Extensive 3' modification of plant small RNAs is modulated by helper component-proteinase expression

RNA silencing is an evolutionarily conserved process in eukaryotes that represses gene expression by using 21- to 24-nt guide RNAs to mediate mRNA cleavage or translational inhibition. Plants have two distinct groups of silencing-associated small RNAs (smRNAs): the micro RNAs (miRNAs) and the small interfering RNAs (siRNAs). A recent report by Yu et al. [Yu, B., Yang, Z., Li, J., Minakhina, S., Yang, M., Padgett, R. W., Steward, R. & Chen, X. (2005) Science 307, 932-935] has shown that plant miRNAs are modified at their 3' termini with a methyl group. Here, we show that a large fraction of all silencing-associated smRNAs in tobacco are modified; this modification occurs on the 2' hydroxyl of the terminal ribose and significantly reduces the cloning efficiency of these modified smRNAs. Expression of the strong silencing suppressor P1/helper-component proteinase results in a marked decrease in the 3'-terminal modification of viral siRNAs but does not significantly affect the modification of endogenous miRNAs and 24-nt siRNAs. The differential modification mediated by helper-component proteinase expression implies that exogenous and endogenous smRNAs are processed through independent pathways that are isolated by subcellular compartmentalization and/or the association with distinct Dicer complexes. The degree of terminal modification may play an important role in regulating the extent to which primary smRNA signals can be amplified by RNA-dependent RNA polymerases.     

AUDIT-C as a brief screen for alcohol misuse in primary care

BACKGROUND: The Alcohol Use Disorders Identification Test Consumption (AUDIT-C) questions have been previously validated as a 3-item screen for alcohol misuse and implemented nationwide in Veterans Affairs (VA) outpatient clinics. However, the AUDIT-C's validity and optimal screening threshold(s) in other clinical populations are unknown. METHODS: This cross-sectional validation study compared screening questionnaires with standardized interviews in 392 male and 927 female adult outpatients at an academic family practice clinic from 1993 to 1994. The AUDIT-C, full AUDIT, self-reported risky drinking, AUDIT question #3, and an augmented CAGE questionnaire were compared with an interview primary reference standard of alcohol misuse, defined as a Diagnostic and Statistical Manual, 4th ed. alcohol use disorder and/or drinking above recommended limits in the past year. RESULTS: Based on interviews with 92% of eligible patients, 128 (33%) men and 177 (19%) women met the criteria for alcohol misuse. Areas under the receiver operating characteristic curves (AUROCs) for the AUDIT-C were 0.94 (0.91, 0.96) and 0.90 (0.87, 0.93) in men and women, respectively (p=0.04). Based on AUROC curves, the AUDIT-C performed as well as the full AUDIT and significantly better than self-reported risky drinking, AUDIT question #3, or the augmented CAGE questionnaire (p-values <0.001). The AUDIT-C screening thresholds that simultaneously maximized sensitivity and specificity were > or =4 in men (sensitivity 0.86, specificity 0.89) and > or =3 in women (sensitivity 0.73, specificity 0.91). CONCLUSIONS: The AUDIT-C was an effective screening test for alcohol misuse in this primary care sample. Optimal screening thresholds for alcohol misuse among men (> or =4) and women (> or =3) were the same as in previously published VA studies.     

Clinical results of a novel wide beam reconstruction method for shortening scan time of Tc-99m cardiac SPECT perfusion studies.

