Persistent HIV-1 transcription in CD4+ T cells from ART-suppressed individuals can originate from biologically competent proviruses.

HIV-1 is able to persist in the face of potent antiretroviral therapy (ART). A number of strategies are being explored to allow ART-free viral remission or viral eradication. In order to gauge the progress of these strategies, assays with which to measure viral reservoir size and activity are needed. In a large percentage of aviremic individuals on suppressive ART, viral transcripts can be detected in peripheral blood CD4+ T cells. While this cell-associated RNA has been considered as a marker of viral reservoir activity, it is unclear whether cell-associated viral transcripts in aviremic individuals originate from biologically competent proviruses as opposed to being a product of abortive transcription from defective proviruses. We assessed whether cell-associated viral RNA in peripheral blood CD4+ T cells from aviremic individuals on ART originated from biologically competent proviruses. We demonstrate that cell-associated viral RNA transcripts were highly related to viral sequences obtained by ex vivo outgrowth. This relationship was also observed when viral transcription in the outgrowth cultures was limited to donor CD4+ T cells. Our study indicates that cell-associated viral RNA warrants further consideration as a viral reservoir surrogate in individuals on suppressive ART.

Measurement of chronic pain in companion animals: Priorities for future research and development based on discussions from the Pain in Animals Workshop (PAW) 2017.

The estimation of long-standing pain in companion animals through the measurement of different dimensions impacted by pain is a fundamental requirement if pain management, and pain therapeutic development, are to advance. Although pain management in veterinary medicine has advanced considerably in the last 20 years, there is much critical work to do in the area of measurement of chronic pain. To date, most work has centered on musculoskeletal pain, and has been focused around the measurement of limb use and the development of owner-completed questionnaires, or clinical metrology instruments (CMI). Recent areas of research have extended to developing measures of activity, sensory function (quantitative sensory testing; nociceptive withdrawal reflexes), and quality of life (QoL). Across all these areas, more data on validity are needed, and studies should be extended to other painful disease states. By necessity, assessing measurement tools requires testing in field studies, which incur considerable time and expense. Facilitating these studies could be optimized with a collaborative (industry, academia and private practice) approach, and the utility of the information produced from all field studies would be enhanced by full and transparent reporting and data sharing, including data already generated by industry in the form of studies submitted to the regulatory authorities.

Measurement of chronic pain in companion animals: Discussions from the Pain in Animals Workshop (PAW) 2017.

In the face of increasing recognition and interest in treating chronic pain in companion animals, we struggle with a lack of therapeutic options. A significant barrier to the development of new therapeutics, or the critical evaluation of current therapies, is our inability to accurately measure chronic pain and its impact on companion animals. Over the last 20 years, much progress has been made in developing methods to measure chronic pain via subjective and objective methods - particularly in owner assessment tools and measurements of limb use and activity. Most work has been focused on chronic joint pain conditions, but there has been relatively little work in other areas of chronic pain, such as neuropathic and cancer pain. Although progress has been made, there is a considerable interest in improving our assessment of chronic pain, as evidenced by the multiple disciplines across industry, academia, and clinical practice from the veterinary and human medical fields that participated in the Pain in Animals Workshop held at the National Institutes of Health in 2017. This review is one product of that meeting and summarizes the current state of knowledge surrounding the measurement of chronic pain (musculoskeletal, cancer, neuropathic), and its impact, in cats and dogs.

The impact of changes in work practice and service delivery on surgical infection rates in a general surgical unit.

Ring-fencing of elective orthopaedic beds has been shown to significantly reduce surgical site infection (SSI) rates. There are fewer studies in general surgical practice. Comparison of overall surgical workload in 2007 and 2011 was performed. Data pertaining to SSI were collected and analysis of this prospectively maintained database was performed on all SSI diagnosed in 2007 and 2011. There was a significant reduction in the crude SSI rate from 117 cases in 2007 (8%) to 42 cases in 2011 (3.5%). A statistically significant reduction in SSI rate for elective surgery was observed, 7.6% vs. 2.5% (p<0.001 Chi-square test). Apart from the introduction of ring fencing, all other contributory variables remained unchanged. Ring-fencing of inpatient general surgical beds has been associated with a significant reduction in SSI rates. These data provide timely supportive evidence that ring-fencing of inpatient beds is an appropriate patient-orientated strategy.

