Nelfinavir inhibition of Kaposi's sarcoma-associated herpesvirus protein expression and capsid assembly.

BACKGROUND: Antiviral therapies that target herpesviruses are clinically important. Nelfinavir is a protease inhibitor that targets the human immunodeficiency virus (HIV) aspartyl protease. Previous studies demonstrated that this drug could also inhibit Kaposi's sarcoma-associated herpesvirus (KSHV) production. Our laboratory demonstrated nelfinavir can effectively inhibit herpes simplex virus type 1 (HSV-1) replication. For HSV-1 we were able to determine that virus capsids were assembled and exited the nucleus but did not mature in the cytoplasm indicating the drug inhibited secondary envelopment of virions. METHODS: For KSHV, we recently derived a tractable cell culture system that allowed us to analyze the virus replication cycle in greater detail. We used this system to further define the stage at which nelfinavir inhibits KSHV replication. RESULTS: We discovered that nelfinavir inhibits KSHV extracellular virus production. This was seen when the drug was incubated with the cells for 3 days and when we pulsed the cells with the drug for 1-5 min. When KSHV infected cells exposed to the drug were examined using ultrastructural methods there was an absence of mature capsids in the nucleus indicating a defect in capsid assembly. Because nelfinavir influences the integrated stress response (ISR), we examined the expression of viral proteins in the presence of the drug. We observed that the expression of many were significantly changed in the presence of drug. The accumulation of the capsid triplex protein, ORF26, was markedly reduced. This is an essential protein required for herpesvirus capsid assembly. CONCLUSIONS: Our studies confirm that nelfinavir inhibits KSHV virion production by disrupting virus assembly and maturation. This is likely because of the effect of nelfinavir on the ISR and thus protein synthesis and accumulation of the essential triplex capsid protein, ORF26. Of interest is that inhibition requires only a short exposure to drug. The source of infectious virus in saliva has not been defined in detail but may well be lymphocytes or other cells in the oral mucosa. Thus, it might be that a "swish and spit" exposure rather than systemic administration would prevent virion production.

Longitudinal analysis of cost and dental utilization patterns for older adults in outpatient and long-term care settings in Minnesota.

BACKGROUND: Dental utilization patterns and costs of providing comprehensive oral healthcare for older adults in different settings have not been examined. METHODS: Retrospective longitudinal cohort data from Apple Tree Dental (ATD) were analyzed (N = 1,159 total; 503 outpatients, 656 long-term care residents) to describe oral health status at presentation, service utilization patterns, and care costs. Generalized estimating equation (GEE) repeated measures analysis identified significant contributors to service cost over the three-year study period. RESULTS: Cohort mean age was 74 years (range = 55-104); the outpatient (OP) group was younger compared to the long-term care (LTC) group. Half (56%) had Medicaid, 22% had other insurance, and 22% self-paid. Most (72%) had functional dentitions (20+ teeth), 15% had impaired dentitions (9-19 teeth), 6% had severe tooth loss (1-8 teeth), and 7% were edentulous (OP = 2%, LTC = 11%). More in the OP group had functional dentition (83% vs. 63% LTC). The number of appointments declined from 5.0 in Year 1 (OP = 5.7, LTC = 4.4) to 3.3 in Year 3 (OP = 3.6, LTC = 3.0). The average cost to provide dental services was $1,375/year for three years (OP = $1,427, LTC = $1,336), and costs declined each year, from an average of $1,959 (OP = $2,068, LTC = $1,876) in Year 1 to $1,016 (OP = $989, LTC = $1,037) by Year 3. Those with functional dentition at presentation were significantly less costly than those with 1-19 teeth, while edentulous patients demonstrated the lowest cost and utilization. Year in treatment, insurance type, dentition type, and problem-focused first exam were significantly associated with year-over-year cost change in both OP and LTC patients. CONCLUSION: Costs for providing comprehensive dental care in OP and LTC settings were similar, modest, and declined over time. Dentate patients with functional dentition and edentulous patients were less costly to treat. LTC patients had lower utilization than OP patients. Care patterns shifted over time to increased preventive care and decreased restorative care visits.

Geriatric Health Experts Validate Oral Neglect Timelines for the Institutionalized Elderly.

The purpose of this follow-up Delphi survey was to have an expert panel of 31 academic geriatric physicians, geriatric nurses, and medical directors of nursing homes evaluate the original timeline set to avoid oral neglect of nursing home residents. The Oral Neglect in Institutionalized Elderly (ONiIE) timelines defined oral neglect as having occurred when >7 days for acute oral diseases/conditions or >34 days for chronic oral disease/conditions had passed between initial diagnosis and offering access to dental care to the long-term care (LTC) nursing home resident. The results of this follow-up Delphi survey validated those originally defined ONiIE timelines as 90% of this panel agreed with the original timelines. This ONiIE definition adds a broad-based validation for the ONiIE timelines for setting an oral health standard of care for institutionalized elderly residents of nursing homes and should now be used to protect the vulnerable elderly residing in LTC nursing homes.

