Screening for functional transcriptional and splicing regulatory variants with GenIE.

Genome-wide association studies (GWAS) have identified numerous genetic loci underlying human diseases, but a fundamental challenge remains to accurately identify the underlying causal genes and variants. Here, we describe an arrayed CRISPR screening method, Genome engineering-based Interrogation of Enhancers (GenIE), which assesses the effects of defined alleles on transcription or splicing when introduced in their endogenous genomic locations. We use this sensitive assay to validate the activity of transcriptional enhancers and splice regulatory elements in human induced pluripotent stem cells (hiPSCs), and develop a software package (rgenie) to analyse the data. We screen the 99% credible set of Alzheimer's disease (AD) GWAS variants identified at the clusterin (CLU) locus to identify a subset of likely causal variants, and employ GenIE to understand the impact of specific mutations on splicing efficiency. We thus establish GenIE as an efficient tool to rapidly screen for the role of transcribed variants on gene expression.

Differences in Quit Attempts, Successful Quits, Methods, and Motivations in a Longitudinal Cohort of Adult Tobacco Users by Sexual Orientation.

INTRODUCTION: Sexual minority (SM) tobacco users are less likely to successfully quit than heterosexuals, yet little evidence describes cessation behaviors in this population over time. AIMS AND METHODS: Our study investigated quit motivations, attempts, and methods in a longitudinal cohort of adult tobacco users by sexual orientation. Participants (N = 1177) completed interviews every 6 months through 48 months and reported quit attempts (24-hour tobacco free), successful quits (7-day point prevalence abstinence), motivations, and methods. Chi-squared and Fisher's exact tests assessed differences by heterosexual and SM orientation, gender, and quit outcome (attempt-only vs. successful quit). RESULTS: Quit rates were similar for heterosexual and SM adults. Over half attempted to quit at least once over 48 months, but few remained abstinent (SM: 16.9%; heterosexual: 12.1%). Most used nicotine replacement therapy (SM: 31.9%; heterosexual: 26.1%) or tobacco product substitution (SM: 27.7%; heterosexual: 21.2%). Few used quitlines (SM: 4.3%; heterosexual: 1.3%) or Internet-based programs (SM: 6.4%; heterosexual: 1.3%). Quit motivations included health concerns, family, and physical fitness. Participants reporting a successful quit were more likely to report a household member quit smoking than 24-hour quit attempters. Among participants reporting a successful quit, more SM than heterosexual participants reported that a coworker quit smoking (55.6% vs. 33.1%, p = .009). CONCLUSIONS: We found few differences between heterosexual and SM tobacco users in our sample. Many repeatedly attempt to quit, yet few used evidence-based methods. Leveraging online quit programs, health messages, and family members in tailored cessation interventions may help SM and heterosexual tobacco users successfully quit. IMPLICATIONS: SM and heterosexual tobacco users evidenced few differences in quit behaviors. Over 4 years, a majority attempted to quit, with over a third making repeated quit attempts. Nicotine replacement therapy and tobacco product substitution were mostly used during quit attempts; however, more SM than heterosexual men reported using web-based quit programs. Personal health and family concerns were universal motivations to quit, yet SM women also cited physical fitness as a primary motivation. Tobacco users reporting that a household member stopped smoking were more likely to successfully quit. More SM than heterosexual men reported that a coworker quit smoking.

The long non-coding RNA Paupar regulates the expression of both local and distal genes.

Although some long noncoding RNAs (lncRNAs) have been shown to regulate gene expression in cis, it remains unclear whether lncRNAs can directly regulate transcription in trans by interacting with chromatin genome-wide independently of their sites of synthesis. Here, we describe the genomically local and more distal functions of Paupar, a vertebrate-conserved and central nervous system-expressed lncRNA transcribed from a locus upstream of the gene encoding the PAX6 transcription factor. Knockdown of Paupar disrupts the normal cell cycle profile of neuroblastoma cells and induces neural differentiation. Paupar acts in a transcript-dependent manner both locally, to regulate Pax6, as well as distally by binding and regulating genes on multiple chromosomes, in part through physical association with PAX6 protein. Paupar binding sites are enriched near promoters and can function as transcriptional regulatory elements whose activity is modulated by Paupar transcript levels. Our findings demonstrate that a lncRNA can function in trans at transcriptional regulatory elements distinct from its site of synthesis to control large-scale transcriptional programmes.

