Single-Shot ChAd3-MARV Vaccine in Modified Formulation Buffer Shows 100% Protection of NHPs.

Marburg virus (MARV) is a virus of high human consequence with a case fatality rate of 24-88%. The global health and national security risks posed by Marburg virus disease (MVD) underscore the compelling need for a prophylactic vaccine, but no candidate has yet reached regulatory approval. Here, we evaluate a replication-defective chimpanzee adenovirus type 3 (ChAd3)-vectored MARV Angola glycoprotein (GP)-expressing vaccine against lethal MARV challenge in macaques. The ChAd3 platform has previously been reported to protect against the MARV-related viruses, Ebola virus (EBOV) and Sudan virus (SUDV), and MARV itself in macaques, with immunogenicity demonstrated in macaques and humans. In this study, we present data showing 100% protection against MARV Angola challenge (versus 0% control survival) and associated production of GP-specific IgGs generated by the ChAd3-MARV vaccine following a single dose of 1 × 1011 virus particles prepared in a new clinical formulation buffer designed to enhance product stability. These results are consistent with previously described data using the same vaccine in a different formulation and laboratory, demonstrating the reproducible and robust protective efficacy elicited by this promising vaccine for the prevention of MVD. Additionally, a qualified anti-GP MARV IgG ELISA was developed as a critical pre-requisite for clinical advancement and regulatory approval.

Bridging Animal and Human Data in Pursuit of Vaccine Licensure.

The FDA Animal Rule was devised to facilitate approval of candidate vaccines and therapeutics using animal survival data when human efficacy studies are not practical or ethical. This regulatory pathway is critical for candidates against pathogens with high case fatality rates that prohibit human challenge trials, as well as candidates with low and sporadic incidences of outbreaks that make human field trials difficult. Important components of a vaccine development plan for Animal Rule licensure are the identification of an immune correlate of protection and immunobridging to humans. The relationship of vaccine-induced immune responses to survival after vaccination and challenge must be established in validated animal models and then used to infer predictive vaccine efficacy in humans via immunobridging. The Sabin Vaccine Institute is pursuing licensure for candidate filovirus vaccines via the Animal Rule and has convened meetings of key opinion leaders and subject matter experts to define fundamental components for vaccine licensure in the absence of human efficacy data. Here, filoviruses are used as examples to review immune correlates of protection and immunobridging. The points presented herein reflect the presentations and discussions during the second meeting held in October 2021 and are intended to address important considerations for developing immunobridging strategies.

Undermining during cutaneous wound closure for wounds less than 3 cm in diameter: a randomized split wound comparative effectiveness trial.

Undermining is thought to improve wound outcomes; however, randomized controlled data regarding its efficacy are lacking in humans. The objective of this randomized clinical trial was to determine whether undermining low to moderate tension wounds improves scar cosmesis compared to wound closure without undermining. Fifty-four patients, 18 years or older, undergoing primary linear closure of a cutaneous defect with predicted postoperative closure length of ≥ 3 cm on any anatomic site were screened. Four patients were excluded, 50 patients were enrolled, and 48 patients were seen in follow-up. Wounds were divided in half and one side was randomized to receive either no undermining or 2 cm of undermining. The other side received the unselected intervention. Three months, patients and 2 masked observers evaluated each scar using the Patient and Observer Scar Assessment Scale (POSAS). A total of 50 patients [mean (SD) age, 67.6 (11.5) years; 31 (64.6%) male; 48 (100%) white] were enrolled in the study. The mean (SD) sum of the POSAS observer component scores was 12.0 (6.05) for the undermined side and 11.1 (4.68) for the non-undermined side (P = .60). No statistically significant difference was found in the mean (SD) sum of the patient component for the POSAS score between the undermined side [15.9 (9.07)] and the non-undermined side [13.33 (6.20)] at 3 months. For wounds under low to moderate perceived tension, no statistically significant differences in scar outcome or total complications were noted between undermined wound halves and non-undermined halves.Trail Registry: Clinical trials.gov Identifier NCT02289859. https://clinicaltrials.gov/ct2/show/NCT02289859 .

GI-19007, a Novel Saccharomyces cerevisiae-Based Therapeutic Vaccine against Tuberculosis.