BACKGROUND: Newly developed reconstruction algorithms enable the acquisition of images at half of the scan time while maintaining image quality. The purpose of this investigation was to evaluate a novel wide beam reconstruction (WBR) method developed by UltraSPECT for decreasing scan times and to compare it with filtered backprojection (FBP), which is the technique routinely used. METHODS AND RESULTS: Phantom and clinical studies were performed. Hot and cold sphere and cardiac phantom acquisitions were reconstructed via WBR, FBP, and ordered-subsets expectation maximization. Fifty patients were prospectively studied by use of both a standard and a short protocol. The short protocol was performed first on 29 of 50 patients via 8-frame gated technetium 99m stress single photon emission computed tomography and low-energy high-resolution collimators. Stress Tc-99m studies (30-45 mCi) were scanned for 20 seconds per frame. For the short protocol, all parameters remained constant except for the time per frame, which was reduced by 50% on Tc-99m studies. All resting Tc-99m scans (36/50 patients) were processed with FBP for the standard full-scan time studies and with WBR for the short scan studies. The images were interpreted by use of a 17-segment model and 5-degree severity score, and the perfusion and functional variables were determined. Distributions including mean, median, and interquartile ranges were examined for all variables. The differences (FBP - WBR) were computed for all variables and were examined by use of nonparametric signed rank tests to determine whether the median difference was 0. The absolute value of the difference was also examined. Spearman rank-order correlation, a nonparametric measure of association, was used for the 2 methods to determine significant correlations between variables. The hot and cold sphere phantom studies demonstrated that WBR had improved contrast recovery and slightly better background uniformity than did the ordered-subsets expectation maximization. The cardiac phantom studies performed with attenuating medium and background activity showed that the half-scan time images processed with WBR had better contrast recovery and background uniformity than did the full-scan time FBP reconstruction. In the clinical studies, highly significant correlations were observed between WBR and FBP for functional as well as perfusion variables (P < .0001). The summed stress score, summed rest scores, and summed difference score were not statistically different for FBP and WBR (P > .05). Left ventricular volumes had a high correlation coefficient but were significantly larger with FBP than with WBR. CONCLUSION: Our study results suggest that cardiac single photon emission computed tomography perfusion studies may be performed with the WBR algorithm using half of the scan time without compromising qualitative or quantitative imaging results.     

Increased longevity of hematopoiesis in continuous bone marrow cultures derived from NOS1 (nNOS, mtNOS) homozygous recombinant negative mice correlates with radioresistance of hematopoietic and marrow stromal cells

OBJECTIVE: Neuronal nitric oxide synthase (NOS1, mitochondrial NOS, neuronal NOS) homozygous deletion recombinant negative mice demonstrate ionizing irradiation resistance in vivo, attributable to the decrease in mitochondrial-localized production of peroxynitrite, a potent lipid toxic free radical species resulting from the combination of nitric oxide and superoxide. The present studies were designed to determine whether reduced mitochondrial generation of toxic radical oxygen species in NOS1-/- mice also increased the longevity of hematopoiesis in continuous bone marrow cultures and conferred radioresistance to cells in vitro. MATERIALS AND METHODS: Long-term bone marrow cultures (LTBMCs) were established from NOS1-/- and NOS1+/+ littermate mice. Radiation resistance of hematopoietic and marrow stromal cells was measured. Cell cycle analysis and measurement of glutathione and glutathione peroxidase were carried out on irradiated clonal bone marrow stromal cell lines. RESULTS: A significant increase in longevity of hematopoiesis was detected in NOS1-/- mouse LTBMCs for over 64 weeks in culture compared to 20 weeks for NOS1+/+ mouse LTBMCs (p < 0.001). Permanent bone marrow stromal cell lines derived from NOS1-/- mouse LTBMCs demonstrated increased radioresistance in vitro reflected by an increased shoulder on the survival curve with n = 32.15 +/- 1.21 compared to NOS1+/+ cells n = 10.47 +/- 3.2 (p = 0.0026), interleukin-3-dependent NOS1-/- hematopoietic progenitor cell lines also demonstrated decreased apoptosis after 10 Gy irradiation. Both pre- and postirradiation stabilization of the cellular antioxidant pool was detected in NOS1-/- cells. NOS1-/- cells showed a prolonged G1 cell cycle arrest after 10 Gy. CONCLUSIONS: Prolonged hematopoiesis in LTBMCs correlates with intrinsic radioresistance of hematopoietic and marrow stromal cells from NOS1-/- mice. The data confirm the importance to hematopoiesis of mitochondrial localized nitric oxide in both radioresistance and longevity of hematopoiesis in continuous bone marrow cultures.     