Infectious Complication Following Midface Reconstruction With Calcified Triglyceride.

This case report describes an infectious complication related to the use of calcified triglyceride (Kryptonite Bone Cement) in post-traumatic midface reconstruction. Ultimately, the infected material required removal, and the facial deformity was repaired with subsequent procedures. The literature suggests that bone cement products should be used with caution when in contact with the paranasal sinuses.

scSNV-seq: high-throughput phenotyping of single nucleotide variants by coupled single-cell genotyping and transcriptomics.

CRISPR screens with single-cell transcriptomic readouts are a valuable tool to understand the effect of genetic perturbations including single nucleotide variants (SNVs) associated with diseases. Interpretation of these data is currently limited as genotypes cannot be accurately inferred from guide RNA identity alone. scSNV-seq overcomes this limitation by coupling single-cell genotyping and transcriptomics of the same cells enabling accurate and high-throughput screening of SNVs. Analysis of variants across the JAK1 gene with scSNV-seq demonstrates the importance of determining the precise genetic perturbation and accurately classifies clinically observed missense variants into three functional categories: benign, loss of function, and separation of function.

Impact of an osteopathic peer recovery coaching model on treatment outcomes in high-risk men entering residential treatment for substance use disorders.

CONTEXT: The United States has witnessed a disproportionate rise in substance use disorders (SUD) and co-occurring mental health disorders, paired with housing instability, especially among racially minoritized communities. Traditional in-patient residential treatment programs for SUD have proven inconsistent in their effectiveness in preventing relapse and maintaining attrition among these patient populations. There is evidence showing that peer recovery programs led by individuals who have lived experience with SUD can increase social support and foster intrinsic motivation within participants to bolster their recovery. These peer recovery programs, when coupled with a standardized training program for peer recovery coaches, may be very efficacious at improving patient health outcomes, boosting performance on Substance Abuse and Mental Health Services Administration (SAMHSA) national outcome measures (NOMs), and helping participants build an overall better quality of life. OBJECTIVES: The goal of this study is to highlight the efficacy of a peer recovery program, the Minority Aids Initiative, in improving health outcomes and associated NOMs in men with SUD and/or co-occurring mental health disorder. METHODS: Participants received six months of peer recovery coaching from trained staff. Sessions were guided by the Manual for Recovery Coaching and focused on 10 different domains of recovery. Participants and coaches set long-term goals and created weekly action plans to work toward them. Standardized assessments (SAMHSA's Government Performance and Results Act [GPRA] tool, Addiction Severity Index [ASI]) were administered by recovery coaches at intake and at the 6-month time point to evaluate participant progress. Analyses of participant recovery were carried out according to SAMHSA's six NOMs and assessed the outcomes of the intervention and their significance. RESULTS: A total of 115 participants enrolled in the program over a 2-year period. Among them, 53 were eligible for 6-month follow-up interviews. In total, 321 sessions were held, with an average of three sessions per participant. Participants showed marked improvement across five of the six NOMs at the end of the 6-month course and across all ASI outcomes, with the exception of three in which participants reported an absence or few symptoms at intake. CONCLUSIONS: Our study shows that participants receive benefits across nearly all NOM categories when paired with recovery coaches who are well trained in medication-assisted treatment (MAT) and medications for opioid use disorder (MOUD) over a 6-month period. We see the following: a higher rate of abstinence; increased housing stability; lower health, behavioral, and social consequences; lower depression and anxiety; longer participant-recovery coach exposure time; and higher follow-up rates. We hope that our results can contribute to advancements and greater acceptance in the implementation of peer recovery coaching as well as an improvement in the lives of the communities affected by substance use.

Solution Conformation of Bovine Leukemia Virus Gag Suggests an Elongated Structure.