As yet, very few vaccine candidates with activity in animals against Mycobacterium tuberculosis infection have been tested as therapeutic postexposure vaccines. We recently described two pools of mycobacterial proteins with this activity, and here we describe further studies in which four of these proteins (Rv1738, Rv2032, Rv3130, and Rv3841) were generated as a fusion polypeptide and then delivered in a novel yeast-based platform (Tarmogen) which itself has immunostimulatory properties, including activation of Toll-like receptors. This platform can deliver antigens into both the class I and class II antigen presentation pathways and stimulate strong Th1 and Th17 responses. In mice this fusion vaccine, designated GI-19007, was immunogenic and elicited strong gamma interferon (IFN-γ) and interleukin-17 (IL-17) responses; despite this, they displayed minimal prophylactic activity in mice that were subsequently infected with a virulent clinical strain. In contrast, in a therapeutic model in the guinea pig, GI-19007 significantly reduced the lung bacterial load and reduced lung pathology, particularly in terms of secondary lesion development, while significantly improving survival in one-third of these animals. In further studies in which guinea pigs were vaccinated with BCG before challenge, therapeutic vaccination with GI-19007 initially improved survival versus that of animals given BCG alone, although this protective effect was gradually lost at around 400 days after challenge. Given its apparent ability to substantially limit bacterial dissemination within and from the lungs, GI-19007 potentially can be used to limit lung damage as well as facilitating chemotherapeutic regimens in infected individuals.

Perceptions of Aesthetic Outcome of Linear vs Multiple Z-Plasty Scars in a National Survey.

IMPORTANCE: The process of Z-plasty scar revision breaks up a linear scar into multiple parts with the purpose of camouflage and improvement of the cosmetic appearance of surgical scars. Although this postulation guides the practices of many reconstructive surgeons, few studies support improved aesthetic outcomes. OBJECTIVE: To compare the perceived cosmetic appearance of linear scars vs zigzag scars by the general public. DESIGN, SETTING, AND PARTICIPANTS: A computer-generated image of a mature scar was designed in linear and zigzag configurations and overlaid on the faces of standardized headshots of 4 white individuals. Twelve sets of images of linear vs zigzag scars were arranged in side-by-side comparisons in an Internet-based national survey. Respondents rated each scar on the 10-point Patient and Observer Scar Assessment Scale, where a lower score indicated likeness with normal skin and a higher score, the worst scar imaginable. Data were collected from May 1 through June 30, 2013, and analyzed from July 31 to September 1, 2013. MAIN OUTCOMES AND MEASURES: Aesthetic rating of scars by the survey respondents. RESULTS: Eight hundred seventy-six participants responded to the survey (24.5% response rate); of these, 810 completed the survey (379 men [46.1%] and 443 women [53.9%]; 148 [18.0%] were 18 to 29 years, 171 [20.8%] were 30 to 44 years, 290 [35.3%] were 45 to 60 years, and 213 [25.9%] were older than 60 years). Significantly lower scores and better perceived cosmetic outcomes were found for linear scars compared with zigzag scars in every assessed group of images on the Patient and Observer Scar Assessment Scale (mean [SD] scores, 2.9 [1.6] vs 4.5 [2.2], respectively; P < .001). CONCLUSIONS AND RELEVANCE: The lay public has a significantly better perception of the appearance of linear scars compared with zigzag scars in 3 facial locations (temple, cheek, and forehead) of white patients in various age groups. LEVEL OF EVIDENCE: NA.

Construction and Immunogenicity Testing of Whole Recombinant Yeast-Based T-Cell Vaccines.

GlobeImmune's Tarmogen(®) immunotherapy platform utilizes recombinant Saccharomyces cerevisiae yeast as a vaccine vector to deliver heterologous antigens for activation of disease-specific, targeted cellular immunity. The vaccines elicit immune-mediated killing of target cells expressing viral and cancer antigens in vivo via a CD8(+) CTL-mediated mechanism. Tarmogens are not neutralized by host immune responses and can be administered repeatedly to boost antigen-specific immunity. Production of the vaccines yields stable off-the-shelf products that avoid the need for patient-specific manufacturing found with other immunotherapeutic approaches. Tarmogens for the treatment of chronic hepatitis B and C and various cancers were well tolerated and immunogenic in phase 1 and 2 clinical trials encompassing >600 subjects. The platform is being widely utilized in basic vaccine research and the most rapid path to success in these endeavors follows from optimal immunoassay selection and execution. This chapter provides detailed methods for the construction and preclinical immunogenicity testing of yeast-based immunotherapeutic products to support the rapid and efficient use of this versatile technology.

Reusing surgical instruments during Mohs micrographic surgery: safe from infection, but not free from risk.