The complete mitochondrial genome of Diadegma semiclausum (hymenoptera: ichneumonidae) indicates extensive independent evolutionary events.

Few complete mitochondrial genomes representing limited families in the order Hymenoptera have been sequenced. Here, we sequenced the complete mitochondrial genome of Diadegma semiclausum (Hymenoptera: Ichneumonidae). This genome is 18 728 bp long, the second largest hexapod mitochondrial genome sequenced in its entirety and that with the highest A+T content at 87.4%. Four tRNAs are rearranged compared with the ancestral arrangement. Gene rearrangement mechanisms are different among all three rearranged regions. Six tRNAs have a large variable loop, which is not found in other metazoan mitochondrial genomes. trnS(AGY) uses the abnormal anticodon TCT but trnK uses the normal CTT. The A+T-rich region is very long (2161 bp). An extremely A+T-rich (99.1%) 1515 bp tandem repeat region with three types of repeat elements is located between cox1 and cox2, and the most likely ancestral element originated from the 3' end of cox1. Independent tandem duplications followed by mutation-insertion-deletion is the best model to explain the formation of this region. These results indicate that independent evolutionary events occurred extensively, such as gene rearrangement events, gene rearrangement mechanisms, derivation of tRNA variable loops, and tandem repeat region evolutionary processes, all of which likely contribute to the diversified features of hymenopteran mitochondrial genomes.

The pharmacogenomics of P-glycoprotein and its role in veterinary medicine.

Despite advancements in pharmacogenetics in human medicine, the incorporation of pharmacogenetics into veterinary medicine is still in its early stages of development. To date, efforts to understand the pharmacologic impact of genetic variation in veterinary species have largely focused on genes encoding for the membrane transporter, P-glycoprotein (P-gp). The emphasis on the role of P-gp is largely because of safety concerns associated with the use of some macrocyclic lactones in dogs. Because of the body of information available on this topic, we use P-gp as a platform for understanding the importance of population diversity in veterinary medicine. The impact of P-gp on drug pharmacokinetics and pharmacodynamics is considered, along with endogenous and exogenous factors that can modulate P-gp activity. The review includes discussion of how population diversity in P-gp activity can lead to susceptibility to certain diseases or alter patient response to environmental stress or pharmaceutical intervention. In addition, phenotypic diversity also needs to be considered, as demonstrated by the impact of P-gp up-regulation and drug resistance. The aim of this review was to set the stage for further exploration into the impact of genetic and phenotypic variability on drug pharmacokinetics, disease propensity, product formulation and drug response in both companion and food-producing animals.

Outcomes of febrile children presumed to be immunocompetent who present with leukopenia or neutropenia to an ambulatory setting.

UNLABELLED: To determine significant medical outcomes in febrile children presenting to an ambulatory setting with neutropenia and/or leukopenia. METHODS: Retrospective medical record review conducted on febrile patients who had a blood culture drawn, with white blood cell counts less than 5,000/mm3 and/or an absolute neutrophil count less than 1000/mm3. Ninety-one patients were identified; 5 with positive blood culture results, 13 with significant non-oncologic disease, and 3 patients with leukemia who had involvement of 2 or more cell lines. CONCLUSIONS: In the majority of patients, clinical judgment, physical findings, and review of all cell lines of the complete blood cell count identified those with significant disease.

Bionomics of Scymnus (Pullus) louisianae J. Chapin (Coleoptera: Coccinellidae) as a predator of the soybean aphid, Aphis glycines Matsumura (Homoptera: Aphididae).

A field population of Scymnus louisianae Chapin (Coccinellidae) was found attacking soybean aphids, Apis glycines Matsumura (Aphidae), a pest recently introduced into Kentucky. This coccinellid had not previously been found in Kentucky. A greenhouse population of S. louisianae was established and its predation on A. glycines studied under laboratory conditions. Total time to develop from egg to adult was about 20 d. About 70% of immatures survived to adulthood and they consumed approximately 100 aphid nymphs per beetle larva during the beetle's four larval instars. Adults lived for an average of 47 d (mated males) and 63 d (mated females) and, during their total adult lifetime, mated males consumed an average of 665 nymphs and mated females consumed 1261 nymphs. All developmental times and predation rates were comparable to those reported for other aphidophagous Scymnus spp. which, in conjunction with reports that Scymnus spp. are effective predators of cotton aphids, Aphis gossypii Glover, suggests that S. louisianae is a potentially important predator of A. glycines in the southern United States.