Bovine leukemia virus (BLV) is a deltaretrovirus that infects domestic cattle. The structural protein Gag, found in all retroviruses, is a polyprotein comprising three major functional domains: matrix (MA), capsid (CA), and nucleocapsid (NC). Previous studies have shown that both mature BLV MA and NC are able to bind to nucleic acids; however, the viral assembly process and packaging of viral genomic RNA requires full-length Gag to produce infectious particles. Compared to lentiviruses, little is known about the structure of the Gag polyprotein of deltaretroviruses. In this work, structural models of full-length BLV Gag and Gag lacking the MA domain were generated based on previous structural data of individual domains, homology modeling, and flexible fitting to SAXS data using molecular dynamics. The models were used in molecular dynamic simulations to determine the relative mobility of the protein backbone. Functional annealing assays revealed the role of MA in the nucleic acid chaperone activity of BLV Gag. Our results show that full-length BLV Gag has an elongated rod-shaped structure that is relatively rigid, with the exception of the linker between the MA and CA domains. Deletion of the MA domain maintains the elongated structure but alters the rate of BLV Gag-facilitated annealing of two complementary nucleic acids. These data are consistent with a role for the MA domain of retroviral Gag proteins in modulating nucleic acid binding and chaperone activity. IMPORTANCE: BLV is a retrovirus that is found worldwide in domestic cattle. Since BLV infection has serious implications for agriculture, and given its similarities to human retroviruses such as HTLV-1, the development of an effective treatment would have numerous benefits. The Gag polyprotein exists in all retroviruses and is a key player in viral assembly. However, the full-length structure of Gag from any virus has yet to be elucidated at high resolution. This study provides structural data for BLV Gag and could be a starting point for modeling Gag-small molecule interactions with the ultimate goal of developing of a new class of pharmaceuticals.

In vivo function of a novel Siah protein in Drosophila.

The Siah proteins, mammalian homologues of the Drosophila Sina protein, function as E3 ubiquitin ligase enzymes and target a wide range of cellular proteins for degradation. Here, I investigate the in vivo function of the fly protein, Sina-Homologue (SinaH), which is highly similar to Sina. Flies that completely lack SinaH are viable and in combination with a mutation in the gene, Ebi, show an extra dorsal central bristle phenotype. I also show that SinaH and Ebi can interact with each other both in vivo and in vitro suggesting that they act in the same physical complex. Flies that lack both Sina and Sina-Homologue were also created and show visible eye and bristle phenotypes, which can be explained by an inability to degrade the neuronal repressor, Tramtrack. I find no evidence for redundancy in the function of Sina and SinaH.

Health Literacy and Attention to Cigarette Health Warning Labels among Rural Smokers.

OBJECTIVES: We evaluated the association of health literacy and attention to the pictorial imagery of 9 health warning labels (HWLs) in a tobacco advertisement among rural US smokers. We hypothesized that lower health literacy would be associated with greater visual attention to pictorial portions of HWLs and evaluated the association between health literacy and recall of advertisement elements. METHODS: Adult smokers from Appalachian Ohio (N = 180) viewed a pictorial HWL encompassing 20% or 33% of a cigarette advertisement while eye tracking software recorded eye movements toward the advertisement. Health literacy was measured with The Short Test of Functional Health Literacy in Adults. RESULTS: Generalized linear regression revealed that every one-unit decrease in health literacy increased viewing time of the pictorial portion of the health warning label by 1.3 percentage points. Logistic regression revealed that the odds of recalling elements of the pictorial portion of the health warning label increased 20% for every one-unit increase in health literacy. CONCLUSIONS: Rural smokers with lower health literacy view pictorial portions of health warning labels longer than those with greater health literacy supporting that health literacy is an important consideration in health communications, including future cigarette warning labels.

Characteristics of the Tobacco User Adult Cohort in Urban and Rural Ohio.

OBJECTIVES: Identifying characteristics associated with the use of new and emerging tobacco products is a priority. The enumeration and baseline characteristics of a new cohort of adult tobacco users are described. METHODS: Residents, ≥18 years of age, in urban Franklin County, or one of 6 rural Appalachian counties, and who were exclusive users of combustible, smokeless (SLT), or electronic nicotine delivery systems (ENDS) tobacco products, or were dual users, were targeted for recruitment. Participants were interviewed in-person at baseline on sociodemographic characteristics, tobacco product use, and cognitive/affective and purchasing factors. RESULTS: We recruited 1210 participants (urban, N = 595; rural, N = 615). Urban participants were less likely to use tobacco daily, began using tobacco later, used tobacco for less time, and had higher cessation interest. ENDS users were significantly less likely to have made a quit attempt than users of other tobacco products. Duration of tobacco use and nicotine dependence also differed by product type. CONCLUSION: This cohort's enumeration allowed us to compare factors associated with tobacco product preferences and the use of novel products. The inclusion of rural Appalachia-a region with high tobacco use and disease burden-may provide additional insights into the implementation of tobacco control interventions.