We report several scenarios of compromise in patient safety owing to the re-use of mis-assigned patient's surgical instruments in Mohs micrographic surgery.We discuss the breaks in universal protocols that others may experience in their practices and describe corrective measures that our institutions employed to avoid such future events.There is a lack of publication in the literature on the topic of mis-assigned instrument use in Mohs surgery. We believe that the  practice of re-using instruments is cost-effective and therefore common. Based on our humbling experience, this publication may initiate important discussion among dermatologist regarding safety protocols at their respective institutions.

Effect of Adhesive Strips and Dermal Sutures vs Dermal Sutures Only on Wound Closure: A Randomized Clinical Trial.

IMPORTANCE: Although applying adhesive strips to a wound closure has been shown to have outcomes equivalent to those with cuticular suturing, it is unknown whether adhesive strips provide additional benefit compared with dermal suturing alone. OBJECTIVE: To determine whether the addition of adhesive strips to a wound closed with buried interrupted subcuticular sutures improves outcomes following wound closure. DESIGN, SETTING, AND PARTICIPANTS: A prospective, randomized split-wound intervention was conducted between November 14, 2013, and May 16, 2014, in patients who underwent cutaneous surgical procedures at the University of California, Davis, outpatient dermatology clinic. Fifty-seven patients 18 years or older with postoperative defects of at least 3 cm, resulting from either Mohs micrographic surgical procedures or surgical excision, were screened for participation. Nine patients were excluded and 48 were enrolled. INTERVENTIONS: Half of each wound was randomized to receive buried interrupted subcuticular sutures and overlying adhesive strips and the other half received buried interrupted subcuticular sutures only. MAIN OUTCOMES AND MEASURES: At 3 months' follow-up, each patient and 2 blinded observers evaluated the wound using the Patient Observer Scar Assessment Scale. RESULTS: The total mean (SD) Patient Observer Scar Assessment Scale score for observers for the side that received a combination of adhesive strips and buried interrupted subcuticular suturing (12.3 [4.8]) and the side that received sutures only (12.9 [6.3]) did not differ significantly at 3 months (P = .32). There was no significant difference in the total patient assessment scale score between the combination closure (14.0 [7.6]) and sutures only (14.7 [7.6]) sides at 3 months (P = .39). There was also no significant difference between the 2 closure methods in terms of mean (SD) scar width (both methods: 1.1 [0.8] mm, P = .89) at follow-up. CONCLUSIONS AND RELEVANCE: Combination closure with adhesive strips and buried interrupted subcuticular suturing was not significantly associated with improved overall scar assessment compared with buried interrupted subcuticular suturing alone when evaluated by blinded observers or the patients themselves. Our results do not support the use of adhesive strips as a means to improve cosmetic outcomes or reduce scar width. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01979497.

Does wound eversion improve cosmetic outcome?: Results of a randomized, split-scar, comparative trial.

BACKGROUND: Wound edge eversion has been hypothesized to improve aesthetic outcomes after cutaneous wound closure. Data supporting this assertion are sparse. OBJECTIVE: We sought to determine if wound eversion, achieved with interrupted subcuticular sutures, improves aesthetic outcome compared with planar closures. METHODS: We undertook a prospective, randomized, split-scar intervention in patients who underwent cutaneous surgery. Half of the wound was randomized to an everted or planar repair; the other side received the opposite one. At 3- and 6-month follow-up, both the patient and 2 blinded observers evaluated the wound using the Patient Observer Self-Assessment Scale (POSAS). RESULTS: The total observer POSAS score for the everted (13.59, 12.26) and planar (12.91, 12.98) sides did not differ significantly at 3 or 6 months, respectively. Similarly, there was not a significant difference in patient assessment between the everted (16.23, 12.84) and planar (15.07, 12.79) sides at 3 or 6 months, respectively. Finally, there was no significant difference between the 2 closure methods in terms of scar height or width at follow-up. LIMITATIONS: This was a single-center trial, which used a validated but still subjective scar assessment instrument. CONCLUSION: Wound eversion was not significantly associated with improved overall scar assessments by blinded observers or patient assessment.

Purse-string suture vs second intention healing: results of a randomized, blind clinical trial.

