Label‐free optical projection tomography for quantitative three‐dimensional anatomy of mouse embryo

Recent progress in three‐dimensional optical imaging techniques allows visualization of many comprehensive biological specimens. Optical clearing methods provide volumetric and quantitative information by overcoming the limited depth of light due to scattering. However, current imaging technologies mostly rely on the synthetic or genetic fluorescent labels, thus limits its application to whole‐body visualization of generic mouse models. Here, we report a label‐free optical projection tomography (LF‐OPT) technique for quantitative whole mouse embryo imaging. LF‐OPT is based on the attenuation contrast of light rather than fluorescence, and it utilizes projection imaging technique similar to computed tomography for visualizing the volumetric structure. We demonstrate this with a collection of mouse embryo morphologies in different stages using LF‐OPT. Additionally, we extract quantitative organ information applicable toward high‐throughput phenotype screening. Our results indicate that LF‐OPT can provide multi‐scale morphological information in various tissues including bone, which can be difficult in conventional optical imaging technique.     

Downregulation of Choline Kinase-Alpha Enhances Autophagy in Tamoxifen-Resistant Breast Cancer Cells

Choline kinase-α (Chk-α) and autophagy have gained much attention, as they relate to the drug-resistance of breast cancer. Here, we explored the potential connection between Chk-α and autophagy in the mechanisms driving to tamoxifen (TAM) resistance, in estrogen receptor positive (ER+) breast cancer cells (BCCs). Human BCC lines (MCF-7 and TAM-resistant MCF-7 (MCF-7/TAM) cells) were used. Chk-α expression and activity was suppressed by the transduction of shRNA (shChk-α) with lentivirus and treatment with CK37, a Chk-α inhibitor. MCF-7/TAM cells had higher Chk-α expression and phosphocholine levels than MCF-7 cells. A specific downregulation of Chk-α by the transduction of shChk-α exhibited a significant decrease in phosphocholine levels in MCF-7 and MCF-7/TAM cells. The autophagy-related protein, cleaved microtubule-associated protein light chain 3 (LC3) and autophagosome-like structures were significantly increased in shChk-α-transduced or CK37-treated MCF-7 and MCF-7/TAM cells. The downregulation of Chk-α attenuated the phosphorylation of AKT, ERK1/2, and mTOR in both MCF-7 and MCF-7/TAM cells. In MCF-7 cells, the downregulation of Chk-α resulted in an induction of autophagy, a decreased proliferation ability and an activation of caspase-3. In MCF-7/TAM cells, despite a significant decrease in proliferation ability and an increase in the percentage of cells in the G0/G1 phase of the cell cycle, the downregulation of Chk-α did not induced caspase-dependent cell death and further enhanced autophagy and G0/G1 phase arrest. An autophagy inhibitor, methyladenine (3-MA) induced death and attenuated the level of elevated LC3 in MCF-7/TAM cells. Elucidating the interplay between choline metabolism and autophagy will provide unique opportunities to identify new therapeutic targets and develop novel treatment strategies that preferentially target TAM-resistance.     

贵州省楠杆自然保护区药用植物多样性调查

贵州德江楠杆自然保护区药用植物资源种类丰富,首次调查植物种类达2 000余种,查阅药用植物文献资料记载,鉴定有药用植物1 079种,隶属于201科603属。其中,真菌类植物19科23属35种,苔藓类14科14属16种,石松和蕨类植物19科42属88种,裸子植物7科11属14种,双子叶植物121科426属792种,单子叶植物21科87属134种,草本多达587种,木本多达438种。对该地区药用植物的组成、丰富度与周边地区进行了比较,提出了保护利用及可持续发展的建议。     

Endocytosis of EphA receptors is essential for the proper development of the retinocollicular topographic map

Endocytosis of Eph-ephrin complexes may be an important mechanism for converting cell-cell adhesion to a repulsive interaction. Here, we show that an endocytosis-defective EphA8 mutant forms a complex with EphAs and blocks their endocytosis in cultured cells. Further, we used bacterial artificial chromosome transgenic (Tg) mice to recapitulate the anterior>posterior gradient of EphA in the superior colliculus (SC). In mice expressing the endocytosis-defective EphA8 mutant, the nasal axons were aberrantly shifted to the anterior SC. In contrast, in Tg mice expressing wild-type EphA8, the nasal axons were shifted to the posterior SC, as predicted for the enhanced repellent effect of ephrinA reverse signalling. Importantly, Rac signalling was shown to be essential for EphA-ephrinA internalization and the subsequent nasal axonal repulsion in the SC. These results indicate that endocytosis of the Eph-ephrin complex is a key mechanism by which axonal repulsion is generated for proper guidance and topographic mapping.     

Monitoring of Leuconostoc population during sauerkraut fermentation by quantitative real-time polymerase chain reaction

A real-time PCR assay method was established to monitor Leuconostoc spp. populations via specific amplification of the dextransucrase gene. Quantification of L. mesenteroides B-512F using both genomic DNA and cell suspensions yielded a log-linear correlation spanning approximately 5 log units. By using this method, monitoring changes of Leuconostoc spp. during sauerkraut fermentation was successfully accomplished with accuracy after inoculation of starter and sugars (sucrose and maltose).     