RNA sequencing and pathway analysis identify tumor necrosis factor alpha driven small proline-rich protein dysregulation in chronic rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis (CRS) is a heterogeneous inflammatory disorder in which many pathways contribute to end-organ disease. Small proline-rich proteins (SPRR) are polypeptides that have recently been shown to contribute to epithelial biomechanical properties relevant in T-helper type 2 inflammation. There is evidence that genetic polymorphism in SPRR genes may predict the development of asthma in children with atopy and, correlatively, that expression of SPRRs is increased under allergic conditions, which leads to epithelial barrier dysfunction in atopic disease. METHODS: RNAs from uncinate tissue specimens from patients with CRS and control subjects were compared by RNA sequencing by using Ingenuity Pathway Analysis (n = 4 each), and quantitative polymerase chain reaction (PCR) (n = 15). A separate cohort of archived sinus tissue was examined by immunohistochemistry (n = 19). RESULTS: A statistically significant increase of SPRR expression in CRS sinus tissue was identified that was not a result of atopic presence. SPRR1 and SPRR2A expressions were markedly increased in patients with CRS (p < 0.01) on RNA sequencing, with confirmation by using real-time PCR. Immunohistochemistry of archived surgical samples demonstrated staining of SPRR proteins within squamous epithelium of both groups. Pathway analysis indicated tumor necrosis factor (TNF) alpha as a master regulator of the SPRR gene products. CONCLUSION: Expression of SPRR1 and of SPRR2A is increased in mucosal samples from patients with CRS and appeared as a downstream result of TNF alpha modulation, which possibly resulted in epithelial barrier dysfunction.

Twenty-Four Hour Ex Vivo Culture of Celiac Duodenal Biopsies.

Organ culture is a valuable technique in celiac disease research. It provides the opportunity to examine interactions between different cell types during the disease process without the need for invasive in vivo studies. Biopsies are maintained in an oxygen-rich environment, in contact with, but not submerged in, culture medium. A very straightforward and successful method of organ culture is described here.

Adaptation of a Cell-Based High Content Screening System for the In-Depth Analysis of Celiac Biopsy Tissue.

The IN Cell Analyzer 1000 possesses several distinguishing features that make it a valuable tool in research today. This fully automated high content screening (HCS) system introduced quantitative fluorescent microscopy with computerized image analysis for use in cell-based analysis. Previous studies have focused on live cell assays, where it has proven to be a powerful and robust method capable of providing reproducible, quantitative data. Using HCS as a tool to investigate antigen expression in duodenal biopsies, we developed a novel approach to tissue positioning and mapping. We adapted IN Cell Analyzer 1000's image acquisition and analysis software for the investigation of tissue transglutaminase (tTG) and smooth muscle alpha-actin (SM α-actin) staining in paraffin-embedded duodenal tissue sections from celiac patients and healthy controls. These innovations allowed a quantitative analysis of cellular structure and protein expression. The results from routine biopsy material indicated the intensity of protein expression was altered in celiac disease compared to normal biopsy material.

Defining the learning curve for laparoscopic splenectomy for immune thrombocytopenia purpura.

BACKGROUND: The current study was undertaken to define the learning curve for laparoscopic splenectomy (LS) in patients with immune thrombocytopenic purpura (ITP). METHODS: The data of 50 patients who underwent LS for ITP between March 1996 and February 2003 were reviewed. Patients were divided into sequential groups of 10. Operative time, estimated blood loss, conversion to open procedure, length of stay (LOS), time to oral intake, complications, and mortality rates were analyzed. RESULTS: The mean OR time in the 3rd, 4th, and 5th groups of 10 were significantly shorter than the 1st and 2nd groups of 10. There were no significant differences in estimated blood loss, LOS, or time to oral intake between the groups. Three conversions to open splenectomy occurred; one each in the 2nd, 3rd, and 4th groups of 10. Complications were evenly distributed between groups. There were no deaths. CONCLUSION: The learning curve for LS in patients with ITP is a minimum of 20 cases.

Androgens and opiates: testosterone interaction with morphine self-administration in male rats.

