In-depth Proteomics Characterization of Embryogenesis of the Honey Bee Worker (Apis mellifera ligustica)

Identifying proteome changes of honey bee embryogenesis is of prime importance for unraveling the molecular mechanisms that they underlie. However, many proteomic changes during the embryonic period are not well characterized. We analyzed the proteomic alterations over the complete time course of honey bee worker embryogenesis at 24, 48, and 72 h of age, using mass spectrometry-based proteomics, label-free quantitation, and bioinformatics. Of the 1460 proteins identified the embryo of all three ages, the core proteome (proteins shared by the embryos of all three ages, accounting for 40%) was mainly involved in protein synthesis, metabolic energy, development, and molecular transporter, which indicates their centrality in driving embryogenesis. However, embryos at different developmental stages have their own specific proteome and pathway signatures to coordinate and modulate developmental events. The young embryos (<24 h) stronger expression of proteins related to nutrition storage and nucleic acid metabolism may correlate with the cell proliferation occurring at this stage. The middle aged embryos (24–48 h) enhanced expression of proteins associated with cell cycle control, transporters, antioxidant activity, and the cytoskeleton suggest their roles to support rudimentary organogenesis. Among these proteins, the biological pathways of aminoacyl-tRNA biosynthesis, β-alanine metabolism, and protein export are intensively activated in the embryos of middle age. The old embryos (48–72 h) elevated expression of proteins implicated in fatty acid metabolism and morphogenesis indicate their functionality for the formation and development of organs and dorsal closure, in which the biological pathways of fatty acid metabolism and RNA transport are highly activated. These findings add novel understanding to the molecular details of honey bee embryogenesis, in which the programmed activation of the proteome matches with the physiological transition observed during embryogenesis. The identified biological pathways and key node proteins allow for further functional analysis and genetic manipulation for both the honey bee embryos and other eusocial insects.Embryogenesis is an important period during which the body plan of adult honey bees (Apis mellifera L.) is formed. This life stage, lasting 72 h, occurs during the egg laid by the queen before bees hatch as young larva. Worker bees are derived from fertilized eggs and develop through four distinct stages until the imago eventually emerges: egg, larva, pupa, and emerging adult (1–4). The worker is the dominate caste and engages in almost all aspects of social life: taking care of larvae, cleaning the hive, guarding the nest, and foraging for nectar and pollen for the colony. Understanding the developmental mechanism of embryogenesis of honey bee workers at the protein level is conducive to gaining a new insight into honey bee embryology, but information about the mechanisms of honey bee embryos at molecular level is still very limited.The embryo is recognized as an ideal model for genetic modification as compared with larva, pupa, and emerged adults (5). The environment for embryonic development requires a constant temperature of 34 °C and 80% relative humidity, which can easily be simulated under laboratory conditions. In contrast, rearing larvae or pupae is more challenging because they demand a specific temperature, humidity, and nutrition in the colony environment (1, 5). Furthermore, the honey bee has adapted an evolutionary strategy for better colony survival that makes it difficult to rear experimentally modified larvae and pupae within the colony (6, 7), nurse bees use acute judgment to identify and remove abnormal eggs or larvae (8). This adaptation makes raising experimentally treated bees, such as genetically manipulated eggs and larvae, very difficult in the honey bee colony (9–11). Because of totipotency and multiple differentiation potential, modified eggs could be hatched out normally and eventually some of them could be induced to morphologically and physiologically normal adult queens (12), increasing their usefulness as a model system. Moreover, the chorion of honey bee egg is more suitable for puncturing a hole for microinjection as it is much thinner than that of the fruit fly (Drosophila melonogastero) or the silk worm (Bombyx mori) (0.1–0.25 μm for honey bees compared with ∼17 μm for silk worm) (13, 14). These superiorities are quite promising for in vivo transgenic research on honey bee embryos.Until now, a number of genetic manipulations of the honey bee embryo have been developed. For example, embryonic cells in the pre-gastrula stage that have been transplanted with nuclear materials have developed into chimeric honey bee larvae (15). RNA interference (RNAi) has been used for honey bee embryos in vivo to characterize the functioning of specific genes (16) and for genetic effects on morphological differentiation (17, 18). Moreover, the cultivation of short-term (19–21), long-term (22), and immortalized cell lines (23), and the expression of non-Apis genes in cultured embryonic cells (24) have opened up a new era for genetic manipulation of honey bee embryos.Like Drosophila, Apis is a long germ insect in which segmentation occurs across the whole body (25). To date, although several studies have examined morphological change (2, 26, 27) and gene expression (25, 28, 29) during the period of embryogenesis in the honey bee, only a few works report on the preliminary results of the unraveling molecular underpinnings of worker (30) and drone (31) embryogenesis at the proteomic level, identifying only 107 proteins. MS-based proteomics is the primary technology that enables a system-wide view of proteomes and their changes. The development of MS with high resolution, high mass accuracy, and high sequencing speed now allows routine identification and quantification of proteins in a comprehensive and unbiased manner in biological samples with high confidence (32). These technological advances in LC-MS now allow the study of protein expression on a system-wide level (33). Therefore, an in-depth characterization of the proteome changes during the honey bee embryogenesis will provide greater understanding of the molecular mechanisms that underlie the process of embryogenesis in honey bee workers, and offers new insights into the embryology of other social insects.     