Activation of NIX-mediated mitophagy by an interferon regulatory factor homologue of human herpesvirus.

Viral control of mitochondrial quality and content has emerged as an important mechanism for counteracting the host response to virus infection. Despite the knowledge of this crucial function of some viruses, little is known about how herpesviruses regulate mitochondrial homeostasis during infection. Human herpesvirus 8 (HHV-8) is an oncogenic virus causally related to AIDS-associated malignancies. Here, we show that HHV-8-encoded viral interferon regulatory factor 1 (vIRF-1) promotes mitochondrial clearance by activating mitophagy to support virus replication. Genetic interference with vIRF-1 expression or targeting to the mitochondria inhibits HHV-8 replication-induced mitophagy and leads to an accumulation of mitochondria. Moreover, vIRF-1 binds directly to a mitophagy receptor, NIX, on the mitochondria and activates NIX-mediated mitophagy to promote mitochondrial clearance. Genetic and pharmacological interruption of vIRF-1/NIX-activated mitophagy inhibits HHV-8 productive replication. Our findings uncover an essential role of vIRF-1 in mitophagy activation and promotion of HHV-8 lytic replication via this mechanism.

CD34 defines melanocyte stem cell subpopulations with distinct regenerative properties.

Melanocyte stem cells (McSCs) are the undifferentiated melanocytic cells of the mammalian hair follicle (HF) responsible for recurrent generation of a large number of differentiated melanocytes during each HF cycle. HF McSCs reside in both the CD34+ bulge/lower permanent portion (LPP) and the CD34- secondary hair germ (SHG) regions of the HF during telogen. Using Dct-H2BGFP mice, we separate bulge/LPP and SHG McSCs using FACS with GFP and anti-CD34 to show that these two subsets of McSCs are functionally distinct. Genome-wide expression profiling results support the distinct nature of these populations, with CD34- McSCs exhibiting higher expression of melanocyte differentiation genes and with CD34+ McSCs demonstrating a profile more consistent with a neural crest stem cell. In culture and in vivo, CD34- McSCs regenerate pigmentation more efficiently whereas CD34+ McSCs selectively exhibit the ability to myelinate neurons. CD34+ McSCs, and their counterparts in human skin, may be useful for myelinating neurons in vivo, leading to new therapeutic opportunities for demyelinating diseases and traumatic nerve injury.

Comparison of Whole Plant and Detached Leaf Screening Techniques for Identifying Anthracnose Resistance in Strawberry Plants.

Anthracnose is a destructive disease of strawberry caused by several Colletotrichum species including C. acutatum, C. fragariae, and C. gloeosporioides. Identification of anthracnose resistant strawberry germplasm has commonly relied on inoculation of whole plants with isolates of these pathogens. In this study, whole plants and detached leaves from 81 germplasm lines were inoculated with a conidial suspension of isolates of C. acutatum, C. fragariae, and C. gloeosporioides, incubated in the dark at 30°C, 100% relative humidity, for 48 h, and assessed for disease severity based on symptoms on inoculated petioles and leaves. The correlation between the disease severity ratings of the whole plants rated 30 days after inoculation and detached leaves rated 5 days after inoculation was determined. Based on leaf symptoms and petiole lesions, the association between the whole plant leaf disease severity rating (DSR) and detached leaf DSR was positive (rp = 0.70), and the association between the whole plant DSR and the detached leaf DSR was also positive (rp = 0.66). Whole plant and detached leaf DSRs were used to assign each germplasm line to a resistance category, and a posthoc Tukey's test showed that the whole plant DSR means and the detached leaf DSR means for each resistance category differed significantly at p < 0.05. This research was used to develop a strawberry detached leaf assay which can reliably and quickly determine the degree of resistance of strawberry germplasm to anthracnose.

Combined effects of aging and inflammation on renin-angiotensin system mediate mitochondrial dysfunction and phenotypic changes in cardiomyopathies.

