Brimonidine as a possible treatment for myopia.

BACKGROUND: Myopia is becoming a huge burden on the world's public health systems. The purpose of this study was to explore the effect of brimonidine in the treatment of form-deprivation myopia (FDM) and the relationship between intraocular pressure (IOP) and myopia development. METHODS: Monocular form deprivation myopia (FDM) was induced in three-week-old pigmented male guinea pigs. They were treated with 3 different methods of brimonidine administration (eye drops, and subconjunctival or intravitreal injections). Four different concentrations of brimonidine were tested for each method (2µg/µL, 4µg/µL, 20µg/µL, and 40µg/µL). All treatments continued for a period of 21 days. Tonometry, retinoscopy, and A-scan ultrasonography were used to monitor intraocular pressure, refractive error and axial length (AL), respectively. RESULTS: Treatment with subconjunctival brimonidine at 40µg/µL, and intravitreal brimonidine at 2µg/µL and 4µg/µL, inhibited the development of FDM. The myopic refraction, excessive axial length, and elevation of IOP were significantly decreased. Brimonidine in eye drops was ineffective. CONCLUSION: Brimonidine at appropriate doses significantly reduced the development of FD myopia in guinea pigs. The IOP may change with FD myopia.

ANN-Based Estimation of Low-Latitude Monthly Ocean Latent Heat Flux by Ensemble Satellite and Reanalysis Products.

Ocean latent heat flux (LHF) is an essential variable for air-sea interactions, which establishes the link between energy balance, water and carbon cycle. The low-latitude ocean is the main heat source of the global ocean and has a great influence on global climate change and energy transmission. Thus, an accuracy estimation of high-resolution ocean LHF over low-latitude area is vital to the understanding of energy and water cycle, and it remains a challenge. To reduce the uncertainties of individual LHF products over low-latitude areas, four machine learning (ML) methods (Artificial Neutral Network (ANN), Random forest (RF), Bayesian Ridge regression and Random Sample Consensus (RANSAC) regression) were applied to estimate low-latitude monthly ocean LHF by using two satellite products (JOFURO-3 and GSSTF-3) and two reanalysis products (MERRA-2 and ERA-I). We validated the estimated ocean LHF using 115 widely distributed buoy sites from three buoy site arrays (TAO, PIRATA and RAMA). The validation results demonstrate that the performance of LHF estimations derived from the ML methods (including ANN, RF, BR and RANSAC) were significantly better than individual LHF products, indicated by R2 increasing by 3.7-46.4%. Among them, the LHF estimation using the ANN method increased the R2 of the four-individual ocean LHF products (ranging from 0.56 to 0.79) to 0.88 and decreased the RMSE (ranging from 19.1 to 37.5) to 11 W m-2. Compared to three other ML methods (RF, BR and RANSAC), ANN method exhibited the best performance according to the validation results. The results of relative uncertainty analysis using the triangle cornered hat (TCH) method show that the ensemble LHF product using ML methods has lower relative uncertainty than individual LHF product in most area. The ANN was employed to implement the mapping of annual average ocean LHF over low-latitude at a spatial resolution of 0.25° during 2003-2007. The ocean LHF fusion products estimated from ANN methods were 10-30 W m-2 lower than those of the four original ocean products (MERRA-2, JOFURO-3, ERA-I and GSSTF-3) and were more similar to observations.

Estimation of Daily Terrestrial Latent Heat Flux with High Spatial Resolution from MODIS and Chinese GF-1 Data.

Reliable estimates of terrestrial latent heat flux (LE) at high spatial and temporal resolutions are of vital importance for energy balance and water resource management. However, currently available LE products derived from satellite data generally have high revisit frequency or fine spatial resolution. In this study, we explored the feasibility of the high spatiotemporal resolution LE fusion framework to take advantage of the Moderate Resolution Imaging Spectroradiometer (MODIS) and Chinese GaoFen-1 Wide Field View (GF-1 WFV) data. In particular, three-fold fusion schemes based on Enhanced Spatial and Temporal Adaptive Reflectance Fusion Model (ESTARFM) were employed, including fusion of surface reflectance (Scheme 1), vegetation indices (Scheme 2) and high order LE products (Scheme 3). Our results showed that the fusion of vegetation indices and further computing LE (Scheme 2) achieved better accuracy and captured more detailed information of terrestrial LE, where the determination coefficient (R2) varies from 0.86 to 0.98, the root-mean-square error (RMSE) ranges from 1.25 to 9.77 W/m2 and the relative RSME (rRMSE) varies from 2% to 23%. The time series of merged LE in 2017 using the optimal Scheme 2 also showed a relatively good agreement with eddy covariance (EC) measurements and MODIS LE products. The fusion approach provides spatiotemporal continuous LE estimates and also reduces the uncertainties in LE estimation, with an increment in R2 by 0.06 and a decrease in RMSE by 23.4% on average. The proposed high spatiotemporal resolution LE estimation framework using multi-source data showed great promise in monitoring LE variation at field scale, and may have value in planning irrigation schemes and providing water management decisions over agroecosystems.

