Development and validation of a loop-mediated isothermal amplification assay for the detection of <Emphasis Type="Italic">Mycoplasma bovis</Emphasis> in mastitic milk

Mycoplasma mastitis is often difficult to control due to a lack of rapid and accurate diagnostic tools. The aim of the current study was to develop a loop-mediated isothermal amplification (LAMP) assay for the detection of Mycoplasma bovis (M. bovis) in mastitic milk. The assay was developed using primers designed for three different target genes: uvrC, 16S rRNA, and gyrB, and validated using mastitic milk samples previously found positive for the target pathogen. Specificity of the developed assay was determined by testing cross-reactivity of LAMP primers against closely related bovine mastitis bacterial pathogens. The sensitivity was found to be higher compared to conventional polymerase chain reaction (PCR). The LAMP assay was also capable of detecting M. bovis in PCR-negative milk samples of cows with clinical mastitis. The uvrC primers were found to be more sensitive, while gyrB primers were more specific; however, 16S rRNA primers were less specific and sensitive compared to either uvrC or gyrB primers. Cohen’s kappa values for uvrC, gyrB, and 16S rRNA primers used in the LAMP assays were 0.940, 0.970, and 0.807, respectively. There was a high level of agreement between the test results and the true-disease status as indicated by the receiver operating characteristic (ROC) curve. Our findings suggest that the newly developed LAMP assays targeting the uvrC and gyrB genes could be a useful tool for rapid and accurate diagnosis of mastitis caused by M. bovis.     

When Color Really Matters: Horticultural Performance and Functional Quality of High-Lycopene Tomatoes

Introgression of spontaneous or induced mutations has been used to increase the levels and diversify the profile of antioxidants in many fruits including tomato. The high-pigment (hp) and old-gold (og) alleles exemplify this approach as attractive genetic resources suitable to inbred elite high-lycopene (HLY) tomato lines with improved color and nutritional attributes. Although several studies have been published on HLY tomatoes, a systematic analysis of the information on their agronomic performances, processing features, and functional quality is lacking, leaving room for the assumption of their poor competitiveness with conventional tomato cultivars and limiting their agricultural diffusion. Therefore, the aim of this study is to critically review the most important agronomic, horticultural, and functional traits of HLY tomatoes, as well as the advances in some emerging (pre)industrial applications. Field experiments performed in different countries showed that most available HLY lines are productive, vigorous, with excellent foliage cover and with morphologically acceptable fruit. Tomato yield of HLY genotypes ranged from ～30 to ～178 t/ha exceeding, in some trials, that of highly productive cultivars. Red-ripe fruits of most HLY lines showed commercially suitable soluble solids and titratable acidity, in addition to increased levels of lycopene (up to 440 mg/kg fw) and other bioactive phytochemicals (mainly flavonoids and vitamin C) compared to their near isogenic conventional counterparts. Innovative (pre)industrial uses of HLY tomato include the following: (1) production of HLY sauces, juices, and powders; (2) supercritical-CO₂ extraction of lycopene containing oleoresins; and (3) preparation of lycopene rich micro- and nano-carriers with improved stability and specific tissue delivery. In turn, the use of these innovative high-quality ingredients in the formulation of lycopene fortified foods, cosmetic products, nutraceuticals, and pharmaceuticals has been proposed as the basis of a novel highly profitable tomato product chain.     

Novel autosomal recessive nonsyndromic hearing impairment locus DFNB90 maps to 7p22.1-p15.3

A novel locus DFNB90 was mapped to 7p22.1-p15.3 by carrying out a genome scan in a multigenerational consanguineous family from Pakistan with autosomal recessive nonsyndromic hearing impairment (ARNSHI).DFNB90 is the eighth ARNSHI locus mapped to chromosome 7. A multipoint LOD score of 4.0 was obtained at a number of SNP marker loci spanning from rs1468996 (chromosome 7: 5.7 Mb) tors957960 (chromosome 7: 18.8 Mb). The 3-unit support interval and the region of homozygosity for DFNB90 spans from markers rs1553960 (chromosome 7: 4.9 Mb) to rs206198 (chromosome 7: 20.3 Mb). Candidate genes ACTB, BZW, OCM, MACC1, NXPH1, PRPS1L1, RAC1 and RPA3, which lie within the DFNB90 region, were sequenced and no potentially causal variants were identified.     

A Lightweight Distributed Framework for Computational Offloading in Mobile Cloud Computing

The latest developments in mobile computing technology have enabled intensive applications on the modern Smartphones. However, such applications are still constrained by limitations in processing potentials, storage capacity and battery lifetime of the Smart Mobile Devices (SMDs). Therefore, Mobile Cloud Computing (MCC) leverages the application processing services of computational clouds for mitigating resources limitations in SMDs. Currently, a number of computational offloading frameworks are proposed for MCC wherein the intensive components of the application are outsourced to computational clouds. Nevertheless, such frameworks focus on runtime partitioning of the application for computational offloading, which is time consuming and resources intensive. The resource constraint nature of SMDs require lightweight procedures for leveraging computational clouds. Therefore, this paper presents a lightweight framework which focuses on minimizing additional resources utilization in computational offloading for MCC. The framework employs features of centralized monitoring, high availability and on demand access services of computational clouds for computational offloading. As a result, the turnaround time and execution cost of the application are reduced. The framework is evaluated by testing prototype application in the real MCC environment. The lightweight nature of the proposed framework is validated by employing computational offloading for the proposed framework and the latest existing frameworks. Analysis shows that by employing the proposed framework for computational offloading, the size of data transmission is reduced by 91%, energy consumption cost is minimized by 81% and turnaround time of the application is decreased by 83.5% as compared to the existing offloading frameworks. Hence, the proposed framework minimizes additional resources utilization and therefore offers lightweight solution for computational offloading in MCC.     