IMPORTANCE: Purse-string suture is a closure method that purportedly reduces the scar area compared with second intention healing. Randomized clinical trials comparing these 2 methods appear to be limited or absent. OBJECTIVE: To determine if purse-string suture improves cosmetic outcome, healing time, and scar to defect area compared with second intention healing for circular defects on the trunk and extremities. DESIGN, SETTING, AND PARTICIPANTS: Prospective, 2-arm, randomized, evaluator-blinded clinical trial in a single-center outpatient academic dermatology center. Patients were eligible if they were older than 18 years, able to give informed consent, and had circular or oval postoperative defects larger than 8 mm on the trunk or extremities. INTERVENTIONS: For the purse-string treatment arm, wounds were sewn in circumferential fashion using polydiaxanone suture. Patients in the other treatment arm were allowed to heal by second intent. MAIN OUTCOMES AND MEASURES: The primary outcome measures were the mean total Patient and Observer Scar Assessment Scale (POSAS) scores ascertained from the patient and 2 blinded observers. Secondary outcomes included the ratio of scar to initial defect size, healing time, pain scores, and complication rates. RESULTS: Fifty-two patients were screened, and a total of 44 patients with 50 surgical sites were enrolled. Forty-two patients with 48 surgical sites completed the study. The mean total observer POSAS score was 18.38 for the purse-string group vs 19.91 for the secondary intention group, a nonsignificant difference (P = .41). Similarly, there were no significant differences for any of the following secondary outcome measures: mean total patient POSAS score (P = .96), mean scar-to-defect area (P = .61), and mean pain level at week 1 (P = .19). Statistical trends toward significance were seen in the mean healing time in favor of purse-string suture (P = .10) and scar relief, which favored second intention healing (P = .07). CONCLUSIONS AND RELEVANCE: The purse-string suture results in similar cosmetic outcomes, scar sizes, and pain levels compared with second intention healing for circular or oval wounds on the trunk and extremities. A larger study might better define the potential differences in our secondary outcome measures of healing time and scar relief. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT02062866.

Set-back versus buried vertical mattress suturing: results of a randomized blinded trial.

BACKGROUND: The set-back suture, an absorbable dermal suturing technique, purportedly improves wound eversion and cosmetic outcomes. OBJECTIVE: We sought to conduct a split-wound, prospective, randomized study to compare the cosmetic outcome and wound eversion achieved with the set-back suture and the buried vertical mattress suture (BVMS). METHODS: A total of 46 surgical elliptical wounds were randomized to subcuticular closure with the set-back suture on half and the BVMS on the other. Maximum eversion height and width were measured immediately postoperatively. At 3 months, 2 blinded observers evaluated each scar using a 7-point Likert physician global scar assessment scale. Subjects and observers also completed the validated Patient and Observer Scar Assessment Scale, where a score of 6 represents normal-appearing skin and 60 represents worst imaginable scar. RESULTS: In all, 42 subjects completed the study. The set-back suture provided statistically significant wound eversion. On the Likert scale, observers rated the set-back suture side 1 point better than the BVMS side. Both patient and observer total Patient and Observer Scar Assessment Scale scores were significantly lower for the set-back suture side (subject mean 13.0 ± 8.7 vs 16.2 ± 12.0 [P = .039]; observer mean 24.5 ± 10.4 vs 27.7 ± 13.6 [P = .028], respectively). LIMITATIONS: Single institution experience and relatively short follow-up are limitations. CONCLUSION: The set-back suture provides superior wound eversion and better cosmetic outcomes than the BVMS.

A whole recombinant yeast-based therapeutic vaccine elicits HBV X, S and Core specific T cells in mice and activates human T cells recognizing epitopes linked to viral clearance.

Chronic hepatitis B infection (CHB) is characterized by sub-optimal T cell responses to viral antigens. A therapeutic vaccine capable of restoring these immune responses could potentially improve HBsAg seroconversion rates in the setting of direct acting antiviral therapies. A yeast-based immunotherapy (Tarmogen) platform was used to make a vaccine candidate expressing hepatitis B virus (HBV) X, surface (S), and Core antigens (X-S-Core). Murine and human immunogenicity models were used to evaluate the type and magnitude of HBV-Ag specific T cell responses elicited by the vaccine. C57BL/6J, BALB/c, and HLA-A*0201 transgenic mice immunized with yeast expressing X-S-Core showed T cell responses to X, S and Core when evaluated by lymphocyte proliferation assay, ELISpot, intracellular cytokine staining (ICS), or tumor challenge assays. Both CD4+ and CD8+ T cell responses were observed. Human T cells transduced with HBc18-27 and HBs183-91 specific T cell receptors (TCRs) produced interferon gamma (IFNγ following incubation with X-S-Core-pulsed dendritic cells (DCs). Furthermore, stimulation of peripheral blood mononuclear cells (PBMCs) isolated from CHB patients or from HBV vaccine recipients with autologous DCs pulsed with X-S-Core or a related product (S-Core) resulted in pronounced expansions of HBV Ag-specific T cells possessing a cytolytic phenotype. These data indicate that X-S-Core-expressing yeast elicit functional adaptive immune responses and supports the ongoing evaluation of this therapeutic vaccine in patients with CHB to enhance the induction of HBV-specific T cell responses.

