeping bentgrass cultivars vary in their susceptibility to these root pathogens.

Comparison of Detection Methods for a Novel Tobamovirus Isolated from Florida Hibiscus

A novel tobamovirus recently was isolated from hibiscus in Florida. Serological and molecular methods, including enzyme-linked immunosorbent assay (ELISA), dot-blot immunoassay (DBIA), tissue-blot immunoassay (TBIA), and immunocapture reverse-transcription poly-merase chain reaction (IC-RT-PCR) were compared to evaluate their usefulness for diagnosis of this virus. Each method was tested with partially purified virus preparations and tissue samples from infected hibiscus and Chenopodium quinoa plants. Indirect ELISA was more sensitive than double-antibody sandwich (DAS)-ELISA with all samples tested. The Florida hibiscus virus was detectable in hibiscus leaves and bark up to 1:12,800 and 1:6,400 dilutions, respectively, by indirect ELISA and up to 1:3,200 and 1:400 dilutions by DAS-ELISA. End-point dilutions of partially purified virus preparations from indirect and DAS-ELISA were 4 and 31 ng/ml, respectively. Florida hibiscus virus was detected by DBIA in sap from hibiscus bark and leaves at dilutions up to 1:400 and 1:800, respectively, showing that DBIA was less sensitive than either ELISA method. The virus also was detected reliably by TBIA from leaves and bark of hibiscus plants. The most sensitive method was IC-RT-PCR, which could detect as little as 500 pg/ml of virus in partially purified preparations and was 16- and 32-fold more sensitive than DAS-ELISA with hibiscus bark and leaf extracts, respectively. Over 600 hibiscus samples were tested by various combinations of these methods to validate their usefulness.

Molecular and Pathogenicity Characterization of Sphaceloma manihoticola Isolates from South-Central Brazil

Isolates of Sphaceloma manihoticola, the asexual stage of Elsinoe brasiliensis, were collected from several regions of south-central Brazil. The isolates were obtained from samples of leaves, stems, and petioles of cassava (Manihot esculenta) and the weedy Euphorbia heterophylla (“amendoim bravo”) by directly plating infected tissue onto acidified potato dextrose agar. For pathogenicity studies, 19 isolates were inoculated onto each of two cassava cultivars, MBRA 703 as a susceptible cultivar and MBRA 12 as a resistant cultivar to S. manihoticola. MBRA 703, with the greatest pathogenicity to 58% (11) of the isolates, showed an intermediate pathogenic reaction to 16% (3) of the isolates, and was less pathogenic to 26% (5) of the isolates. MBRA 12, with a less pathogenic reaction to 63% (12) of the isolates, showed an intermediate pathogenic reaction to 16% (3) of the isolates, and was highly pathogenic to 21% (4) of the isolates. The isolates were verified as belonging to the genus Sphaceloma based on their morphological characteristics, including conidia and hyphae of monoconidial isolate. Conidia of isolates were small, thin-walled, ellipsoid to (rarely) globose, commonly with one or two gut-tules. Conidiophores were phialides, hyaline to slightly pigmented 0-to-1 septate; conidiophores from the weedy specie were phialides, hyaline to brown 0-to-2 septate producing hyaline conidia. The isolates also were verified as belonging to the genus Sphaceloma by using a poly-merase chain reaction (PCR) assay, which detected a 645-bp band in all isolates except two (1 and 6) for which the PCR product had 600 bp. Digestion of the amplified product with the enzymes MspI and CfoI allowed differences to be detected in restriction patterns among isolates. A homogeneous banding pattern was obtained for 17 of the isolates but a different restriction pattern was obtained for isolates 1 and 6 of E. heterophylla. This suggests the possibility of another species within this group of isolates. The results indicate the presence of pathogenic variation among isolates of the fungus and an isolate-host interaction, because statistically significant differences were observed between the two cassava cultivars in response to inoculation with the isolates of S. manihoticola.

The Xanthomonas Type III Effector Protein AvrBs3 Modulates Plant Gene Expression and Induces Cell Hypertrophy in the Susceptible Host

Xanthomonas campestris pv. vesicatoria bacteria expressing the type III effector protein AvrBs3 induce a hypersensitive response in pepper plants carrying the resistance gene Bs3. Here, we report that infection of susceptible pepper and tomato plants leads to an AvrBs3-dependent hypertrophy of the mesophyll tissue. Agrobacterium-mediated transient expression of the avrBs3 gene in tobacco and potato plants resulted in a similar phenotype. Induction of hypertrophy was shown to depend on the repeat region, nuclear localization signals, and acidic transcription activation domain (AAD) of AvrBs3, suggesting that the effector modulates the host's transcriptome. To search for host genes regulated by AvrBs3 in an AAD-dependent manner, we performed a cDNA-amplified fragment length polymorphism analysis of pepper mRNA populations. Thirteen AvrBs3-induced transcripts were identified and confirmed by reverse transcriptase-poly-merase chain reaction. Sequence analysis revealed homologies to auxin-induced and expansinlike genes, which play a role in cell enlargement. These results suggest that some of the AvrBs3-induced genes may be involved in hypertrophy development and that xanthomonads possess type III effectors that steer host gene expression.

Geographic Distribution and Molecular Variation of Isolates of Three Whitefly-Borne Closteroviruses of Cucurbits: Lettuce Infectious Yellows Virus, Cucurbit Yellow Stunting Disorder Virus, and Beet Pseudo-Yellows Virus

The geographic incidence and molecular variation of three whitefly-borne closteroviruses (lettuce infectious yellows virus [LIYV], cucurbit yellow stunting disorder virus [CYSDV], and beet pseudo-yellows virus [BPYV]) were studied in cucurbits collected from several distinct geographic locations. Of 498 samples analyzed, none were found to be infected by LIYV. Sixty-nine samples collected in the Middle East and Mediterranean Europe were found infected by CYSDV, and twelve samples from Crete and Italy were infected by BPYV. Reverse-transcription poly-merase chain reaction of a portion of the heat shock protein 70 homolog coding region, followed by single-strand conformation polymorphism and nucleotide sequence analysis, was used to estimate the intra- and inter-isolate molecular variability. These analyses showed that each BPYV and CYSDV isolate was composed of a population of sequence variants with a nucleotide identity greater than 98%. CYSDV isolates could be divided into two divergent groups. Group I was only composed of isolates from Spain, Jordan, and Turkey, and group II isolates were predominantly found in Saudi Arabia. Nucleotide identity between isolates of the same group was greater than 99%, whereas identity between both groups was less than 92%. All BPYV isolates showed a nucleotide identity greater than 98%.