Regenerative Therapeutic Potential of Adipose Stromal Cells in Early Stage Diabetic Retinopathy

Diabetic retinopathy (DR) is the leading cause of blindness in working-age adults. Early stage DR involves inflammation, vascular leakage, apoptosis of vascular cells and neurodegeneration. In this study, we hypothesized that cells derived from the stromal fraction of adipose tissue (ASC) could therapeutically rescue early stage DR features. Streptozotocin (STZ) induced diabetic athymic nude rats received single intravitreal injection of human ASC into one eye and saline into the other eye. Two months post onset of diabetes, administration of ASC significantly improved “b” wave amplitude (as measured by electroretinogram) within 1–3 weeks of injection compared to saline treated diabetic eyes. Subsequently, retinal histopathological evaluation revealed a significant decrease in vascular leakage and apoptotic cells around the retinal vessels in the diabetic eyes that received ASC compared to the eyes that received saline injection. In addition, molecular analyses have shown down-regulation in inflammatory gene expression in diabetic retina that received ASC compared to eyes that received saline. Interestingly, ASC were found to be localized near retinal vessels at higher densities than seen in age matched non-diabetic retina that received ASC. In vitro, ASC displayed sustained proliferation and decreased apoptosis under hyperglycemic stress. In addition, ASC in co-culture with retinal endothelial cells enhance endothelial survival and collaborate to form vascular networks. Taken together, our findings suggest that ASC are able to rescue the neural retina from hyperglycemia-induced degeneration, resulting in importantly improved visual function. Our pre-clinical studies support the translational development of adipose stem cell-based therapy for DR to address both retinal capillary and neurodegeneration.     

Whole Mitochondrial and Plastid Genome SNP Analysis of Nine Date Palm Cultivars Reveals Plastid Heteroplasmy and Close Phylogenetic Relationships among Cultivars

Date palm is a very important crop in western Asia and northern Africa, and it is the oldest domesticated fruit tree with archaeological records dating back 5000 years. The huge economic value of this crop has generated considerable interest in breeding programs to enhance production of dates. One of the major limitations of these efforts is the uncertainty regarding the number of date palm cultivars, which are currently based on fruit shape, size, color, and taste. Whole mitochondrial and plastid genome sequences were utilized to examine single nucleotide polymorphisms (SNPs) of date palms to evaluate the efficacy of this approach for molecular characterization of cultivars. Mitochondrial and plastid genomes of nine Saudi Arabian cultivars were sequenced. For each species about 60 million 100 bp paired-end reads were generated from total genomic DNA using the Illumina HiSeq 2000 platform. For each cultivar, sequences were aligned separately to the published date palm plastid and mitochondrial reference genomes, and SNPs were identified. The results identified cultivar-specific SNPs for eight of the nine cultivars. Two previous SNP analyses of mitochondrial and plastid genomes identified substantial intra-cultivar ( = intra-varietal) polymorphisms in organellar genomes but these studies did not properly take into account the fact that nearly half of the plastid genome has been integrated into the mitochondrial genome. Filtering all sequencing reads that mapped to both organellar genomes nearly eliminated mitochondrial heteroplasmy but all plastid SNPs remained heteroplasmic. This investigation provides valuable insights into how to deal with interorganellar DNA transfer in performing SNP analyses from total genomic DNA. The results confirm recent suggestions that plastid heteroplasmy is much more common than previously thought. Finally, low levels of sequence variation in plastid and mitochondrial genomes argue for using nuclear SNPs for molecular characterization of date palm cultivars.     

