Site-Specific Inter-Strand Cross-Links of DNA Duplexes

We report the development of technology that allows inter-strand coupling across various positions within one turn of DNA. Four 2\u27-modified nucleotides were synthesized as protected phosphoramidites and incorporated into DNA oligonucleotides. The modified nucleotides contain either 5-atom or 16-atom linker components, with either amine or carboxylic acid functional groups at their termini, forming 10 or 32 atom (11 or 33 bond) linkages. Chemical coupling of the amine and carboxylate groups in designed strands resulted in the formation of an amide bond. Coupling efficiency as a function of trajectory distance between the individual linker components was examined. For those nucleotides capable of forming inter-strand cross-links (ICLs), coupling yields were found to depend on temperature, distance, and linker length, enabling several approaches that can control regioselective linkage. In the most favorable cases, the coupling yields are quantitative. Spectroscopic measurements of strands that were chemically cross-linked indicate that the global structure of the DNA duplex does not appear to be distorted from the B form after coupling. Thermal denaturing profiles of those strands were shifted to somewhat higher temperatures than those of their respective control duplexes. Thus, the robust amide ICLs formed by this approach are site-specific, do not destabilize the rest of the duplex, and only minimally perturb the secondary structure

Evaluating the Effects of Cerebrospinal Fluid Protein Content on the Performance of Differential Pressure Valves and Antisiphon Devices Using a Novel Benchtop Shunting Model

BACKGROUNDHydrocephalus is managed by surgically implanting flow-diversion technologies such as differential pressure valves and antisiphoning devices; however, such hardware is prone to failure. Extensive research has tested them in flow-controlled settings using saline or de-aerated water, yet little has been done to validate their performance in a setting recreating physiologically relevant parameters, including intracranial pressures, cerebrospinal fluid (CSF) protein content, and body position.OBJECTIVETo more accurately chart the episodic drainage characteristics of flow-diversion technology. A gravity-driven benchtop model of flow was designed and tested continuously during weeks-long trials.METHODSUsing a hydrostatic pressure gradient as the sole driving force, interval flow rates of 6 valves were examined in parallel with various fluids. Daily trials in the upright and supine positions were run with fluid output collected from distal catheters placed at alternating heights for extended intervals.RESULTSSignificant variability in flow rates was observed, both within specific individual valves across different trials and among multiple valves of the same type. These intervalve and intravalve variabilities were greatest during supine trials and with increased protein. None of the valves showed evidence of overt obstruction during 30 d of exposure to CSF containing 5 g/L protein.CONCLUSIONDay-to-day variability of ball-in-cone differential pressure shunt valves may increase overdrainage risk. Narrow-lumen high-resistance flow control devices as tested here under similar conditions appear to achieve more consistent flow rates, suggesting their use may be advantageous, and did not demonstrate any blockage or trend of decreasing flow over the 3 wk of chronic use

DNA Topoisomerase III from the Hyperthermophilic Archaeon Sulfolobus solfataricus with Specific DNA Cleavage Activity

We report the production, purification, and characterization of a type IA DNA topoisomerase, previously designated topoisomerase I, from the hyperthermophilic archaeon Sulfolobus solfataricus. The protein was capable of relaxing negatively supercoiled DNA at 75°C in the presence of Mg(2+). Mutation of the putative active site Tyr318 to Phe318 led to the inactivation of the protein. The S. solfataricus enzyme cleaved oligonucleotides in a sequence-specific fashion. The cleavage occurred only in the presence of a divalent cation, preferably Mg(2+). The cofactor requirement of the enzyme was partially satisfied by Cu(2+), Co(2+), Mn(2+), Ca(2+), or Ni(2+). It appears that the enzyme is active with a broader spectrum of metal cofactors in DNA cleavage than in DNA relaxation (Mg(2+) and Ca(2+)). The enzyme-catalyzed oligonucleotide cleavage required at least 7 bases upstream and 2 bases downstream of the cleavage site. Analysis of cleavage by the S. solfataricus enzyme on a set of oligonucleotides revealed a consensus cleavage sequence of the enzyme: 5′-G(A/T)CA(T)AG(T)G(A)X↓XX-3′. This sequence bears more resemblance to the preferred cleavage sites of topoisomerases III than to those of topoisomerases I. Based on these data and sequence analysis, we designate the enzyme S. solfataricus topoisomerase III

Identification of a Natural Hybrid between Castanopsis sclerophylla and Castanopsis tibetana (Fagaceae) Based on Chloroplast and Nuclear DNA Sequences

