Image-based closed-loop feedback for highly mono-dispersed microdroplet production.

Micron-scale droplets isolated by an immiscible liquid can provide miniaturised reaction vessels which can be manipulated in microfluidic networks, and has seen a rapid growth in development. In many experiments, the precise volume of these microdroplets is a critical parameter which can be influenced by many external factors. In this work, we demonstrate the combination of imaging-based feedback and pressure driven pumping to accurately control the size of microdroplets produced in a microfluidic device. The use of fast-response, pressure-driving pumps allows the microfluidic flow to be quickly and accurately changed, while directly measuring the droplet size allows the user to define the more meaningful parameters of droplet size and generation frequency rather than flow rates or pressures. The feedback loop enables the drift correction of pressure based pumps, and leads to a large increase in the mono-dispersity of the droplets produced over long periods. We also show how this can be extended to control multiple liquid flows, allowing the frequency of droplet formation or the average concentration of living cells per droplet to be controlled and kept constant.

Inactivation of the ubiquitin conjugating enzyme UBE2Q2 causes a prophase arrest and enhanced apoptosis in response to microtubule inhibiting agents.

A putative ubiquitin conjugating enzyme known as UBE2Q2 was previously identified in a microarray screen for mitotic regulatory proteins. UBE2Q2 is very similar to another human protein, UBE2Q1 and orthologs from other higher eukaryotic species. In these studies, we demonstrate that UBE2Q2 can covalently bind ubiquitin on the active site cysteine in vitro and show that inhibition of this protein in vivo causes an early mitotic arrest and increased cytotoxicity when cells are treated with microtubule inhibiting agents (MIAs). Changes in cell cycle progression and viability are not observed in the absence of MIA treatment, indicating that UBE2Q2 is involved in the response to MIAs rather than performing a more general function in mitosis. Inhibition of the UBE2Q2 protein causes cells to undergo a prolonged prophase arrest suggesting that UBE2Q2 normally functions to antagonize an early mitotic checkpoint. Furthermore, UBE2Q2 inhibition sensitizes cells to the cytotoxic effects of MIAs through caspase-mediated apoptosis that is correlated with PARP-1 cleavage. These data provide insights into the cellular response to MIAs and demonstrate that inhibition of UBE2Q2 protein function may be useful in the treatment of malignancies.

The G(2) DNA damage checkpoint delays expression of genes encoding mitotic regulators.

Transcriptional control of gene expression contributes to the regulation of diverse cellular processes including cell cycle progression and the cellular response to DNA damage. Global gene expression profiling was performed using p53-deficient human cells to identify genes with G(2)/M-specific and DNA damage-responsive expression. Numerous cell cycle-regulated genes were identified, but surprisingly the analysis failed to identify genes activated by ionizing radiation. Instead, significant delays in expression of G(2)/M-specific genes, including known mitotic regulators, were observed following DNA damage. Thus, in the absence of p53, gene induction does not contribute to the G(2) arrest following DNA damage. Rather, the DNA damage checkpoint elicits a G(2) cell cycle arrest, in part, by delaying accumulation of proteins required in mitosis.

T gamma/delta hepatosplenic lymphoma in a heart transplant patient after an Epstein-Barr virus positive lymphoproliferative disorder: a case report.

