Resolving the titer of murine cytomegalovirus by plaque assay using the M2-10B4 cell line and a low viscosity overlay.

BACKGROUND: Murine cytomegalovirus (MCMV) is increasingly used as an infectious model to investigate host-pathogen interactions in mice. Detailed methods have been published for using primary murine embryonic fibroblasts (MEFs) for preparing stocks and determining viral titers of MCMV. For determining the titer of MCMV by plaque assay, these methods rely on a high viscosity media that restricts viral spreading through the supernatant of the culture, but is also usually too viscous to pipet. Moreover, MEFs must be repeatedly generated and can vary widely from batch-to-batch in purity, proliferation rates, and the development of senescence. In contrast, the M2-10B4 bone marrow stromal cell line (ATCC # CRL-1972), which is also permissive for MCMV, has been reported to produce high-titer stocks of MCMV and has the considerable advantages of growing rapidly and consistently. However, detailed methods using these cells have not been published. METHODS: We modified existing protocols to use M2-10B4 cells for measuring MCMV titers by plaque assay. RESULTS: We found that MCMV plaques could be easily resolved on monolayers of M2-10B4 cells. Moreover, plaques formed normally even when cultures of M2-10B4 cells were less than 50% confluent on the day of infection, as long as we also used a reduced viscosity overlay. CONCLUSIONS: Overall, our protocol enabled us to use a consistent cell line to assess viral titers, rather than repeatedly producing primary MEFs. It also allowed us to start the assay with 4-fold fewer cells than would be required to generate a confluent monolayer, reducing the lead-time prior to the start of the assay. Finally, the reduced viscosity CMC could be handled by pipet and did not need to be pre-mixed with media, thus increasing its shelf-life and ease-of-use. We describe our results here, along with detailed protocols for the use of the M2-10B4 cell lines to determine the titer and grow stocks of MCMV

Applying naive bayesian networks to disease prediction: A systematic review

Introduction: Naive Bayesian networks (NBNs) are one of the most effective and simplest Bayesian networks for prediction. Objective: This paper aims to review published evidence about the application of NBNs in predicting disease and it tries to show NBNs as the fundamental algorithm for the best performance in comparison with other algorithms. Methods: PubMed was electronically checked for articles published between 2005 and 2015. For characterizing eligible articles, a comprehensive electronic searching method was conducted. Inclusion criteria were determined based on NBN and its effects on disease prediction. A total of 99 articles were found. After excluding the duplicates (n= 5), the titles and abstracts of 94 articles were skimmed according to the inclusion criteria. Finally, 38 articles remained. They were reviewed in full text and 15 articles were excluded. Eventually, 23 articles were selected which met our eligibility criteria and were included in this study. Result: In this article, the use of NBN in predicting diseases was described. Finally, the results were reported in terms of Accuracy, Sensitivity, Specificity and Area under ROC curve (AUC). The last column in Table 2 shows the differences between NBNs and other algorithms. Discussion: This systematic review (23 studies, 53,725 patients) indicates that predicting diseases based on a NBN had the best performance in most diseases in comparison with the other algorithms. Finally in most cases NBN works better than other algorithms based on the reported accuracy. Conclusion: The method, termed NBNs is proposed and can efficiently construct a prediction model for disease. � 2016 Mostafa Langarizadeh and Fateme Moghbeli

Memory T Cells Specific for Murine Cytomegalovirus Re-Emerge after Multiple Challenges and Recapitulate Immunity in Various Adoptive Transfer Scenarios.

Reconstitution of CMV-specific immunity after transplant remains a primary clinical objective to prevent CMV disease, and adoptive immunotherapy of CMV-specific T cells can be an effective therapeutic approach. Because of viral persistence, most CMV-specific CD8(+) T cells become terminally differentiated effector phenotype CD8(+) T cells (TEFF). A minor subset retains a memory-like phenotype (memory phenotype CD8(+) T cells [TM]), but it is unknown whether these cells retain memory function or persist over time. Interestingly, recent studies suggest that CMV-specific CD8(+) T cells with different phenotypes have different abilities to reconstitute sustained immunity after transfer. The immunology of human CMV infections is reflected in the murine CMV (MCMV) model. We found that human CMV- and MCMV-specific T cells displayed shared genetic programs, validating the MCMV model for studies of CMV-specific T cells in vivo. The MCMV-specific TM population was stable over time and retained a proliferative capacity that was vastly superior to TEFF. Strikingly, after transfer, TM established sustained and diverse T cell populations even after multiple challenges. Although both TEFF and TM could protect Rag(-/-) mice, only TM persisted after transfer into immune replete, latently infected recipients and responded if recipient immunity was lost. Interestingly, transferred TM did not expand until recipient immunity was lost, supporting that competition limits the Ag stimulation of TM. Ultimately, these data show that CMV-specific TM retain memory function during MCMV infection and can re-establish CMV immunity when necessary. Thus, TM may be a critical component for consistent, long-term adoptive immunotherapy success

The Masticatory Contractile Load Induced Expression and Activation of Akt1/PKBα in Muscle Fibers at the Myotendinous Junction within Muscle-Tendon-Bone Unit