Abuse of anabolic androgenic steroids (AAS) and opioids intersects in athletics. Evidence from humans and animals suggests that AAS may act in the brain through opioidergic mechanisms, and may potentiate effects of opioids. To determine whether AAS enhance motivation for opioid intake, in this study, male rats were treated chronically for 6 weeks with high levels of testosterone (7.5 mg/kg) or vehicle subcutaneously, and they were tested for morphine self-administration under fixed-ratio (FR) and progressive-ratio (PR) schedules. Initially, rats received chronic morphine infusion (16.8-50 mg/kg/day) over 7 days. Subsequently, rats were tested for morphine self-administration (3.2 mg/kg) 6 h/day for 3 days under an FR1 schedule, and for 7 days under a PR 9-4 schedule. Under the FR1 schedule, controls self-administered more morphine (95.9±8.5 mg/kg) than testosterone-treated rats (63.2±7.2 mg/kg; P<0.05). Under the PR schedule, there was no effect of testosterone on morphine intake or operant responding (26.7±5.7 responses vs. 30.9±5.9 responses for vehicle; NS). To determine whether testosterone enhances morphine sedation, additional rats were treated with testosterone or vehicle and evaluated for locomotor behavior and rearing activity over 30 min in response to saline or 10 mg/kg morphine. Morphine inhibited locomotor activity and rearing; testosterone selectively reduced rearing behavior, but did not alter locomotor behavior. These results suggest that testosterone does not increase motivation for morphine.

Testosterone enhances risk tolerance without altering motor impulsivity in male rats.

Anabolic-androgenic steroids (AAS) increase impulsive and uncontrolled aggressive ('roid rage) in humans and enhance agonistic behavior in animals. However, the underlying mechanisms for AAS-induced aggression remain unclear. Potential contributing elements include an increase risk-taking and/or motor impulsivity due to AAS. This study addressed the effects of chronic high-dose testosterone on risk tolerance using a risky decision-making task (RDT) and motor impulsivity with a go/no-go task in operant chambers. Male Long-Evans rats were treated for at least 4 weeks with testosterone (7.5mg/kg) or vehicle beginning in late adolescence. Testosterone was used because it is popular among human AAS users. In RDT testing, one lever was paired with delivery of a small "safe" food reward, while the other was paired with a large "risky" reward associated with an increasing risk of footshock (0%, 25%, 50%, 75%, 100%) in successive test blocks. Three shock intensities were used: 1.0, 1.2, and 1.4mA/kg. As shock intensity and risk of shock increased, preference for the lever signifying a large reward significantly declined for both vehicle- and testosterone-treated rats (p<0.05). There was also a significant effect of drug (p<0.05), where testosterone-treated rats showed greater preference for the large reward, compared to vehicle-treated controls. Increased preference for the large reward, despite risk of footshock, is consistent with increased risk tolerance. In go/no-go testing, rats were trained to press a single lever if the go cue was presented (stimulus light) or to refrain from pressing during the no-go cue (tone). There was no effect of testosterone on pre-cue responses, or performance in go and no-go trials. These results suggest that AAS may increase risk-tolerance without altering motor impulsivity.

Solitary chemosensory cells and bitter taste receptor signaling in human sinonasal mucosa.

BACKGROUND: Solitary chemosensory cells (SCCs) are specialized cells in the respiratory epithelium that respond to noxious chemicals including bacterial signaling molecules. SCCs express components of bitter taste transduction including the taste receptor type 2 (TAS2R) bitter taste receptors and downstream signaling effectors: α-Gustducin, phospholipase Cß2 (PLCß2), and transient receptor potential cation channel subfamily M member 5 (TRPM5). When activated, SCCs evoke neurogenic reflexes, resulting in local inflammation. The purpose of this study was to test for the presence SCCs in human sinonasal epithelium, and to test for a correlation with inflammatory disease processes such as allergic rhinitis and chronic rhinosinusitis. METHODS: Patient demographics and biopsies of human sinonasal mucosa were obtained from control patients (n = 7) and those with allergic rhinitis and/or chronic rhinosinusitis (n = 15). Reverse transcription polymerase chain reaction (RT-PCR), quantitative PCR (qPCR), and immunohistochemistry were used to determine whether expression of signaling effectors was altered in diseased patients. RESULTS: RT-PCR demonstrated that bitter taste receptors TAS2R4, TAS2R14, and TAS2R46, and downstream signaling effectors α-Gustducin, PLCß2, and TRPM5 are expressed in the inferior turbinate, middle turbinate, septum, and uncinate of both control and diseased patients. PLCß2/TRPM5-immunoreactive SCCs were identified in the sinonasal mucosa of both control and diseased patients. qPCR showed similar expression of α-Gustducin and TRPM5 in the uncinate process of control and diseased groups, and there was no correlation between level of expression and 22-item Sino-Nasal Outcomes Test (SNOT-22) or pain scores. CONCLUSION: SCCs are present in human sinonasal mucosa in functionally relevant areas. Expression level of signaling effectors was similar in control and diseased patients and did not correlate with measures of pain and inflammation. Further study into these pathways may provide insight into nasal inflammatory diseases and may offer potential therapeutic targets.