BACKGROUND: An unusual case of a peripheral T-cell lymphoma of T gamma/delta hepatosplenic type (Tgamma/deltaHSL) that arose in a child 5 years after she received a heart transplant and 9 months after she developed Epstein-Barr virus (EBV) positive, B-cell lymphoid hyperplasia involving the tonsils is presented. The majority of the reported cases of Tgamma/deltaHSL have been described in young adult men without antecedent immunodeficiency; several well documented cases of Tgamma/deltaHSL in the posttransplant setting have been described previously, but none has been described in a child (or an adult) with a previously diagnosed EBV+ B-cell lymphoid hyperplasia. METHODS: Standard histologic, immunohistochemical, flow cytometric, and molecular genetic techniques were used in the evaluation of diagnostic material. RESULTS: The patient's Tgamma/deltaHSL involved the spleen in a predominantly cordal pattern, and infiltrated the liver in an exclusively sinusoidal distribution. Bone marrow involvement was focal and interstitial. In all locations, malignant cells were of intermediate or large size and had oval nuclei with coarse chromatin, with a scant or moderate amount of eosinophilic cytoplasm. This Tgamma/deltaHSL expressed the characteristic CD2+, CD3+, [CD4- CD8-], Tdelta1+ phenotype, and malignant cells also expressed the natural killer cell marker CD56. Cytogenetic studies demonstrated isochromosome 7q with the addition of trisomy 8 as the tumor progressed. Southern blot analysis demonstrated clonal rearrangements of the gamma, delta, and beta loci of the T-cell receptor but did not identify EBV DNA within the tumor cells. CONCLUSIONS: This case highlights the fact that a full range of lymphoid proliferations is possible in the posttransplantation period, and that a prior diagnosis of a B-cell disorder does not preclude the development of a subsequent T-cell posttransplant lymphoproliferative disorder (PTLD), which should be formally evaluated, especially if clinical circumstances appear atypical for a PTLD of the "usual" (EBV-related, B-cell) type.

Psychosocial outcomes of HIV illness in male and female African American clients.

With the rapid growth of HIV infection among African Americans, the issue of how medical problems relate to psychological functioning in the black community population has acquired new meaning and urgency for health care policy. To develop effective strategies to meet the mental health needs of infected African Americans we need a better understanding of the pattern of Association between HIV and psychological distress. The objective of this study is to test several hypotheses that predict depression and anxiety in black adults infected with HIV. Our conceptual model is derived from learned helplessness theory (Seligman, 1975), the concept of perceived coherence (Antonovsky, 1980; Lewis & Gallison, 1989), and social support theory (Cohen & Willis, 1985). Instruments used in the study include: The Center for Epidemiological Studies-Depression (CES-D) Scale (Radloff, 1977), the Anxiety Scale (Lewis, Firsich, and Parsell, 1979), and the Perceived Coherence Scale (Lewis, 1989). Data were obtained from 255 HIV infected black males and females (age > or = 18) who sought support, counseling, and maintenance services from one of three HIV care and referral centers in the Mid-South. The results of the study emphasize the relative importance of perceived physical symptoms over stage of illness for psychological functioning among African American adults with HIV. Further, the findings also demonstrate the potential importance of perceived coherence for psychological functioning. Black clients who reported higher perceived coherence, regardless of the stage of illness or level of HIV symptoms, had lower anxiety and depression. Significant gender differences in depression are also observed and implications are drawn for strategies to address HIV related mental health care needs of African Americans.

Isolation patterns of the human immunodeficiency virus from cervical secretions during the menstrual cycle of women at risk for the acquired immunodeficiency syndrome.

Human immunodeficiency virus (HIV) has been isolated from both male and female genital secretions. We evaluated the pattern of female genital carriage during the menstrual cycle, and its relationship to HIV viremia. Seven menstruating seropositive women and one seronegative control had cervical secretions and venous blood samples cultured at weekly intervals during a single menstrual cycle. The virus was isolated from cervical secretions in four of seven women. No specific cycle pattern was seen, and positivity for HIV at one site (blood or cervical) did not correlate with positivity at another site. Blood cultures generally, but not always, became positive earlier than cultures from cervical specimens, suggesting higher titers of virus in blood. Thus, HIV secretion may be intermittent. These findings, together with earlier reports, suggest that seropositive women may transmit HIV at any time during the menstrual cycle.

Isolation of HTLV-III/LAV from cervical secretions of women at risk for AIDS.

Cervical secretions from 14 women seropositive for HTLV-III/LAV were obtained between days 7 and 21 of the menstrual cycle and cultured for virus. HTLV-III/LAV was isolated from cervical secretions in 4 of 14 women, as well as from blood of 7 of 13 women tested. Female genital secretions may therefore be a source for sexual transmission of the virus to men.