Although the effects of aging and inflammation on the health of the cardiac muscle are well documented, the combined effects of aging and chronic inflammation on cardiac muscle are largely unknown. The renin-angiotensin system (RAS) has been linked independently to both aging and inflammation, but is understudied in the context of their collective effect. Thus, we investigated localized cardiac angiotensin II type I and type II receptors (AT(1)R, AT(2)R), downstream effectors, and phenotypic outcomes using mouse models of the combination of aging and inflammation and compared it to a model of aging and a model of inflammation. We show molecular distinction in the combined effect of aging and inflammation as compared to each independently. The combination maintained an increased AT(1)R:AT(2)R and expression of Nox2 and exhibited the lowest activity of antioxidants. Despite signaling pathway differences, the combined effect shared phenotypic similarities with aging including oxidative damage, fibrosis, and hypertrophy. These phenotypic similarities have dubbed inflammatory conditions as premature aging, but they are, in fact, molecularly distinct. Moreover, treatment with an AT(1)R blocker, losartan, selectively reversed the signaling changes and ameliorated adverse phenotypic effects in the combination of aging and inflammation as well as each independently.

Modulation of keratocyte phenotype by collagen fibril nanoarchitecture in membranes for corneal repair.

Type I collagen membranes with tailored fibril nanoarchitectures were fabricated through a vitrification processing, which mimicked, to a degree, the collagen maturation process of corneal stromal extracellular matrix in vivo. Vitrification was performed at a controlled temperature of either 5 °C or 39 °C at a constant relative humidity of 40% for various time periods from 0.5 wk up to 8 wk. During vitrification, the vitrified collagen membranes (collagen vitrigels, CVs) exhibited a rapid growth in fibrillar density through the evaporation of water and an increase in fibrillar stiffness due to the formation of new and/or more-stable interactions. On the other hand, the collagen fibrils in CVs maintained their D-periodicity and showed no significant difference in fibrillar diameter, indicating preservation of the native states of the collagen fibrils during vitrification. Keratocyte phenotype was maintained on CVs to varying degrees that were strongly influenced by the collagen fibril nanoarchitectures. Specifically, the vitrification time of CVs mainly governed the keratocyte morphology, showing significant increases in the cell protrusion number, protrusion length, and cell size along with CV vitrification time. The CV vitrification temperature affected the regulation of keratocyte fibroblasts' gene expressions, including keratocan and aldehyde dehydrogenase (ALDH), demonstrating a unique way to control the expression of specific genes in vitro.

Identification and characterization of a functional mitochondrial angiotensin system.

The renin-angiotensin (Ang) system regulates multiple physiological functions through Ang II type 1 and type 2 receptors. Prior studies suggest an intracellular pool of Ang II that may be released in an autocrine manner upon stretch to activate surface membrane Ang receptors. Alternatively, an intracellular renin-Ang system has been proposed, with a primary focus on nuclear Ang receptors. A mitochondrial Ang system has not been previously described. Here we report that functional Ang II type 2 receptors are present on mitochondrial inner membranes and are colocalized with endogenous Ang. We demonstrate that activation of the mitochondrial Ang system is coupled to mitochondrial nitric oxide production and can modulate respiration. In addition, we present evidence of age-related changes in mitochondrial Ang receptor expression, i.e., increased mitochondrial Ang II type 1 receptor and decreased type 2 receptor density that is reversed by chronic treatment with the Ang II type 1 receptor blocker losartan. The presence of a functional Ang system in human mitochondria provides a foundation for understanding the interaction between mitochondria and chronic disease states and reveals potential therapeutic targets for optimizing mitochondrial function and decreasing chronic disease burden with aging.

Oral healthcare access and adequacy in alternative long-term care facilities.

This study was undertaken to determine practices and perceived barriers to access related to oral health by surveying administrators in Michigan alternative long-term care facilities (ALTCF). A 24-item questionnaire was mailed to all 2,275 Michigan ALTCF serving residents aged 60+. Facility response rate was 22% (n = 508). Eleven percent of facilities had a written dental care plan; 18% stated a dentist examined new residents; and 19% of facilities had an agreement with a dentist to come to the facility, with 52% of those being for emergency care only. The greatest perceived barriers were willingness of general and specialty dentists to treat residents at the nursing facility and/or private offices as well as financial concerns. Substantial barriers to care were uniformly perceived. Oral health policies and practices within Michigan ALTCF vary, as measured by resources, attitudes, and the availability of professional care. There is limited involvement by dental professionals in creating policy and providing consultation and service.

Defining oral neglect in institutionalized elderly: a consensus definition for the protection of vulnerable elderly people.