Castanopsis × kuchugouzhui Huang et Y. T. Chang was recorded in Flora Reipublicae Popularis Sinicae (FRPS) as a hybrid species on Yuelushan mountain, but it is treated as a hybrid between Castanopsis sclerophylla (Lindl.) Schott. and Castanopsis tibetana Hance in Flora of China. After a thorough investigation on Yuelushan mountain, we found a population of C. sclerophylla and one individual of C. × kuchugouzhui, but no living individual of C. tibetana. We collected C. × kuchugouzhui, and we sampled 42 individuals of C. sclerophylla from Yuelushan and Xiushui and 43 individuals of C. tibetana from Liangyeshan and Xiushui. We used chloroplast DNA sequences and 29 nuclear microsatellite markers to investigate if C. × kuchugouzhui is a natural hybrid between C. sclerophylla and C. tibetana. The chloroplast haplotype analysis showed that C. × kuchugouzhui shared haplotype H2 with C. sclerophylla on Yuelushan. The STRUCTURE analysis identified two distinct genetic pools that corresponded well to C. sclerophylla and C. tibetana, revealing the genetic admixture of C. × kuchugouzhui. Furthermore, the NewHybrids analysis suggested that C. × kuchugouzhui is an F2 hybrid between C. sclerophylla and C. tibetana. Our results confirm that C. × kuchugouzhui recorded in FRPS is a rare hybrid between C. sclerophylla and C. tibetana

Genetic Diversity, Population Structure, and Conservation Units of <i>Castanopsis sclerophylla</i> (Fagaceae)

Castanopsis sclerophylla (Lindl.) Schott. is a canopy tree species of evergreen broad-leaved forests in subtropical China. In this study, the genetic diversity and population structure of C. sclerophylla were investigated by using chloroplast DNA sequences and nuclear microsatellite markers. Permutation tests with chloroplast DNA sequences indicated the presence of phylogeographic structure in C. sclerophylla. Based on nuclear microsatellite markers, Bayesian clustering analysis revealed eastern-to-western differentiation in C. sclerophylla, and the analysis of molecular variance suggested population divergence has arisen along the Xuefeng, Luoxiao, and Wuyi mountain ranges. The approximate Bayesian computation demonstrated that the genetic diversity pattern of C. sclerophylla could be explained by geographic isolation followed by secondary contact. Ecological niche modelling showed that distribution of C. sclerophylla shrank southward at the Last Glacial Maximum and expanded northward at the Mid Holocene. These results suggested that the uplift of the Xuefeng, Luoxiao, and Wuyi mountain ranges and the interglacial–glacial climate change shaped the genetic diversity of C. sclerophylla. The Luoxiao mountain range should be considered as a key conservation unit of C. sclerophylla due to its higher level of genetic diversity. Our study supplies important information for prioritizing the conservation and sustainable utilization of C. sclerophylla, and provides insight on the dynamics of evergreen broad-leaved forests in subtropical China

Myeloid-like tumor hybrid cells in bone marrow promote progression of prostate cancer bone metastasis

Abstract Background Bone metastasis is the leading cause of death in patients with prostate cancer (PCa) and currently has no effective treatment. Disseminated tumor cells in bone marrow often obtain new characteristics to cause therapy resistance and tumor recurrence. Thus, understanding the status of disseminated prostate cancer cells in bone marrow is crucial for developing a new treatment. Methods We analyzed the transcriptome of disseminated tumor cells from a single cell RNA-sequencing data of PCa bone metastases. We built a bone metastasis model through caudal artery injection of tumor cells, and sorted the tumor hybrid cells by flow cytometry. We performed multi-omics analysis, including transcriptomic, proteomic and phosphoproteomic analysis, to compare the difference between the tumor hybrid cells and parental cells. In vivo experiments were performed to analyze the tumor growth rate, metastatic and tumorigenic potential, drug and radiation sensitivity in hybrid cells. Single cell RNA-sequencing and CyTOF were performed to analyze the impact of hybrid cells on tumor microenvironment. Results Here, we identified a unique cluster of cancer cells in PCa bone metastases, which expressed myeloid cell markers and showed a significant change in pathways related to immune regulation and tumor progression. We found that cell fusion between disseminated tumor cells and bone marrow cells can be source of these myeloid-like tumor cells. Multi-omics showed the pathways related to cell adhesion and proliferation, such as focal adhesion, tight junction, DNA replication, and cell cycle, were most significantly changed in these hybrid cells. In vivo experiment showed hybrid cells had a significantly increased proliferative rate, and metastatic potential. Single cell RNA-sequencing and CyTOF showed tumor-associated neutrophils/monocytes/macrophages were highly enriched in hybrid cells-induced tumor microenvironment with a higher immunosuppressive capacity. Otherwise, the hybrid cells showed an enhanced EMT phenotype with higher tumorigenicity, and were resistant to docetaxel and ferroptosis, but sensitive to radiotherapy. Conclusion Taken together, our data demonstrate that spontaneous cell fusion in bone marrow can generate myeloid-like tumor hybrid cells that promote the progression of bone metastasis, and these unique population of disseminated tumor cells can provide a potential therapeutic target for PCa bone metastasis

