Cascades of Genetic Instability Resulting from Compromised Break-Induced Replication

Break-induced replication (BIR) is a mechanism to repair double-strand breaks (DSBs) that possess only a single end that can find homology in the genome. This situation can result from the collapse of replication forks or telomere erosion. BIR frequently produces various genetic instabilities including mutations, loss of heterozygosity, deletions, duplications, and template switching that can result in copy-number variations (CNVs). An important type of genomic rearrangement specifically linked to BIR is half-crossovers (HCs), which result from fusions between parts of recombining chromosomes. Because HC formation produces a fused molecule as well as a broken chromosome fragment, these events could be highly destabilizing. Here we demonstrate that HC formation results from the interruption of BIR caused by a damaged template, defective replisome or premature onset of mitosis. Additionally, we document that checkpoint failure promotes channeling of BIR into half-crossover-initiated instability cascades (HCC) that resemble cycles of non-reciprocal translocations (NRTs) previously described in human tumors. We postulate that HCs represent a potent source of genetic destabilization with significant consequences that mimic those observed in human diseases, including cancer.     

Changes in satellite‐derived spring vegetation green‐up date and its linkage to climate in China from 1982 to 2010: a multimethod analysis

The change in spring phenology is recognized to exert a major influence on carbon balance dynamics in temperate ecosystems. Over the past several decades, several studies focused on shifts in spring phenology; however, large uncertainties still exist, and one understudied source could be the method implemented in retrieving satellite‐derived spring phenology. To account for this potential uncertainty, we conducted a multimethod investigation to quantify changes in vegetation green‐up date from 1982 to 2010 over temperate China, and to characterize climatic controls on spring phenology. Over temperate China, the five methods estimated that the vegetation green‐up onset date advanced, on average, at a rate of 1.3 ± 0.6 days per decade (ranging from 0.4 to 1.9 days per decade) over the last 29 years. Moreover, the sign of the trends in vegetation green‐up date derived from the five methods were broadly consistent spatially and for different vegetation types, but with large differences in the magnitude of the trend. The large intermethod variance was notably observed in arid and semiarid vegetation types. Our results also showed that change in vegetation green‐up date is more closely correlated with temperature than with precipitation. However, the temperature sensitivity of spring vegetation green‐up date became higher as precipitation increased, implying that precipitation is an important regulator of the response of vegetation spring phenology to change in temperature. This intricate linkage between spring phenology and precipitation must be taken into account in current phenological models which are mostly driven by temperature.     

Metabolite profiles and the risk of developing diabetes

Emerging technologies allow the high-throughput profiling of metabolic status from a blood specimen (metabolomics). We investigated whether metabolite profiles could predict the development of diabetes. Among 2,422 normoglycemic individuals followed for 12 years, 201 developed diabetes. Amino acids, amines and other polar metabolites were profiled in baseline specimens by liquid chromatography-tandem mass spectrometry (LC-MS). Cases and controls were matched for age, body mass index and fasting glucose. Five branched-chain and aromatic amino acids had highly significant associations with future diabetes: isoleucine, leucine, valine, tyrosine and phenylalanine. A combination of three amino acids predicted future diabetes (with a more than fivefold higher risk for individuals in top quartile). The results were replicated in an independent, prospective cohort. These findings underscore the potential key role of amino acid metabolism early in the pathogenesis of diabetes and suggest that amino acid profiles could aid in diabetes risk assessment.     

Hydrodynamic Determinants of Cell Necrosis and Molecular Delivery Produced by Pulsed Laser Microbeam Irradiation of Adherent Cells

Time-resolved imaging, fluorescence microscopy, and hydrodynamic modeling were used to examine cell lysis and molecular delivery produced by picosecond and nanosecond pulsed laser microbeam irradiation in adherent cell cultures. Pulsed laser microbeam radiation at λ = 532 nm was delivered to confluent monolayers of PtK₂ cells via a 40×, 0.8 NA microscope objective. Using laser microbeam pulse durations of 180–1100 ps and pulse energies of 0.5–10.5 μJ, we examined the resulting plasma formation and cavitation bubble dynamics that lead to laser-induced cell lysis, necrosis, and molecular delivery. The cavitation bubble dynamics are imaged at times of 0.5 ns to 50 μ  s after the pulsed laser microbeam irradiation, and fluorescence assays assess the resulting cell viability and molecular delivery of 3 kDa dextran molecules. Reductions in both the threshold laser microbeam pulse energy for plasma formation and the cavitation bubble energy are observed with decreasing pulse duration. These energy reductions provide for increased precision of laser-based cellular manipulation including cell lysis, cell necrosis, and molecular delivery. Hydrodynamic analysis reveals critical values for the shear-stress impulse generated by the cavitation bubble dynamics governs the location and spatial extent of cell necrosis and molecular delivery independent of pulse duration and pulse energy. Specifically, cellular exposure to a shear-stress impulse J?0.1$J\mathrm{?}0.1$ Pa s ensures cell lysis or necrosis, whereas exposures in the range of 0.035?J?0.1$0.035\mathrm{?}J\mathrm{?}0.1$ Pa s preserve cell viability while also enabling molecular delivery of 3 kDa dextran. Exposure to shear-stress impulses of J?0.035$J\mathrm{?}0.035$ Pa s leaves the cells unaffected. Hydrodynamic analysis of these data, combined with data from studies of 6 ns microbeam irradiation, demonstrates the primacy of shear-stress impulse in determining cellular outcome resulting from pulsed laser microbeam irradiation spanning a nearly two-orders-of-magnitude range of pulse energy and pulse duration. These results provide a mechanistic foundation and design strategy applicable to a broad range of laser-based cellular manipulation procedures.     