BACKGROUND: The authors administered surveys to develop an operational definition of oral neglect in institutionalized elderly (ONiIE) in the United States. METHODS: The authors administered a Delphi technique survey involving three rounds to a panel of 19 geriatric dental experts in 1995 to arrive at a definition of ONiIE. The authors validated the 1995 ONiIE definition by administering a Delphi technique survey involving four rounds to a subset of eight experts from the 1995 panel. RESULTS: The panelists in the 2009 survey validated the 28 oral diseases or conditions that were part of the 1995 ONiIE definition and added one new oral condition-bleeding. They also reached consensus agreement for each of the 29 listed oral diseases and conditions regarding both the diagnostic stage at which those diseases and conditions should be included in a definition (mild, moderate, or severe) and the specified time period required to constitute neglect (that is, total "time to qualify as neglect" was eight days for acute conditions and 35 days for chronic conditions). CONCLUSIONS: An expert-driven consensus ONiIE definition was established. It consists of 29 oral diseases and conditions, each of which has been associated with a diagnostic stage and a specified time period required to constitute neglect. CLINICAL IMPLICATIONS: Since federal legislation that funds payments to nursing homes for the care and housing of their residents requires that there shall be no oral neglect, this validated consensus ONiIE definition provides a utilitarian means to enforce that legislative expectation.

Managing older patients who have neurologic disease: Alzheimer disease and cerebrovascular accident.

Neurologic diseases represent some of the most common disabling and costly conditions in older age. Alzheimer disease and cerebrovascular accidents (strokes) are two of the most common neurologic conditions, and represent the leading causes of nursing home placement. Dental professionals will be caring for older patients who have age-associated neurologic diseases, including Alzheimer disease and stroke because of the increased longevity of the United States population coupled with improved survivorship of these conditions as a result of advanced medical diagnosis and treatment. Understanding the clinical manifestations of these two common, but distinctly different, neurologic conditions will enable dental professionals to provide safe and rational dental care.

Perceptions of oral health adequacy and access in Michigan nursing facilities.

OBJECTIVE: To determine practices and perceived access barriers (facility resources, attitudes and professional dental involvement) related to oral health by surveying directors of nursing (DONs) in Michigan nursing homes (NHs). BACKGROUND: DONs are crucial to NH practice and policy, so understanding their perceptions of oral health care is vital. METHODS: A 27-item questionnaire exploring aspects of oral health was mailed to all 402 Michigan NH. Descriptive statistics were calculated for response items. RESULTS: Facility response rate was 32% (n = 129). Sixty-three per cent of facilities had a written dental care plan primarily co-ordinated by nursing staff and social workers. Stationary dental equipment was available in 3% of facilities. Thirty-eight per cent stated an examination by a dentist was provided to new residents. Seventy-five per cent of residents identified as needing dental treatment were likely to receive it. Of the 28% of residents receiving dental treatment beyond an examination in the past year, 28% received emergent care. Over 50% of responding DONs indicated satisfaction with how oral hygiene needs were met in their facilities. The greatest perceived barriers were willingness of general and specialty dentists to treat residents at the nursing facility and/or their private offices as well as financial concerns of the resident and/or family. Generally, greater resources were available in urban facilities, but substantial barriers to care were uniformly perceived. CONCLUSION: Oral health policies and practices within Michigan NH vary, as measured by resources, attitudes, and the availability of professional care. Dental involvement in policy creation, provision of consultation, and service is limited.

Improved Microassays Used to Test Natural Product-Based and Conventional Fungicides on Plant Pathogenic Fungi.

Seven important plant pathogenic fungi (Botrytis cinerea, Colletotrichum acutatum, C. fragariae, C. gloeosporioides, Fusarium oxysporum, Phomopsis obscurans, and P. viticola) valuable in screening fungicides were tested. Our procedure included washing conidia to reduce germination times, incorporating Roswell Park Memorial Institute 1640 as a medium of known composition, and using coverslips in the 24-well cell culture clusters to document the effect of fungicides on fungal morphology. The natural product-based fungicide, sampangine, a sampangine analog, 4-bromosampangine, plus seven conventional fungicides (benomyl, captan, cyprodinil, fenbuconazole, fenhexamid, iprodione, and kresoxim-methyl) were tested in vitro for their ability to inhibit germination and growth of the seven fungal species. Sampangine inhibited germination in all fungi except C. acutatum. Comparison of results of germination and morphology microbioassays with results of microtiter assays suggests that some fungicides stop fungal germination, whereas others only slow down fungal growth. We hypothesize that sampangine, except against C. acutatum, has the same physical mode of action, germination inhibition, as the conventional fungicides captan, iprodione, and kresoxim-methyl. 4-Bromosampangine caused morphological anomalies including excessive branching of germ tubes of C. fragariae and splaying and branching of germ tubes of B. cinerea.

Nosocomial pneumonia and oral health.