The association between liver cirrhosis and fracture risk: A systematic review and meta-analysis.

OBJECTIVE: Liver cirrhosis (LC) was associated with an increased risk of osteoporosis; however, the association between LC and fracture risk was inconclusive. Therefore, this systematic review and meta-analysis aims to explore the association between LC and fracture risk. DESIGN: To identify related literature, a systematic search of PubMed, EMBASE, Web of science and the Cochrane Library from 1965 to July 2017 without language limitation was performed. The random-effects model described by DerSimonian and Laird was used to calculate pooled odds ratios (ORs) and 95% confidence intervals (CIs). RESULTS: Eventually, 5 cohort and 3 case-control studies were identified, which included 321 035 subjects and 31 272 fracture cases. The pooled OR of the association between LC and any fracture risk, hip fracture, spine/trunk fracture and limb fracture was 1.94 (95% CI, 1.59-2.37), 2.11 (95% CI, 1.34-3.32), 2.00 (95% CI, 1.50-2.67) and 1.82 (95% CI, 1.65-2.01), respectively. CONCLUSION: In conclusion, this study indicates that cirrhotic patients have an increased risk of fracture. Preventive measures should be instituted as early as possible.

The Contrasting Effects of Alum-Treated Chicken Manures and KH2PO4 on Phosphorus Behavior in Soils.

Alum [KAl(SO)⋅12HO] is often added to chicken manure to limit P solubility after land application. This is generally ascribed to the formation of Al-PO complexes. However, Al-PO complex formation could be affected by the matrix of chicken manure, which varies with animal diet. Alum was added to KHPO (as a reference material) and two manures from typical chicken farms in China, one from an intensive farm (CMIF) and another from free-ranging chickens (CMFR). These were subsequently incubated with soils for 100 d to investigate P transformations. Alum reduced water-soluble colorimetrically reactive phosphorus (RP) from soils amended with manure more effectively than in soils amended with KHPO. Alum addition lowered Mehlich-3 RP in soils with CMFR but had no influence on Mehlich-3 RP in CMIF- or KHPO-amended soils. A comparison of P in digested Mehlich-3 extracts with RP in undigested samples showed significantly increased P in digests of alum-treated CMFR only. Fractionation data indicated that alum treatment increased P in the NHF-RP (Al-P) fraction only in soils with KHPO, but not in soils with manure treatments. Furthermore, NaOH-extracted nonreactive P was markedly higher in soil with alum-treated CMFR relative to normal CMFR. The CMFR manure was assumed to contain higher concentrations of organic P because these chickens were fed grains only. These results suggest that the formation of alum-organic P complexes may reduce P solubility. By comparing alum-treated KHPO and manures, it appears that organic matter in manure could interfere with the formation of Al-PO complexes.

Intrahepatic Transcriptional Signature Associated with Response to Interferon-α Treatment in the Woodchuck Model of Chronic Hepatitis B.