Glucocorticoid receptor-mediated protection from apoptosis is associated with induction of the serine/threonine survival kinase gene, sgk-1.

We previously demonstrated that activation of the glucocorticoid receptor (GR) initiates an antiapoptotic signal in the immortalized human mammary epithelial cell line MCF10A that is dependent on the GR's transcriptional activity. In this study, we show that the survival role of GR activation extends to protecting human breast cancer cells undergoing apoptosis after growth factor deprivation. Serum and glucocorticoid-regulated kinase-1 (sgk), a gene previously identified as a direct transcriptional target of the activated GR in a rat mammary tumor cell line, was rapidly induced after GR activation in human mammary epithelial cells. Furthermore, in the absence of all growth factors, ectopic sgk expression inhibited apoptosis, suggesting that SGK is a survival kinase. Finally, kinase-dead SGK expression inhibited the protection from apoptosis usually seen after GR activation. These findings suggest that SGK is an important downstream target of GR-mediated survival signaling and that it is distinct from other survival kinases because it can be primarily regulated at the level of transcription.

Two long terminal repeat circles and persistent HIV-1 replication.

Building on the success of combination antiretroviral drug therapy will require a better understanding of the underlying basis for viral persistence. Characterization of the therapeutic, viral, and immunological factors that influence the size and stability of viral reservoirs will foster the development of strategies to control or eliminate HIV-1 from infected individuals. Here we review recent work aimed at delineating the complex interplay between viral replication, the immune system, and viral reservoirs. Finally, we address the implications and clinical significance of the residual replication that persists in infected individuals on potent antiretroviral therapy to evaluate both the possible risks and benefits of ongoing HIV-1 replication.

Early therapy of vertical human immunodeficiency virus type 1 (HIV-1) infection: control of viral replication and absence of persistent HIV-1-specific immune responses.

Studies of potent antiretroviral combination regimens were undertaken in young infants to evaluate the potential for long-term suppression of viral replication and to evaluate the immune consequences of such therapies. Early combination antiretroviral therapy led to a loss of plasma viremia, cultivable virus, and labile extrachromosomal replication intermediates. Despite preservation of immune function, persistent human immunodeficiency type 1 (HIV-1)-specific immune responses were not detected in most infants. The absence of detectable, persisting immune responses in most HIV-1-infected infants treated early contrasts with what is typically seen in adults who are treated early. These results are consistent with the notion that early combination antiretroviral therapy of HIV-1-infected infants allows the long-term suppression of viral replication.

Persistence of episomal HIV-1 infection intermediates in patients on highly active anti-retroviral therapy.

Treatment of HIV-1-infected individuals with a combination of anti-retroviral agents results in sustained suppression of HIV-1 replication, as evidenced by a reduction in plasma viral RNA to levels below the limit of detection of available assays. However, even in patients whose plasma viral RNA levels have been suppressed to below detectable levels for up to 30 months, replication-competent virus can routinely be recovered from patient peripheral blood mononuclear cells and from semen. A reservoir of latently infected cells established early in infection may be involved in the maintenance of viral persistence despite highly active anti-retroviral therapy. However, whether virus replication persists in such patients is unknown. HIV-1 cDNA episomes are labile products of virus infection and indicative of recent infection events. Using episome-specific PCR, we demonstrate here ongoing virus replication in a large percentage of infected individuals on highly active anti-retroviral therapy, despite sustained undetectable levels of plasma viral RNA. The presence of a reservoir of 'covert' virus replication in patients on highly active anti-retroviral therapy has important implications for the clinical management of HIV-1-infected individuals and for the development of virus eradication strategies.

Potential strategies utilised by papillomavirus to evade host immunity.