This article will critically review the evidence linking pneumonia to the aspiration of microbe-laden oropharyngeal secretions and tie that to the predisposition for these processes to affect dependent, medically compromised individuals. The goal of this review is to alert the reader to the role that oral disease and oral health play in fostering and preventing, respectively, widespread and potentially fatal pulmonary disease among high-risk individuals.

What predicts oral health stability in a long-term care population?

The purpose of this study was to determine whether oral health stability was achievable over time for institutionalized elderly who routinely received comprehensive care and to examine the factors associated with stability. Records of 868 dentate nursing home residents, each with a minimum of 24 months continuous care, were analyzed to determine the number of services by type for each time period between periodic examinations. Presenting dental condition, age, gender, functional status, payer source and facility characteristics were tested as explanatory variables. Oral health status was considered stable when a resident had a "good checkup" (needing no further treatment), otherwise it was coded as unstable. Generalized estimating equations (GEE) were used to analyze predictors of stability over time. Stability over time was achieved in 44% of the study group and negatively associated with male gender, advanced age, and more initial treatment needs. The data show that high levels of initial unmet needs were associated with difficulty achieving oral health stability for institutionalized elderly who routinely received comprehensive care.

Rapid identification of germline mutations in retinoblastoma by protein truncation testing.

OBJECTIVE: To demonstrate the utility of protein truncation testing (PTT) for rapid detection and sequencing of germline mutations in the retinoblastoma tumor suppressor gene (RB1). METHODS: We performed PTT, a technique based on the in vitro synthesis of protein from amplified RNA, on 27 probands from 27 kindreds with hereditary retinoblastoma. In 4 kindreds, PTT was also performed on 1 additional affected relative. Ten unrelated patients without retinoblastoma were included as negative control subjects. All PTT-detected mutations were further analyzed by focused sequencing of genomic DNA. When no mutation was detected by PTT, we performed exon-by-exon sequencing, as well as cytogenetic analysis by Giemsa-trypsin-Giemsa banding and by fluorescent in situ hybridization for RB1. The results of proband testing were used for direct genetic testing by polymerase chain reaction and sequencing in 11 relatives from 7 of the 27 kindreds. RESULTS: Of the probands tested, 19 (70%) of 27 tested positive for germline mutations by PTT. In 1 kindred, the proband had negative PTT results but an additional affected relative had positive PTT results. Focused DNA sequencing of 1 patient with positive PTT results from each of the 20 kindreds with positive PTT results revealed truncating mutations in 19 kindreds. Four demonstrated frameshift deletions, 6 had splice site mutations, and 9 showed nonsense mutations. Further analysis by genomic exon-by-exon sequencing and karyotype analysis of the 8 probands who tested negative for germline mutations by PTT revealed 1 splice site mutation, 2 missense mutations, and 1 chromosomal deletion. Focused sequencing based on positive PTT results was successfully used to confirm shared truncating mutations in additional affected family members in 2 kindreds. Using a multitiered approach to genetic testing, 23 (85%) of 27 kindreds had mutations identified and those detected by PTT received a positive result in as few as 7 days. In control subjects, PTT produced no false-positive results. CONCLUSIONS: Protein truncation testing is an effective, rapid single-modality screen for germline mutations in patients with retinoblastoma. When used as an initial screen, PTT can increase the yield of additional testing modalities, such as sequencing and chromosomal analysis, providing a timely and cost-effective approach for the diagnosis of heritable germline mutations in patients with retinoblastoma.Clinical Relevance The clinical application of PTT in retinoblastoma will improve detection of germline retinoblastoma mutations, which will supply critical information for prognosis, treatment planning, follow-up care, and genetic counseling.

Effect of Cercosporella rubi on Blackberry Floral Bud Development.

Rosette, caused by the fungus Cercosporella rubi, is an important blackberry disease in the southeastern United States. This disease severely reduces fruit production, and its management has been erratic due to a limited understanding of the host-pathogen relationship. In this study, we expand on previous histological investigations of the development of C. rubi on blackberry at tissue and cellular levels from floral bud initiation through senescence of the flower. Symptomatic and asymptomatic floral buds were examined with light microscopy and both scanning and transmission electron microscopy (SEM and TEM, respectively). Fungal development on the surface of floral buds was examined with SEM. Previous light microscope histological studies were unclear about whether C. rubi penetrated host tissue. With TEM, we demonstrated an intimate association between fungal and host cells with no penetration prior to death of the host tissue. C. rubi was present on symptomatic floral buds before development and through senescence. No morphological differences were seen between healthy and diseased floral buds ≤5.0 mm in diameter other than the presence of C. rubi. Necrosis was observed in symptomatic buds at the 6.0-mm-diameter stage and progressed through floral senescence.