The effect of protein a cycle number on the performance and lifetime of an anion exchange polishing step

Most mAb platform purification processes consist of an affinity capture step followed by one or two polishing steps. An understanding of the performance linkages between the unit operations can lead to robust manufacturing processes. In this study, a weak‐partitioning anion‐exchange chromatography polishing step used in a mAb purification process was characterized through high‐throughput screening (HTS) experiments, small‐scale experiments including a cycling study performed on qualified scale‐down models, and large‐scale manufacturing runs. When material from a Protein A column that had been cycled <10× was loaded on the AEX resin, early breakthrough of impurities and premature loss of capacity was observed. As the cycle number on the Protein A resin increased, the capacity of the subsequent AEX step increased. Different control strategies were considered for preventing impurity breakthrough and improving AEX resin lifetimes. Depth filtration of the Protein A peak pool significantly improved the AEX resin capacity, robustness, and lifetime. Further, the turbidity of the Protein A pool has the potential for use as an in‐process control parameter for monitoring the performance of the AEX step. Biotechnol. Bioeng. 2013; 110: 1142–1152. © 2012 Wiley Periodicals, Inc.     

Synthesis and antifungal activities of miltefosine analogs

Miltefosine is an alkylphosphocholine that shows broad-spectrum in vitro antifungal activities and limited in vivo efficacy in mouse models of cryptococcosis. To further explore the potential of this class of compounds for the treatment of systemic mycoses, nine analogs (3a?3i) were synthesized by modifying the choline structural moiety and the alkyl chain length of miltefosine. In vitro testing of these compounds against the opportunistic fungal pathogens Candida albicans, Candida glabrata, Candida krusei, Aspergillus fumigatus, and Cryptococcus neoformans revealed that N-benzyl-N,N-dimethyl-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3a), N,N-dimethyl-N-(4-nitrobenzyl)-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3d), and N-(4-methoxybenzyl)-N,N-dimethyl-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3e) exhibited minimum inhibitory concentrations (MIC) of 2.5–5.0 μg/mL against all tested pathogens, when compared to miltefosine with MICs of 2.5–3.3 μg/mL. Compound 3a showed low in vitro cytotoxicity against three mammalian cell lines similar to miltefosine. In vivo testing of 3a and miltefosine against C. albicans in a mouse model of systemic infection did not demonstrate efficacy. The results of this study indicate that further investigation will be required to determine the potential usefulness of the alkylphosphocholines in the treatment of invasive fungal infections.     

Human gingival derived neuronal cells in the optimized caffeic acid hydrogel for hemitransection spinal cord injury model

Spinal cord injury induces scar formation causes axonal damage that leads to the degeneration of axonal function. Still, there is no robust conceptual design to regenerate the damaged axon after spinal injury. Therefore, the present study demonstrates that human gingival derived neuronal stem cells (GNSCs) transplants in the injectable caffeic acid bioconjugated hydrogel (CBGH) helps to bridge the cavity and promote the engraftment and repopulation of transplants in the injured spinal tissue. Our study reports that the bioluminescence imaging in vivo imaging system (IVIS) provides a satisfactory progression in CBGH-GNSCs transplants compare to lesion control and CBGH alone. Immune regulators interleukin-6 (IL-6), tumor necrosis factor-α, neutrophil elastase are decreased, IL-10 is increased. Likewise, immunostaining (TAU/TUJ-1, SOX-2/NeuN, MAP-2/PSD93, NSE, S100b, and GFAP) shown repopulated cells. Also, TRA-1-81 expression confirms the absence of immune rejection in the CBGH-GNSCs transplants. However, locomotor recovery test, gene (IL-6, CASPASE3, p14-ARF, VEGF, LCAM, BDNF, NT3, NGN2, TrKc, FGF2, Sox-2, TUJ-1, MAP-2, Nestin, and NeuN) and protein expression (TAU, TUJ-1, SOX-2 MAP-2, PSD93, NeuN, TRA-1-81, GFAP, TAU, and MBP) shows functional improvements in the CBGH-GNSCs group. Further, GABA and glutamine level demonstrates the new synaptic vesicle formation. Hence, the CBGH scaffold enhances GNSCs transplants to restore the injured spinal tissue.