Adenosine signaling in normal and sickle erythrocytes and beyond

Sickle cell disease (SCD) is a debilitating hemolytic genetic disorder with high morbidity and mortality affecting millions of individuals worldwide. Although SCD was discovered more than a century ago, no effective mechanism-based prevention and treatment are available due to poorly understood molecular basis of sickling, the fundamental pathogenic process of the disease. SCD patients constantly face hypoxia. One of the best-known signaling molecules to be induced under hypoxic conditions is adenosine. Recent studies demonstrate that hypoxia-mediated elevated adenosine signaling plays an important role in normal erythrocyte physiology. In contrast, elevated adenosine signaling contributes to sickling and multiple life threatening complications including tissue damage, pulmonary dysfunction and priapism. Here, we summarize recent research on the role of adenosine signaling in normal and sickle erythrocytes, progression of the disease and therapeutic implications. In normal erythrocytes, both genetic and pharmacological studies demonstrate that adenosine can enhance 2,3-bisphosphoglycerate (2,3-BPG) production via A(2B) receptor (ADORA2B) activation, suggesting that elevated adenosine has an unrecognized role in normal erythrocytes to promote O(2) release and prevent acute ischemic tissue injury. However, in sickle erythrocytes, the beneficial role of excessive adenosine-mediated 2,3-BPG induction becomes detrimental by promoting deoxygenation, polymerization of sickle hemoglobin and subsequent sickling. Additionally, adenosine signaling via the A(2A) receptor (ADORA2A) on invariant natural killer T (iNKT) cells inhibits iNKT cell activation and attenuates pulmonary dysfunction in SCD mice. Finally, elevated adenosine coupled with ADORA2BR activation is responsible for priapism, a dangerous complication seen in SCD. Overall, the research reviewed here reveals a differential role of elevated adenosine in normal erythrocytes, sickle erythrocytes, iNK cells and progression of disease. Thus, adenosine signaling represents a potentially important therapeutic target for the treatment and prevention of disease.     

Deep Genome-Wide Measurement of Meiotic Gene Conversion Using Tetrad Analysis in Arabidopsis thaliana

Gene conversion, the non-reciprocal exchange of genetic information, is one of the potential products of meiotic recombination. It can shape genome structure by acting on repetitive DNA elements, influence allele frequencies at the population level, and is known to be implicated in human disease. But gene conversion is hard to detect directly except in organisms, like fungi, that group their gametes following meiosis. We have developed a novel visual assay that enables us to detect gene conversion events directly in the gametes of the flowering plant Arabidopsis thaliana. Using this assay we measured gene conversion events across the genome of more than one million meioses and determined that the genome-wide average frequency is 3.5×10⁻⁴ conversions per locus per meiosis. We also detected significant locus-to-locus variation in conversion frequency but no intra-locus variation. Significantly, we found one locus on the short arm of chromosome 4 that experienced 3-fold to 6-fold more gene conversions than the other loci tested. Finally, we demonstrated that we could modulate conversion frequency by varying experimental conditions.     

Metabolic disturbances associated with systemic lupus erythematosus

The metabolic disturbances that underlie systemic lupus erythematosus are currently unknown. A metabolomic study was executed, comparing the sera of 20 SLE patients against that of healthy controls, using LC/MS and GC/MS platforms. Validation of key differences was performed using an independent cohort of 38 SLE patients and orthogonal assays. SLE sera showed evidence of profoundly dampened glycolysis, Krebs cycle, fatty acid β oxidation and amino acid metabolism, alluding to reduced energy biogenesis from all sources. Whereas long-chain fatty acids, including the n3 and n6 essential fatty acids, were significantly reduced, medium chain fatty acids and serum free fatty acids were elevated. The SLE metabolome exhibited profound lipid peroxidation, reflective of oxidative damage. Deficiencies were noted in the cellular anti-oxidant, glutathione, and all methyl group donors, including cysteine, methionine, and choline, as well as phosphocholines. The best discriminators of SLE included elevated lipid peroxidation products, MDA, gamma-glutamyl peptides, GGT, leukotriene B4 and 5-HETE. Importantly, similar elevations were not observed in another chronic inflammatory autoimmune disease, rheumatoid arthritis. To sum, comprehensive profiling of the SLE metabolome reveals evidence of heightened oxidative stress, inflammation, reduced energy generation, altered lipid profiles and a pro-thrombotic state. Resetting the SLE metabolome, either by targeting selected molecules or by supplementing the diet with essential fatty acids, vitamins and methyl group donors offers novel opportunities for disease modulation in this disabling systemic autoimmune ailment.     

The phytochelatin transporters AtABCC1 and AtABCC2 mediate tolerance to cadmium and mercury

Heavy metals such as cadmium (Cd) and mercury (Hg) are toxic pollutants that are detrimental to living organisms. Plants employ a two-step mechanism to detoxify toxic ions. First, phytochelatins bind to the toxic ion, and then the metal-phytochelatin complex is sequestered in the vacuole. Two ABCC-type transporters, AtABCC1 and AtABCC2, that play a key role in arsenic detoxification, have recently been identified in Arabidopsis thaliana. However, it is unclear whether these transporters are also implicated in phytochelatin-dependent detoxification of other heavy metals such as Cd(II) and Hg(II). Here, we show that atabcc1 single or atabcc1 atabcc2 double knockout mutants exhibit a hypersensitive phenotype in the presence of Cd(II) and Hg(II). Microscopic analysis using a Cd-sensitive probe revealed that Cd is mostly located in the cytosol of protoplasts of the double mutant, whereas it occurs mainly in the vacuole of wild-type cells. This suggests that the two ABCC transporters are important for vacuolar sequestration of Cd. Heterologous expression of the transporters in Saccharomyces cerevisiae confirmed their role in heavy metal tolerance. Over-expression of AtABCC1 in Arabidopsis resulted in enhanced Cd(II) tolerance and accumulation. Together, these results demonstrate that AtABCC1 and AtABCC2 are important vacuolar transporters that confer tolerance to cadmium and mercury, in addition to their role in arsenic detoxification. These transporters provide useful tools for genetic engineering of plants with enhanced metal tolerance and accumulation, which are desirable characteristics for phytoremediation.