Vaccine-mediated immunotherapy directed against a transcription factor driving the metastatic process.

Numerous reports have now demonstrated that the epithelial-to-mesenchymal transition (EMT) process is involved in solid tumor progression, metastasis, and drug resistance. Several transcription factors have been implicated as drivers of EMT and metastatic progression, including Twist. Overexpression of Twist has been shown to be associated with poor prognosis and drug resistance for many carcinomas and other tumor types. The role of Twist in experimental cancer metastases has been principally studied in the 4T1 mammary tumor model, where silencing of Twist in vitro has been shown to greatly reduce in vivo metastatic spread. Transcription factors such as Twist are generally believed to be "undruggable" because of their nuclear location and lack of a specific groove for tight binding of a small molecule inhibitor. An alternative approach to drug therapy targeting transcription factors driving the metastatic process is T-cell-mediated immunotherapy. A therapeutic vaccine platform that has been previously characterized consists of heat-killed recombinant Saccharomyces cerevisiae (yeast) capable of expressing tumor-associated antigen protein. We report here the construction and characterization of a recombinant yeast expressing the entire Twist protein, which is capable of inducing both CD8(+) and CD4(+) Twist-specific T-cell responses in vivo. Vaccination of mice reduced the size of primary transplanted 4T1 tumors and had an even greater antitumor effect on lung metastases of the same mice, which was dependent on Twist-specific CD8(+) T cells. These studies provide the rationale for vaccine-induced T-cell-mediated therapy of transcription factors involved in driving the metastatic process.

Combination therapy with a second-generation androgen receptor antagonist and a metastasis vaccine improves survival in a spontaneous prostate cancer model.

PURPOSE: Enzalutamide, a second-generation androgen antagonist, was approved by the U.S. Food and Drug Administration (FDA) for castration-resistant prostate cancer (CRPC) treatment. Immunotherapy has been shown to be a promising strategy for prostate cancer. This study was performed to provide data to support the combination of enzalutamide and immunotherapy for CRPC treatment. EXPERIMENTAL DESIGN: Male C57BL/6 or TRAMP (transgenic adenocarcinoma of the mouse prostate) prostate cancer model mice were exposed to enzalutamide and/or a therapeutic vaccine targeting Twist, an antigen involved in epithelial-to-mesenchymal transition and metastasis. The physiologic and immunologic effects of enzalutamide were characterized. The generation of Twist-specific immunity by Twist-vaccine was assessed. Finally, the combination of enzalutamide and Twist-vaccine to improve TRAMP mice overall survival was evaluated. RESULTS: Enzalutamide mediated immunogenic modulation in TRAMP-C2 cells. In vivo, enzalutamide mediated reduced genitourinary tissue weight, enlargement of the thymus, and increased levels of T-cell excision circles. Because no changes were seen in T-cell function, as determined by CD4(+) T-cell proliferation and regulatory T cell (Treg) functional assays, enzalutamide was determined to be immune inert. Enzalutamide did not diminish the ability of Twist-vaccine to generate Twist-specific immunity. Twist was confirmed as a valid tumor antigen in TRAMP mice by immunohistochemistry. The combination of enzalutamide and Twist-vaccine resulted in significantly increased overall survival of TRAMP mice compared with other treatment groups (27.5 vs. 10.3 weeks). Notably, the effectiveness of the combination therapy increased with disease stage, i.e., the greatest survival benefit was seen in mice with advanced-stage prostate tumors. CONCLUSIONS: These data support the combination of enzalutamide and immunotherapy as a promising treatment strategy for CRPC. Clin Cancer Res; 19(22); 6205-18. ©2013 AACR.

To cut or not to cut? A review of aesthetic approaches to the facial subcutaneous cyst.

The treatment of facial cysts often entails some thorny decision making for the dermatologist. We offer a review of several approaches for their removal or modification and examine the outcome evidence for some common techniques and some that are seen less often. Finally, we offer our recommendations based on this trial evidence.