Intrapulmonary Pharmacokinetics and Pharmacodynamics of Posaconazole at Steady State in Healthy Subjects

We evaluated the pharmacokinetics (PK) and pharmacodynamics (PD) of posaconazole (POS) in a prospective, open-label study. Twenty-five healthy adults received 14 doses of POS oral suspension (400 mg twice daily) with a high-fat meal over 8 days. Pulmonary epithelial lining fluid (ELF) and alveolar cell (AC) samples were obtained via bronchoalveolar lavage, and blood samples were collected during the 24 h after the last dose. POS concentrations were determined using liquid chromatography with tandem mass spectrometry parameters. The maximum concentrations (C_max) (mean ± standard deviation) in plasma, ELF, and ACs were 2.08 ± 0.93, 1.86 ± 1.30, and 87.7 ± 65.0 μg/ml. The POS concentrations in plasma, ELF, and ACs did not decrease significantly, indicating slow elimination after multiple dosing. The mean concentrations of POS in plasma, ELF, and ACs were above the MIC₉₀ (0.5 μg/ml) for Aspergillus spp. over the 12-h dosing interval and for 24 h following the last dose. Area under the curve from 0 to 12 h (AUC_0-12) ratios for ELF/plasma and AC/plasma were 0.84 and 33. AUC_0-24/MIC₉₀ ratios in plasma, ELF, and AC were 87.6, 73.2, and 2,860. Nine (36%) of 25 subjects had treatment-related adverse events during the course of the study, which were all mild or moderate. We conclude that a dose of 400 mg twice daily resulted in sustained plasma, ELF, and AC concentrations above the MIC₉₀ for Aspergillus spp. during the dosing interval. The intrapulmonary PK/PD of POS are favorable for treatment or prevention of aspergillosis, and oral POS was well tolerated in healthy adults.     

Myeloma and the t(11;14)(q13;q32); evidence for a biologically defined unique subset of patients

The t(11;14)(q13;q32) results in up-regulation of cyclin D1 and is the most common translocation detected in multiple myeloma, where it is also associated with a lymphoplasmacytic morphology. We performed an interphase fluorescent in situ hybridization (FISH) study to determine the clinical and biologic significance of the abnormality when testing a large cohort of myeloma patients. Bone marrow slides from multiple myeloma patients entered into the Eastern Cooperative Oncology Group phase III clinical trial E9486 and associated laboratory correlative study E9487 were analyzed using interphase FISH combined with immune-fluorescent (cytoplasmic immunoglobulin-FISH) detection of clonal plasma cells. We used FISH probes that hybridize to the 14q32 and 11q13 chromosomal loci. The t(11;14)(q13;q32) was correlated with known biologic and prognostic factors. Of 336 evaluable patients, 53 (16%) had abnormal FISH patterns compatible with the t(11;14)(q13;q32). These patients appeared to be more likely to have a serum monoclonal protein of less than 10 g/L (1 g/dL) (28% vs 15%, P =.029) and a lower plasma cell labeling index (P =.09). More strikingly, patients were less likely to be hyperdiploid by DNA content analysis (n = 251, 14% vs 62%, P <.001). Patients with the t(11;14)(q13;q32) appeared to have better survival and response to treatment, although this did not reach statistical significance. Multiple myeloma with the t(11;14)(q13;q32) is a unique subset of patients, not only characterized by cyclin D1 up-regulation and a lymphoplasmacytic morphology, but is also more frequently associated with small serum monoclonal proteins and is much less likely to be hyperdiploid. These patients do not have a worsened prognosis as previously thought.     

BORIS,a novel male germ-line-specific protein associated with epigenetic reprogramming events,shares the same 11-zinc-finger domain with CTCF,the insulator protein involved in reading imprinting marks in the soma

CTCF, a conserved, ubiquitous, and highly versatile 11-zinc-finger factor involved in various aspects of gene regulation, forms methylation-sensitive insulators that regulate X chromosome inactivation and expression of imprinted genes. We document here the existence of a paralogous gene with the same exons encoding the 11-zinc-finger domain as mammalian CTCF genes and thus the same DNA-binding potential, but with distinct amino and carboxy termini. We named this gene BORIS for Brother of the Regulator of Imprinted Sites. BORIS is present only in the testis, and expressed in a mutually exclusive manner with CTCF during male germ cell development. We show here that erasure of methylation marks during male germ-line development is associated with dramatic up-regulation of BORIS and down-regulation of CTCF expression. Because BORIS bears the same DNA-binding domain that CTCF employs for recognition of methylation marks in soma, BORIS is a candidate protein for the elusive epigenetic reprogramming factor acting in the male germ line.     