FTSH4 and OMA1 mitochondrial proteases reduce moderate heat stress-induced protein aggregation

The threat of global warming makes uncovering mechanisms of plant tolerance to long-term moderate heat stress particularly important. We previously reported that Arabidopsis (Arabidopsis thaliana) plants lacking mitochondrial proteases FTSH4 or OMA1 suffer phenotypic changes under long-term stress of 30°C, while their growth at 22°C is not affected. Here we found that these morphological and developmental changes are associated with increased accumulation of insoluble mitochondrial protein aggregates that consist mainly of small heat-shock proteins (sHSPs). Greater accumulation of sHSPs in ftsh4 than oma1 corresponds with more severe phenotypic abnormalities. We showed that the proteolytic activity of FTSH4, and to a lesser extent of OMA1, as well as the chaperone function of FTSH4, is crucial for protecting mitochondrial proteins against aggregation. We demonstrated that HSP23.6 and NADH dehydrogenase subunit 9 present in aggregates are proteolytic substrates of FTSH4, and this form of HSP23.6 is also a substrate of OMA1 protease. In addition, we found that the activity of FTSH4 plays an important role during recovery from elevated to optimal temperatures. Isobaric tags for relative and absolute quantification (iTRAQ)-based proteomic analyses, along with identification of aggregation-prone proteins, implicated mitochondrial pathways affected by protein aggregation (e.g. assembly of complex I) and revealed that the mitochondrial proteomes of ftsh4 and oma1 plants are similarly adapted to long-term moderate heat stress. Overall, our data indicate that both FTSH4 and OMA1 increase the tolerance of plants to long-term moderate heat stress by reducing detergent-tolerant mitochondrial protein aggregation.

Mitochondrial proteases prevent accumulation of insoluble protein aggregates and protect Arabidopsis plants against long-term moderate heat stress.     

ImShot: An Open-Source Software for Probabilistic Identification of Proteins In Situ and Visualization of Proteomics Data

Imaging mass spectrometry (IMS) has developed into a powerful tool allowing label-free detection of numerous biomolecules in situ. In contrast to shotgun proteomics, proteins/peptides can be detected directly from biological tissues and correlated to its morphology leading to a gain of crucial clinical information. However, direct identification of the detected molecules is currently challenging for MALDI–IMS, thereby compelling researchers to use complementary techniques and resource intensive experimental setups. Despite these strategies, sufficient information could not be extracted because of lack of an optimum data combination strategy/software. Here, we introduce a new open-source software ImShot that aims at identifying peptides obtained in MALDI–IMS. This is achieved by combining information from IMS and shotgun proteomics (LC–MS) measurements of serial sections of the same tissue. The software takes advantage of a two-group comparison to determine the search space of IMS masses after deisotoping the corresponding spectra. Ambiguity in annotations of IMS peptides is eliminated by introduction of a novel scoring system that identifies the most likely parent protein of a detected peptide in the corresponding IMS dataset. Thanks to its modular structure, the software can also handle LC–MS data separately and display interactive enrichment plots and enriched Gene Ontology terms or cellular pathways. The software has been built as a desktop application with a conveniently designed graphic user interface to provide users with a seamless experience in data analysis. ImShot can run on all the three major desktop operating systems and is freely available under Massachusetts Institute of Technology license.     

Mutations in <Emphasis Type="Italic">WDR62</Emphasis> gene in Pakistani families with autosomal recessive primary microcephaly

Background  

Autosomal recessive primary microcephaly is a disorder of neurogenic mitosis that causes reduction in brain size. It is a rare heterogeneous condition with seven causative genes reported to date. Mutations in WD repeat protein 62 are associated with autosomal recessive primary microcephaly with cortical malformations. This study was initiated to screen WDR62 mutations in four consanguineous Pakistani families with autosomal recessive primary microcephaly.     

A novel cyclic opioid peptide analog showing high preference for mu-receptors

The side-chain to side-chain cyclized opioid peptide analogs H-Tyr-D-Orn-Phe-Asp-NH2 (I) and H-Tyr-D-Lys-Phe-Glu-NH2 (II) were synthesized and tested in the guinea pig ileum and mouse vas deferens assays and in binding assays based on displacement of mu- and delta-opioid receptor-selective radioligands from rat brain membranes. The more rigid cyclic analog I containing a 13-membered ring structure showed very high preference for mu-receptors over delta-receptors, whereas the more flexible cyclic peptide II (15-membered ring) was non-selective. These results indicate that variation in the degree of conformational restriction of opioid peptides can produce drastic shifts in their receptor selectivity profile. Because of its high mu-receptor selectivity and rigidity cyclic analog I will be useful for determining the conformational requirements of mu-opioid receptors.