The co-evolution of papillomaviruses (PV) and their mammalian hosts has produced mechanisms by which PV might avoid specific and non-specific host immune responses. Low level expression of PV proteins in infected basal epithelial cells, together with an absence of inflammation and of virus-induced cell lysis, restricts the opportunity for effective PV protein presentation to immunocytes by dendritic cells. Additionally, PV early proteins, by a range of mechanisms, may restrict the efficacy of antigen presentation by these cells. Should an immune response be induced to PV antigens, resting keratinocytes (KC) appear resistant to interferon-gamma-enhanced mechanisms of cytotoxic T-lymphocyte (CTL)-mediated lysis, and expression of PV antigens by resting KC can tolerise PV-specific CTL. Thus, KC, in the absence of inflammation, may represent an immunologically privileged site for PV infection. Together, these mechanisms play a part in allowing persistence of PV-induced proliferative skin lesions for months to years, even in immunocompetent hosts.

Vpx is required for dissemination and pathogenesis of SIV(SM) PBj: evidence of macrophage-dependent viral amplification.

The viral accessory protein Vpx is required for productive in vitro infection of macrophages by simian immunodeficiency virus from sooty mangabey monkeys (SIV(SM)). To evaluate the roles of Vpx and macrophage infection in vivo, we inoculated pigtailed macaques intravenously or intrarectally with the molecularly cloned, macrophage tropic, acutely pathogenic virus SIV(SM) PBj 6.6, or accessory gene deletion mutants (deltaVpr or deltaVpx) of this virus. Both wild-type and SIV(SM) PBj deltaVpx viruses were readily transmitted across the rectal mucosa. A subsequent 'stepwise' process of local amplification of infection and dissemination was observed for wild-type virus, but not for SIV(SM) PBj deltaVpx, which also showed considerable impairment of the overall kinetics and extent of its replication. In animals co-inoculated with equivalent amounts of wild-type and SIV(SM) Pbj deltaVpx intravenously or intrarectally, the deltaVpx mutant was at a strong competitive disadvantage. Vpx-dependent viral amplification at local sites of initial infection, perhaps through a macrophage-dependent mechanism, may be a prerequisite for efficient dissemination of infection and pathogenic consequences after exposure through either mucosal or intravenous routes.

Differential association of uracil DNA glycosylase with SIVSM Vpr and Vpx proteins.

The HIV-1 Vpr protein is a virion-associated protein which has been shown to facilitate infection of nondividing macrophages and additionally to alter cell cycle and proliferation status of the infected host cell. HIV-1 Vpr also was recently shown to associate with the DNA repair enzyme uracil DNA glycosylase (UDG). This association with a DNA repair enzyme is intriguing given that nonprimate lentiviruses encode a dUTPase, which, like UDG, minimizes the misincorporation of uracil into DNA and is important for virus replication in primary nondividing macrophages but not in dividing cells. This raises the possibility that the dependence upon Vpr for infection of nondividing macrophages may relate to its ability to interact with UDG. Members of the HIV-2/SIVSM group encode, in addition to Vpr, a related protein called Vpx. We previously demonstrated (Fletcher et al., 1996) that Vpx of HIV-2/SIVSM is necessary and sufficient for infection of primary macaque macrophages, while Vpr is not required for macrophage infection but governs cell cycle arrest. Here, we extend on these observations by demonstrating that Vpr, but not Vpx of HIV-2/SIVSM, associates with UDG, which suggests that Vpx facilitates infection of macrophages by a UDG-independent mechanism.

SH3-mediated Hck tyrosine kinase activation and fibroblast transformation by the Nef protein of HIV-1.

Tyrosine kinases of the Src family are regulated via their Src homology 2 (SH2) and SH3 domains. The Nef protein of human immunodeficiency virus-1 (HIV-1) has previously been shown to bind with high affinity and specificity in vitro to the SH3 domain of Hck, a Src family member expressed primarily in myeloid cells. However, the effect of Nef on Hck activity in living cells is unknown. Here we show that Rat-2 fibroblasts co-expressing Hck and Nef rapidly developed transformed foci, whereas control cells expressing either protein alone did not. Nef formed a stable complex with Hck and stimulated its tyrosine kinase activity in vivo. Mutagenesis of the Nef proline-rich motif essential for SH3 binding completely blocked complex formation, kinase activation, and transformation, indicating that the Nef SH3-binding function is required for its effects on Hck. These results provide direct evidence that SH3 engagement is sufficient to activate a Src family kinase in vivo and suggest that Hck may be activated by Nef in HIV-infected macrophages.

Hox genes in evolution: protein surfaces and paralog groups.

