George J. Klarmann

Human Immunodeficiency Virus Type 1 Recombination: Rate, Fidelity, and Putative Hot Spots

ABSTRACT Previously, we reported that human immunodeficiency virus type 1 (HIV-1) recombines approximately two to three times per genome per replication cycle, an extremely high rate of recombination given the relatively small genome size of HIV-1. However, a recombination hot spot involving sequence of nonretroviral origin was identified in the vector system utilized, raising the possibility that this hot spot skewed the rate of recombination, and the rate of recombination observed was an overestimation. To address this issue, an HIV-1-derived vector system was used to examine the rate of recombination between autologous HIV-1 sequences after restricting replication to a single cycle in the absence of this hot spot. Viral DNA and RNA were analyzed by a combination of the heteroduplex tracking assay, restriction enzyme analysis, DNA sequencing, and reverse transcription-PCR. The results indicate that HIV-1 undergoes recombination at a minimum rate of 2.8 crossovers per genome per cycle. Again, this is a very high rate given the small size of the HIV-1 genome. The results also suggested that there might be local hot spots of recombination at different locations throughout the genome since 13 of the 33 strand transfers identified by DNA sequencing shared the same site of recombination with one or two other clones. Furthermore, identification of crossover segments also allowed examination of mutations at the point of recombination, since it has been predicted from some studies of cell-free systems that mutations may occur with a frequency of 30 to 50% at crossover junctions. However, DNA sequence analysis of crossover junctions indicated that homologous recombination during viral replication was not particularly mutagenic, indicating that there are other factors or conditions not yet reproduced in cell-free systems which contribute to fidelity during retroviral recombination.

Functional roles of carboxylate residues comprising the DNA polymerase active site triad of Ty3 reverse transcriptase

Aspartic acid residues comprising the -D-(aa)n-Y-L-D-D- DNA polymerase active site triad of reverse transcriptase from the Saccharomyces cerevisiae long terminal repeat-retrotransposon Ty3 (Asp151, Asp213 and Asp214) were evaluated via site-directed mutagenesis. An Asp151→Glu substitution showed a dramatic decrease in catalytic efficiency and a severe translocation defect following initiation of DNA synthesis. In contrast, enzymes harboring the equivalent alteration at Asp213 and Asp214 retained DNA polymerase activity. Asp151→Asn and Asp213→Asn substitutions eliminated both polymerase activities. However, while Asp214 of the triad could be replaced by either Asn or Glu, introducing Gln seriously affected processivity. Mutants of the carboxylate triad at positions 151 and 213 also failed to catalyze pyrophosphorolysis. Finally, alterations to the DNA polymerase active site affected RNase H activity, suggesting a close spatial relationship between these N- and C-terminal catalytic centers. Taken together, our data reveal a critical role for Asp151 and Asp213 in catalysis. In contrast, the second carboxylate of the Y-L-D-D motif (Asp214) is not essential for catalysis, and possibly fulfills a structural role. Although Asp214 was most insensitive to substitution with respect to activity of the recombinant enzyme, all alterations at this position were lethal for Ty3 transposition.

Identification of mesenchymal stem cell differentiation state using dual-micropore microfluidic impedance flow cytometry

As stem cell therapies become more common in the clinic, there is a greater need for real-time, label-free monitoring of the differentiation status of the cells. In this paper, we present a dual-micropore-based, high-throughput microfluidic electrical impedance flow cytometer for non-invasive identification of the differentiation state of mesenchymal stem cells. The mesenchymal stem cells were induced to differentiate into osteoblasts over a 21 day period. Samples of mesenchymal stem cells and osteoblasts were flowed through the device, and impedance measurements were acquired over a frequency range from 50 kHz to 10 MHz. The opacity and relative angle, which shed light on the membrane capacitance and interior dielectric properties of cells, were used as interrogation parameters to analyze collected impedance data. Specifically, identification of mesenchymal stem cells and osteoblasts in a mixed population was optimized using a combination of opacity signature at 500 kHz and relative angle at 3 MHz. Identification of both cell populations in a mixed sample was successfully achieved with an accuracy of 87%. The results show a progressive increase in the number of osteoblasts throughout the 21 day differentiation process, with 36% more mesenchymal stem cells differentiated after 14 days of induction compared to after just 7 days. The dual-micropore microfluidic impedance flow cytometer system may become an important non-invasive tool for assessing stem cell quality and differentiation stages for future regenerative medicine applications.

Label-free mesenchymal stem cell enrichment from bone marrow samples by inertial microfluidics

Isolation of pure populations of mesenchymal stem cells from bone marrow aspirate is a critical need in regenerative medicine such as orthopedic and cartilage reconstruction with important clinical and therapeutic implications. Currently available stem cell isolation systems mainly rely on intrusive immuno-labeling techniques. Mesenchymal stem cells in bone marrow samples are typically larger than other cells, which can be used as a distinctive biophysical cue for non-invasive isolation. In this work, a spiral-shaped inertial microfluidic sorter was developed to isolate mesenchymal stem cells from mouse bone marrow samples with minimal sample preparation steps. To characterize the sorting performance, cultured mesenchymal stem cells were spiked into tissue-digested mouse bone marrow cells. At a flow rate of 1.6 mL min−1, an average enrichment of 6.0× and a recovery of 73% were demonstrated. About 3 × 106 tissue-digested bone marrow cells can be processed in 1 minute with a single microfluidic run. The recovered mesenchymal stem cells after microfluidic sorting retained high (>95%) viability and similar immuno-phenotype expressions and multi-potencies in tri-differentiation lineages to unprocessed cells. This rapid, label-free and non-invasive inertial microfluidic sorter has practical applications in target stem cell enrichment in stem cell therapy.