State-of-the-art behavioral and pharmacological treatments for alcohol use disorder

Background: Alcohol use disorder (AUD) and its associated consequences remain significant public health concerns. Given that AUD represents a spectrum of severity, treatment options represent a continuum of care, ranging from single-session brief interventions to more intensive, prolonged, and specialized treatment modalities. Objective: This qualitative literature review seeks to describe the best practices for AUD by placing a particular emphasis on identifying those practices which have received the most empirical support. Method: This review summarizes psychological and pharmacological intervention options for AUD treatment, with a focus on the relapse prevention phase of recovery. Psychological and pharmacological treatments are summarized in terms of the empirical evidence favoring each approach and the level of AUD severity for which they are most indicated. Scientific significance: One of the broad assertions from this review is that while AUD is highly prevalent, seeking treatment for AUD is not. There are a myriad of behavioral and pharmacological treatments that have shown compelling evidence of efficacy for the treatment of AUD. In the behavioral treatment literature, cognitive behavioral therapy has received the most consistent support. Opioid antagonism (via naltrexone) has been the most widely studied pharmacotherapy and has produced moderate effect sizes. While none of the treatments reviewed herein represents a so-called silver bullet for AUD, they each have the potential to significantly improve the odds of recovery. Precision medicine, or the identification of best treatment matches for individual patients, looms as an important overarching goal for the field, although specific matches are not yet sufficiently reliable in their empirical evidence to warrant clinical dissemination.     

Institutional distrust among gay,bisexual, and other men who have sex with men as a barrier to accessing pre-exposure prophylaxis (PrEP)

Populations at highest risk for acquiring HIV are more likely to pass through criminal justice (CJ) settings, and CJ-involved individuals are often at the intersection of multiple overlapping risk factors. The present study explored interest in, knowledge of, and barriers to PrEP uptake among gay, bisexual, and other men who have sex with men involved in the criminal justice system. Using semi-structured interviews, 26 participants who identified as MSM were asked about PrEP knowledge and interest, HIV risk, and incarceration experience. One theme that emerged across interviews was how institutional distrust in CJ settings may instill lack of trust in medical care after perceived mistreatment. Participants explained how lack of privacy fostered feelings that medical care was not confidential, care received was tied to status as an incarcerated person, and feelings of dehumanization led to distrust. Findings explore how distrust may hinder PrEP uptake and other HIV prevention efforts in CJ settings as well as after release. They highlight the need for greater privacy efforts and cultural humility, and explore how medical settings may function as spaces for people who are incarcerated to disclose HIV risk status. Few studies to our knowledge have examined the role of institutional distrust on men who have sex with men (MSM) in the context of pre-exposure prophylaxis (PrEP) interventions. The present study has implications for creating best practices to structure HIV prevention interventions in CJ settings.     

Toward the development of peptide nanofilaments and nanoropes as smart materials

Protein design studies using coiled coils have illustrated the potential of engineering simple peptides to self-associate into polymers and networks. Although basic aspects of self-assembly in protein systems have been demonstrated, it remains a major challenge to create materials whose large-scale structures are well determined from design of local protein-protein interactions. Here, we show the design and characterization of a helical peptide, which uses phased hydrophobic interactions to drive assembly into nanofilaments and fibrils ("nanoropes"). Using the hydrophobic effect to drive self-assembly circumvents problems of uncontrolled self-assembly seen in previous approaches that used electrostatics as a mode for self-assembly. The nanostructures designed here are characterized by biophysical methods including analytical ultracentrifugation, dynamic light scattering, and circular dichroism to measure their solution properties, and atomic force microscopy to study their behavior on surfaces. Additionally, the assembly of such structures can be predictably regulated by using various environmental factors, such as pH, salt, other molecular crowding reagents, and specifically designed "capping" peptides. This ability to regulate self-assembly is a critical feature in creating smart peptide biomaterials.