The clustered Hox genes, which encode homeodomain transcription factors, control cell fates along the anterior-posterior axis. Differences between Hox proteins cause differences between body parts. Vertebrates have 13 Hox subgroups, called paralog groups, which can be correlated with some of the insect and Amphioxus genes, and have remained distinctive for hundreds of millions of years. We identify characteristic residues that define the different paralog groups. Some paralog groups can be recognized by the homeodomain sequence alone; others only by using characteristic residues outside the homeodomain. Mapping characteristic residues onto the known homeodomain crystal structure reveals that most of the homeodomain amino acids that distinguish paralog groups are oriented away from the DNA, in positions where they might engage in protein-protein interactions.

On the bioactive conformation of a small peptide and its set of thermodynamically accessible conformations.

In order to investigate the relationship between the bioactive conformation of a peptide and its set of thermodynamically accessible structures in solution, the conformational profile of the tetrapeptide Ac-Pro-Ala-Pro-Tyr-OH was characterized by computational methods. Search of the conformational space was performed within the molecular mechanics frame-work using the AMBER4.0 force field with an effective dielectric constant of 80. Unique structures of the peptide were compared with its bioactive conformation for the protein Streptomyces griseus Protease A, as taken from the crystal structure of the enzyme-peptide complex. The results show that the bound conformation is close to one of the unique conformations characterized in the conformational search of the isolated peptide. Moreover, the lowest energy minimum characterized in the conformational search exhibits large deviations when compared to the bound conformation of the crystal structure.

Gaucher's disease in pregnancy.

Gaucher's disease is an autosomal recessive lysosomal storage disease, resulting from a deficiency of the enzyme glucocerebrosidase, important for the physiologic recycling of cell membrane lipids. The clinical symptoms and disease presentations of Gaucher's disease are heterogeneous, including hepatosplenomegaly, bone "crisis" and fracture, anemia, thrombocytopenia and in some forms, rapid neurological decompensation. Similarly, the genetic variability of Gaucher's disease is diverse, and in some aspects affects phenotypic expression. Type 1 Gaucher's disease, however, usually present with less severe symptoms, at more advanced age, and is particularly amenable to enzyme replacement therapy with alglucerase. In type 1 patients with Gaucher's disease reproductive age is commonly reached and childbearing frequently desired with need for appropriate prenatal diagnosis, counseling and careful obstetrical surveillance. Although pregnancy concurrent with Gaucher's disease has been reported in the medical literature, only one small series of alglucerase treated Gaucher's disease during pregnancy exists. Without treatment, pregnancy concurrent with Gaucher's disease has several risks including an increased severity of anemia and thrombocytopenia that can potentiate postpartum bleeding, significant increases in organomegaly and possibly an increased spontaneous abortion rate. It is yet to be shown whether alglucerase reduces the risk of these complications during pregnancy and whether its use has any adverse effect on fetal development.

Matrix metalloproteinase expression during mouse peri-implantation development.

PROBLEM: The purpose of this study was to define the temporal expression and to quantitate the mRNA levels of collagenase, 72 kDa, 92 kDa, and membrane-type matrix metalloproteinase during the peri-implantation period of pregnancy in the mouse uterus. Embryonic expression of 72 kDa and 92 kDa matrix metalloproteinases, as well as interleukin 1 alpha, was also investigated. METHODS: Uterine matrix metalloproteinases were detected using gelatin substrate gel electrophoresis (zymography) and reverse-transcription polymerase chain reaction methodology was used to detect and quantitate different mRNA species in the mouse uterus and blastocyst. RESULTS: Collagenase, 72 kDa, and 92 kDa matrix metalloproteinases are developmentally regulated during the peri-implantation period of pregnancy, but membrane-type matrix metalloproteinase appears to be expressed constitutively. Matrix metalloproteinase mRNA levels have been quantitated and confirm the observed developmental expression patterns. Prominent expression of bot 92 kDa matrix metalloproteinase and interleukin 1 alpha was observed in blastocysts during outgrowth while weak expression of the 72 kDa matrix metalloproteinase was detected. CONCLUSIONS: The date provide evidence of matrix metalloproteinase expression in vivo and substantiate their potential role in tissue remodeling prior to and during blastocyst implantation. Expression of interleukin 1 alpha, 92 kDa, and 72 kDa matrix metalloproteinases suggests that these proteins are important for trophoblast invasion associated with implantation of the early embryo.