Soft cheese supplemented with black cumin oil: Impact on food borne pathogens and quality during storage

Black cumin seed oil (BCSO) was tested for its inhibitory effect against some pathogenic bacteria (Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, Listeria monocytogenes Scott A and Salmonella enteritidis PT4) in Domiati cheese during cold storage. Physical, chemical and sensorial changes in cheese during storage were recorded. Pasteurized milk was inoculated before renneting with a mixed culture of bacteria at ca. 4 log CFU mL⁻¹. In vitro and in situ supplementation with BCSO showed antimicrobial impact on the growth of S. aureus, E. coli, L. monocytogenes and S. enteritidis inoculated into media and cheese samples. Supplementing of cheese with BCSO (0.1% or 0.2%, w/w) significantly reduced the counts of the inoculated pathogens by ca. 1.3 log and 1.5 log CFU g⁻¹ after 21 days of storage. In addition, BCSO controlled the development of titratable acidity, limited the changes in ripening indices, flavor components and kept considerable physicochemical and sensorial properties of cheese.     

Carnosol Induces ROS-Mediated Beclin1-Independent Autophagy and Apoptosis in Triple Negative Breast Cancer

Background

In this study we investigated the in vitro and in vivo anticancer effect of carnosol, a naturally occurring polyphenol, in triple negative breast cancer.

Results

We found that carnosol significantly inhibited the viability and colony growth induced G2 arrest in the triple negative MDA-MB-231. Blockade of the cell cycle was associated with increased p21/WAF1 expression and downregulation of p27. Interestingly, carnosol was found to induce beclin1-independent autophagy and apoptosis in MDA-MB-231 cells. The coexistence of both events, autophagy and apoptosis, was confirmed by electron micrography. Induction of autophagy was found to be an early event, detected within 3 h post-treatment, which subsequently led to apoptosis. Carnosol treatment also caused a dose-dependent increase in the levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2). Moreover, we show that carnosol induced DNA damage, reduced the mitochondrial potential and triggered the activation of the intrinsic and extrinsic apoptotic pathway. Furthermore, we found that carnosol induced a dose-dependent generation of reactive oxygen species (ROS) and inhibition of ROS by tiron, a ROS scavenger, blocked the induction of autophagy and apoptosis and attenuated DNA damage. To our knowledge, this is the first report to identify the induction of autophagy by carnosol.

Conclusion

In conclusion our findings provide strong evidence that carnosol may be an alternative therapeutic candidate against the aggressive form of breast cancer and hence deserves more exploration.     

Anticancer Efficacy of Self-Nanoemulsifying Drug Delivery System of Sunitinib Malate

Sunitinib malate (SM) is reported as a weakly soluble drug in water due to its poor dissolution rate and oral bioavailability. Hence, in the current study, various “self-nanoemulsifying drug delivery systems (SNEDDS)” of SM were prepared, characterized and evaluated for the enhancement of its in vitro dissolution rate and anticancer efficacy. On the basis of solubilization potential of SM in various excipients, “Lauroglycol-90 (oil), Triton-X100 (surfactant) and Transcutol-P (cosurfactant)” were selected for the preparation of SM SNEDDS. SM-loaded SNEDDS were developed by spontaneous emulsification method, characterized and evaluated for “thermodynamic stability, self-nanoemulsification efficiency, droplet size, polydispersity index (PDI), zeta potential (ZP), surface morphology, refractive index (RI), the percent of transmittance (% T) and drug release profile.” In vitro dissolution rate of SM was significantly enhanced from an optimized SNEDDS in comparison with SM suspension. The optimized SNEDDS of SM with droplet size of 42.3 nm, PDI value of 0.174, ZP value of ?36.4 mV, RI value of 1.339, % T value of 97.3%, and drug release profile of 95.4% (after 24 h via dialysis membrane) was selected for in vitro anticancer efficacy in human colon cancer cells (HT-29) by MTT assay. MTT assay indicated significant anticancer efficacy of optimized SM SNEDDS against HT-29 cells in comparison with free SM. The results of this study showed the great potential of SNEDDS in the enhancement of in vitro dissolution rate and anticancer efficacy of poorly soluble drug such as SM.     

First report of detection of the putative receptor of Bacillus thuringiensis toxin Vip3Aa from black cutworm (Agrotis ipsilon)

Black cutworm (BCW) Agrotis ipsilon, an economically important lepidopteran insect, has attracted a great attention. Bacillus thuringiensis (Bt) is spore forming soil bacteria and is an excellent environment-friendly approach for the control of phytophagous and disease-transmitting insects. In fact, bio-pesticide formulations and insect resistant transgenic plants based on the bacterium Bt delta-endotoxin have attracted worldwide attention as a safer alternative to harmful chemical pesticides. The major objective of the current study was to understand the mechanism of interaction of Bt toxin with its receptor molecule(s). The investigation involved the isolation, identification, and characterization of a putative receptor – vip3Aa. In addition, the kinetics of vip toxin binding to its receptor molecule was also studied. The present data suggest that Vip3Aa toxin bound specifically with high affinity to a 48-kDa protein present at the brush border membrane vesicles (BBMV) prepared from the midgut epithelial cells of BCW larvae.