Sex differences in adult rat insulin and glucose responses to arginine: programming effects of neonatal separation, hypoxia, and hypothermia

Acute neonatal hypoxia, a common stressor, causes a spontaneous decrease in body temperature which may be protective. There is consensus that hypothermia should be prevented during acute hypoxia in the human neonate; however, this may be an additional stress with negative consequences. We hypothesize that maintaining body temperature during hypoxia in the first week of postnatal life alters the subsequent insulin, glucose, and glucagon secretion in adult rats. Rat pups were separated from their dam daily from postnatal days (PD) 2-6 for the following 90 min experimental treatments: (1) normoxic separation (control), (2) hypoxia (8% O2) allowing spontaneous hypothermia, (3) normoxic hypothermia with external cold, and (4) exposure to 8% O2 while maintaining body temperature using external heat. An additional normoxic non-separated control group was performed to determine if separation per se changed the adult phenotype. Plasma insulin, glucose, and glucagon responses to arginine stimulation were evaluated from PD105 to PD133. Maternal separation (compared to non-separated neonates) had more pronounced effects on the adult response to arginine compared to the hypoxic, hypothermic, and hypoxic-isothermic neonatal treatments. Adult males exposed to neonatal maternal separation had augmented insulin and glucose responses to arginine compared to unseparated controls. Additionally, neonatal treatment had a significant effect on body weight gain; adults exposed to neonatal maternal separation were significantly heavier. Female adults had significantly smaller insulin and glucose responses to arginine regardless of neonatal treatment. Neonatal maternal separation during the first week of life significantly altered adult beta-cell function in a sexually dimorphic manner

Role of the Unfolded Protein Response Regulator GRP78/BiP in Development, Cancer, and Neurological Disorders

GRP78/BiP is a major endoplasmic reticulum (ER) chaperone protein critical for protein quality control of the ER, as well as controlling the activation of the ER-transmembrane signaling molecules. Through creation of mouse models targeting the Grp78 allele, the function of GRP78 in development and disease has been investigated. These led to the discovery that GRP78 function is obligatory for early embryonic development. However, in adult animals, GRP78 is preferably required for cancer cell survival under pathologic conditions such as tumor progression and drug resistance. The discovery of surface localization of GRP78 in cancer cells reveals potential novel function, interaction with cell-surface receptors, and possible therapeutic implications. Mouse models also reveal that GRP78 controls maturation and secretion of neuronal factors for proper neural migration and offers neuroprotection. Antioxid. Redox Signal. 11, 2307–2316

Autonomous interconversion between adult pancreatic α-cells and β-cells after differential metabolic challenges

Background: Evidence hints at the ability of β-cells to emerge from non-β-cells upon genetic or pharmacological interventions. However, their quantitative contributions to the process of autonomous β-cell regeneration without genetic or pharmacological manipulations remain to be determined. Methods & results: Using PANIC-ATTAC mice, a model of titratable, acute β-cell apoptosis capable of autonomous, and effective islet mass regeneration, we demonstrate that an extended washout of residual tamoxifen activity is crucial for β-cell lineage tracing studies using the tamoxifen-inducible Cre/loxP systems. We further establish a doxycycline-inducible system to label different cell types in the mouse pancreas and pursued a highly quantitative assessment to trace adult β-cells after various metabolic challenges. Beyond proliferation of pre-existing β-cells, non-β-cells contribute significantly to the post-challenge regenerated β-cell pool. α-cell trans-differentiation is the predominant mechanism upon post-apoptosis regeneration and multiparity. No contributions from exocrine acinar cells were observed. During diet-induced obesity, about 25% of α-cells arise de novo from β-cells. Ectopic expression of Nkx6.1 promotes α-to-β conversion and insulin production. Conclusions: We identify the origins and fates of adult β-cells upon post-challenge upon autonomous regeneration of islet mass and establish the quantitative contributions of the different cell types using a lineage tracing system with high temporal resolution. Author Video: Author Video Watch what authors say about their articles Keywords: Lineage tracing, Adult β-cell origins, Nkx6.1, Tamoxifen artifact