Recombinant interferon-alpha (IFN-α) is an approved therapy for chronic hepatitis B (CHB), but the molecular basis of treatment response remains to be determined. The woodchuck model of chronic hepatitis B virus (HBV) infection displays many characteristics of human disease and has been extensively used to evaluate antiviral therapeutics. In this study, woodchucks with chronic woodchuck hepatitis virus (WHV) infection were treated with recombinant woodchuck IFN-α (wIFN-α) or placebo (n = 12/group) for 15 weeks. Treatment with wIFN-α strongly reduced viral markers in the serum and liver in a subset of animals, with viral rebound typically being observed following cessation of treatment. To define the intrahepatic cellular and molecular characteristics of the antiviral response to wIFN-α, we characterized the transcriptional profiles of liver biopsies taken from animals (n = 8-12/group) at various times during the study. Unexpectedly, this revealed that the antiviral response to treatment did not correlate with intrahepatic induction of the majority of IFN-stimulated genes (ISGs) by wIFN-α. Instead, treatment response was associated with the induction of an NK/T cell signature in the liver, as well as an intrahepatic IFN-γ transcriptional response and elevation of liver injury biomarkers. Collectively, these data suggest that NK/T cell cytolytic and non-cytolytic mechanisms mediate the antiviral response to wIFN-α treatment. In summary, by studying recombinant IFN-α in a fully immunocompetent animal model of CHB, we determined that the immunomodulatory effects, but not the direct antiviral activity, of this pleiotropic cytokine are most closely correlated with treatment response. This has important implications for the rational design of new therapeutics for the treatment of CHB.

Efficacy and Safety of Ledipasvir/Sofosbuvir With and Without Ribavirin in Patients With Chronic HCV Genotype 1 Infection Receiving Opioid Substitution Therapy: Analysis of Phase 3 ION Trials.

BACKGROUND: Interferon-based hepatitis C virus (HCV) therapy is safe and effective among people receiving opioid substitution therapy (OST), but treatment uptake remains low. Our aim was to evaluate the impact of OST and drug use during therapy on completion, adherence, sustained virologic response (SVR12), and safety of ledipasvir/sofosbuvir ± ribavirin. METHODS: The phase 3 ION studies evaluated a fixed-dose combination of ledipasvir/sofosbuvir ± ribavirin administered for 8, 12, or 24 weeks in patients with chronic HCV genotype 1. People with clinically significant drug use (prior 12 months) or noncannabinoids detected at screening by urine drug tests (not explained by prescriptions) were ineligible. Stored samples were available from ION-1 for retrospective testing for illicit drugs by enzyme-linked immunosorbent assay. RESULTS: Among 1952 patients enrolled in the ION studies, 4% (n = 70) were receiving OST. Among those receiving (n = 70) and not receiving OST (n = 1882), there was no difference in treatment completion (97% vs 98%; P = .40), ≥80% adherence (93% vs 92%; P = 1.00), SVR12 (94% vs 97%; P = .28), and serious adverse events (4% vs 3%; P = .43), respectively. Among participants in the ION-1 trial, 23% (n = 196) used illicit drugs during therapy (15% cannabinoids alone; 8% other illicit drugs ± cannabinoids). There was no difference in treatment completion, ≥80% adherence, SVR12, or serious AEs in those with no drug use during treatment compared with those who used cannabinoids and/or other illicit drugs. No cases of HCV reinfection were observed in the 24 weeks following treatment. CONCLUSIONS: OST and drug use during HCV therapy did not impact treatment completion, adherence, SVR12, or safety. CLINICAL TRIALS REGISTRATION: ION-1 (NCT01701401); ION-2 (NCT01768286); and ION-3 (NCT01851330).

Liver-directed gene therapy of chronic hepadnavirus infection using interferon alpha tethered to apolipoprotein A-I.

BACKGROUND & AIMS: Current hepatitis B virus (HBV) management is challenging as treatment with nucleos(t)ide analogues needs to be maintained indefinitely and because interferon (IFN)-α therapy is associated with considerable toxicity. Previously, we showed that linking IFNα to apolipoprotein A-I generates a molecule (IA) with distinct antiviral and immunostimulatory activities which lacks the hematological toxicity of IFNα. METHODS: Here, we analyse the antiviral potential of an adeno-associated vector encoding IFNα fused to apolipoprotein A-I (AAV-IA) in comparison to a vector encoding only IFNα (AAV-IFN) in two animal models of chronic hepadnavirus infection. RESULTS: In HBV transgenic mice, we found that both vectors induced marked reductions in serum and liver HBV DNA and in hepatic HBV RNA but AAV-IFN caused lethal pancytopenia. Woodchucks with chronic hepatitis virus (WHV) infection that were treated by intrahepatic injection of vectors encoding the woodchuck sequences (AAV-wIFN or AAV-wIA), experienced only a slight reduction of viremia which was associated with hematological toxicity and high mortality when using AAV-wIFN, while AAV-wIA was well tolerated. However, when we tested AAV-wIA or a control vector encoding woodchuck apolipoprotein A-I (AAV-wApo) in combination with entecavir, we found that AAV-wApo-treated animals exhibited an immediate rebound of viral load upon entecavir withdrawal while, in AAV-wIA-treated woodchucks, viremia and antigenemia remained at low levels for several weeks following entecavir interruption. CONCLUSIONS: Treatment with AAV-IA is safe and elicits antiviral effects in animal models with difficult to treat chronic hepadnavirus infection. AAV-IA in combination with nucleos(t)ide analogues represents a promising approach for the treatment of HBV infection in highly viremic patients.