Maternal and neonatal outcomes of successful Kielland's rotational forceps delivery.

OBJECTIVE: To estimate the rates of early neonatal and maternal complications in a consecutive series of successful Kielland's rotational forceps deliveries. METHODS: This was a retrospective cohort study of consecutive cases of successful rotational forceps deliveries performed in singleton pregnancies at 36 weeks of gestation or more in a tertiary referral center in Scotland, UK, from 2001 to 2008 (n=873). We also compared outcomes associated with successful rotational forceps deliveries in 2008 (n=150) with those of nonrotational forceps delivery (n=873), ventouse delivery (n=159), spontaneous vertex delivery (n=3,494), and emergency cesarean delivery (n=947). RESULTS: There was one stillbirth associated with a rotational forceps delivery. This was diagnosed before application of forceps. After rotational forceps deliveries, 58 of 872 (6.7%) of live-born neonates were admitted to the neonatal unit. Twenty-seven of 872 (3.1%) neonates had one or more complications that could be attributable to traumatic delivery and seven neonates (0.8%) had a diagnosis of neonatal encephalopathy. When compared with alternative methods of delivery over a single year, neonatal admission rates after delivery by rotational forceps deliveries (5 of 150 [3.3%]) were not significantly different from spontaneous vertex delivery (128 of 3,494 [3.7%; P=1.00]) or ventouse delivery (6 of 159 [3.8%; P=1.00]) and lower than emergency cesarean delivery (106 of 947 [11.2%; P=.002). Postpartum hemorrhage rates after rotational forceps deliveries (8 of 150 [5.3%; P=.008]) were lower than those associated with emergency cesarean delivery (142 of 947 [15.0%; P=.008]). CONCLUSION: Rates of short-term neonatal and maternal complications after successful rotational forceps deliveries are low. LEVEL OF EVIDENCE: II.

Direct cauterization of the nasal septal artery for epistaxis.

Emergency management of epistaxis may include the use of local pressure and vasoconstrictors, chemical or electric cautery, hemostatic agents, nasal packing, embolization, and surgical arterial ligation. There is no definitive protocol for the management of epistaxis, although various protocols have been proposed in the literature. As approaches to surgical ligation of the arterial supply of the nasal cavity have evolved from external carotid ligation to minimally invasive approaches, surgical management of epistaxis has become more effective than embolization and may be less risky. In the surgical management of epistaxis, arterial ligation immediately proximal to the bleeding site is preferred. We propose a simple variation of the endoscopic sphenopalatine artery ligation that may be used to manage epistaxis arising from the nasal septum and floor.

Two modes of degradation of the tramtrack transcription factors by Siah homologues.

The Siah proteins, mammalian homologues of the Drosophila Sina protein, function as ubiquitin-protein isopeptide ligase enzymes to target a wide range of cellular proteins for degradation. We report here a novel Drosophila protein that is homologous to Sina, named Sina-Homologue (SinaH). We show that it can direct the degradation of the transcriptional repressor Tramtrack (Ttk) using two different mechanisms. One is similar to Sina and requires the adaptor Phyllopod, and the other is a novel mechanism of recognition. This novel mode of targeting for degradation is specific for the 69-kDa Ttk isoform, Ttk69. Ttk69 contains a region that is required for binding of SinaH and for SinaH-directed degradation. This region contains an AXVXP motif, which is the consensus sequence found in Siah substrate proteins. These results suggest that degradation directed by SinaH differs from that directed by Sina and is more similar to that found in vertebrates. We speculate that SinaH may be involved in regulating the levels of developmentally important transcription factors.