Strong dependence between functional domains in a dual-function snoRNA infers coupling of rRNA processing and modification events.

Most small nucleolar RNAs (snoRNAs) guide rRNA nucleotide modifications, some participate in pre-rRNA cleavages, and a few have both functions. These activities involve direct base-pairing of the snoRNA with pre-rRNA using different domains. It is not known if the modification and processing functions occur independently or in a coordinated manner. We address this question by mutational analysis of a yeast box H/ACA snoRNA that mediates both processing and modification. This snoRNA (snR10) contains canonical 5'- and 3'-hairpin structures with a guide domain for pseudouridylation in the 3' hairpin. Our functional mapping results show that: (i) processing requires the 5' hairpin exclusively, in particular a 7-nt element; (ii) loss of the 3' hairpin or pseudouridine does not affect rRNA processing; (iii) a single nucleotide insertion in the guide domain shifts modification to an adjacent uridine in rRNA, and severely impairs both processing and cell growth; and (iv) the deleterious effects of the insertion mutation depend on the presence of the processing element in the 5' hairpin, but not modification of the novel site. Together, the results suggest that the snoRNA hairpins function in a coordinated manner and that their interactions with pre-rRNA could be coupled.

Whole recombinant yeast-based immunotherapy induces potent T cell responses targeting HCV NS3 and Core proteins.

Control of primary infection with hepatitis C virus (HCV) is associated with robust and broad T cell immunity. In contrast, chronic infection is characterized by weak T cell responses suggesting that an approach that boosts these responses could be a therapeutic advance. Saccharomyces cerevisiae is an effective inducer of innate and adaptive cellular immunity and we have generated recombinant yeast cells (GI-5005) that produce an HCV NS3-Core fusion protein. Pre-clinical studies in mice showed that GI-5005 induced potent antigen-specific proliferative and cytotoxic T cell responses that were associated with Th1-type cytokine secretion. In studies in which GI-5005 was administered up to 13 times, no detectable vector neutralization or induction of tolerance was observed. Prophylactic as well as therapeutic administration of GI-5005 in mice led to eradication of tumor cells expressing HCV NS3 protein. Immunotherapy with GI-5005 is being evaluated in chronic HCV infected individuals in a Phase 1 clinical trial.

Yeasts encoding tumour antigens in cancer immunotherapy.

Immunotherapy for cancer represents an attractive therapeutic target because of its specificity and lack of toxicity, but products investigated so far have been limited by neutralisation, complexity of manufacturing and requirement for patient-specific products. Recombinant yeast cells are capable of stimulating the immune system to produce highly specific and potent cellular responses against target protein antigens with little toxicity. Data from animal models suggest that Tarmogens (yeast-based immunotherapeutics) can elicit protective immunity against xenografted and chemically induced tumours. This concept is now being tested in a Phase I trial in patients with colorectal, pancreatic and non-small cell lung cancers.

Antisense and RNAi: powerful tools in drug target discovery and validation.

Drug target discovery and validation are complex processes that require significant resource investments and impose a substantial economic burden on the pharmaceutical industry. Technologies that accelerate or enhance the precision of target selection are, therefore, in high demand. Traditional antisense and RNA interference (RNAi) technologies are powerful tools with applications in multiple phases of drug target discovery and validation. These approaches elicit potent and highly selective cleavage of a target mRNA, permitting evaluation of the role of the corresponding protein based on a loss-of-function phenotype. Incorporation of these technologies into high-throughput screens, in vitro biological assays and in vivo disease models provides valuable insight into gene function. Efforts are also underway to develop these agents as drugs. This review presents recent studies involving antisense and RNAi, and discusses how these technologies are facilitating target selection at various stages of the drug development process.

Ribosome structure and activity are altered in cells lacking snoRNPs that form pseudouridines in the peptidyl transferase center.

One of the oldest questions in RNA science is the role of nucleotide modification. Here, the importance of pseudouridine formation (Psi) in the peptidyl transferase center of rRNA was examined by depleting yeast cells of 1-5 snoRNAs that guide a total of six Psi modifications. Translation was impaired substantially with loss of a conserved Psi in the A site of tRNA binding. Depletion of other Psis had subtle or no apparent effect on activity; however, synergistic effects were observed in some combinations. Pseudouridines are proposed to enhance ribosome activity by altering rRNA folding and interactions, with some Psis having greater effects than others. The possibility that modifying snoRNPs might affect ribosome structure in other ways is also discussed.