The cell specific detection of enzyme activation in response to the physiological contractile load within muscle-tendon-bone unit is essential for understanding of the mechanical forces transmission from muscle cells via tendon to the bone. The hypothesis that the physiological mechanical loading regulates activation of Akt1/PKBα at Thr308 and at Ser473 in muscle fibers within muscle-tendon-bone unit was tested using quantitative immunohistochemistry, confocal double fluorescence analysis, and immunoblot analysis. In comparison to the staining intensities in peripheral regions of the muscle fibers, Akt1/PKBα was detected with a higher staining intensity in muscle fibers at the myotendinous junction (MTJ) areas. In muscle fibers at the MTJ areas, Akt1/PKBα is dually phosphorylated at Thr308 and Ser473. The immunohistochemical results were confirmed by immunoblot analysis. We conclude that contractile load generated by masticatory muscles induces local domain-dependent expression of Akt1/PKBα as well as activation by dually phosphorylation at Thr308 and Ser473 in muscle fibers at the MTJ areas within muscle-tendon-bone unit

Role of volatile compounds emitted from the first and second trophic levels in host finding behavior of Tamarixia radiata (Hym.: Eulophidae)

Odors play an important role in the seeking behavior of wasp parasitoids and enhances their performance in biological control programs. This study was intended to determine the olfactory response of Tamarixia radiata (Waterston) to chemical cues that emanates from various stages of Asian citrus psyllid, Diaphorina citri Kuwayama, in Y-tube olfactometer in laboratory conditions (27 ± 1°C, 60 ± 10% RH, 1600 lux light intensity and constant airflow of 300 ml/min). The effect of green leaf volatiles (GLVs) and herbivore-induced plant volatiles (HIPVs) of four varieties of the rutaceous plants, Mexican lime, Citrus aurantifolia, Campbell Valencia orange, C. sinensis, Marsh grapefruit, C. Paradisi, and sour orange, C. aurantium, were also evaluated based on separately release of 30 females and 30 males of T. radiata. The results indicated that the females were significantly attracted to the volatiles emanating from D. citri nymphs and HIPVs resulted from the feeding of nymphs and adults of D. citri. The males were not responsive to the odors emanating from various stages of D. citri, GLVs or HIPVs. It is found that the female parasitoid wasps use chemical cues emanating from the nymphs of Asian citrus psyllids and HIPVs for host seeking. Using the synthetic volatile compounds could increase the viability of the biological control agents in integrated pest management programs

A method for body fat composition analysis in abdominal magnetic resonance images via self-organizing map neural network

Introduction: The present study aimed to suggest an unsupervised method for the segmentation of visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) in axial magnetic resonance (MR) images of the abdomen. Materials and Methods: A self-organizing map (SOM) neural network was designed to segment the adipose tissue from other tissues in the MR images. The segmentation of SAT and VAT was accomplished using a new level set method called distance regularized level set evolution (DRLSE). To evaluate the suggested method, the whole-body abdominal MRI was performed on 23 subjects, and three slices were selected for each case. Results: The results of the automatic segmentation were compared with those of the manual segmentation and previous artificial intelligent methods. According to the results, there was a significant correlation between the automatic and manual segmentation results of VAT and SAT. Conclusion: As the findings indicated, the suggested method improved detection of body fat. In this study, a fully automated abdominal adipose tissue segmentation algorithm was suggested, which used the SOM neural network and DRLSE level set algorithm. The proposed methodology was concluded to be accurate and robust with a significant advantage over the manual and previous segmentation methods in terms of speed and accuracy. © 2018, Mashhad University of Medical Sciences

Bi-allelic truncating variants in CASP2 underlie a neurodevelopmental disorder with lissencephaly.

Lissencephaly (LIS) is a malformation of cortical development due to deficient neuronal migration and abnormal formation of cerebral convolutions or gyri. Thirty-one LIS-associated genes have been previously described. Recently, biallelic pathogenic variants in CRADD and PIDD1, have associated with LIS impacting the previously established role of the PIDDosome in activating caspase-2. In this report, we describe biallelic truncating variants in CASP2, another subunit of PIDDosome complex. Seven patients from five independent families presenting with a neurodevelopmental phenotype were identified through GeneMatcher-facilitated international collaborations. Exome sequencing analysis was carried out and revealed two distinct novel homozygous (NM_032982.4:c.1156delT (p.Tyr386ThrfsTer25), and c.1174 C > T (p.Gln392Ter)) and compound heterozygous variants (c.[130 C > T];[876 + 1 G > T] p.[Arg44Ter];[?]) in CASP2 segregating within the families in a manner compatible with an autosomal recessive pattern. RNA studies of the c.876 + 1 G > T variant indicated usage of two cryptic splice donor sites, each introducing a premature stop codon. All patients from whom brain MRIs were available had a typical fronto-temporal LIS and pachygyria, remarkably resembling the CRADD and PIDD1-related neuroimaging findings. Other findings included developmental delay, attention deficit hyperactivity disorder, hypotonia, seizure, poor social skills, and autistic traits. In summary, we present patients with CASP2-related ID, anterior-predominant LIS, and pachygyria similar to previously reported patients with CRADD and PIDD1-related disorders, expanding the genetic spectrum of LIS and lending support that each component of the PIDDosome complex is critical for normal development of the human cerebral cortex and brain function