Development and Characterization of Novel EST-SSRs from Larix gmelinii and Their Cross-Species Transferability.

A set of 899 L. gmelinii expression sequence tags (ESTs), available at the National Center of Biotechnology Information (NCBI), was employed to address the feasibility on development of simple sequence repeat (SSR) markers for Larch species. Totally, 634 non-redundant unigenes including 145 contigs and 489 singletons were finally identified and mainly involved in biosynthetic, metabolic processes and response to stress according to BLASTX results, gene ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) maps. Approximately 11.7% (74) unigenes contained 90 candidate SSRs, which were mainly trinucleotides (29, 32.2%) and dinucleotides (26, 28.9%). A relatively high frequency of SSRs was respectively found in the Open Reading Frame (ORF, about 54.4%) and 5'-untranslated region (5'-UTR, 31.2%), while a low frequency was observed in the 3'-untranslated region (3'-UTR, about 14.4%). Of the 45 novel EST-SSRs markers, nine were found to be polymorphic at two L. gmelinii populations. The number of alleles per locus (Na) ranged from two to four, and the observed (Ho) and expected (He) heterozygosity values were 0.200-0.733 and 0.408-0.604, respectively. The inbreeding coefficients (FIS) for all loci were more than zero except Lg41. Most of these 9EST-SSR markers were transferable to its related species L. kaempferi, L. principis-rupprechtii and L. olgensis. These novel EST-SSRs will be useful for further research on comparative genomics, genetic resources conservation and molecular breeding in larch trees.

Rheological behaviors of nylon 6/silica modified melamine cyanurate non-halogen flame retardant composites.

Nylon 6 (PA 6)/silica modified melamine cyanurate (MCA) flame retardant (FR) composites were prepared by melt blending method. The morphology and rheological behaviors of composites were characterized using scanning electron microscopy and capillary rheometer. The results show that the presence of silica can improve the dispersion of mMCA in PA 6. The rheological behaviors are significantly affected by temperature, shear rate and the structure of composites. With increase of temperature and shear rate the apparent viscosities of composites decrease. All composites are pesudoplastic fluids. At same temperature and shear rate PA 6 composites, which contain different flame retardants (FRs) and different loadings, exhibit different viscosities. The presence of MCA can enhance the non-Newtonian index of composites. The flow activation energies (E) of PA 6/silica modified MCA (S-mMCA) composites are larger than that of PA 6/pure MCA composites, and, with the increasing shear rate the flow activation energies of composites decrease. At a fixed FRs content, with the increase of silica loading, E of PA 6/S-mMCA composites increases firstly and then decreases. These results would be useful for the processing of FR PA 6 composites and for further understanding their flame retardant mechanisms.

Ocular Posterior Segment Distribution and Pharmacokinetics of Brimonidine After Intravitreal Administration in Guinea Pigs.

Purpose: Brimonidine is a highly alpha-2 adrenergic agonist, which provides a potential myopia control effect. This study aimed to examine the pharmacokinetics and concentration of brimonidine in the posterior segment tissue of eyes in guinea pigs. Methods: A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was successfully used for brimonidine pharmacokinetics and tissue distribution research in guinea pigs following intravitreal administration (20 µg/eye). Results: Brimonidine concentrations in the retina and sclera were maintained at a high level (>60 ng/g) at 96 h postdosing. Brimonidine concentration peaked in the retina (377.86 ng/g) at 2.41 h and sclera (306.18 ng/g) at 6.98 h. The area under curve (AUC0-∞) was 27,179.99 ng h/g in the retina and 39,529.03 ng h/g in the sclera. The elimination half-life (T1/2e) was 62.43 h in the retina and 67.94 h in the sclera. Conclusions: The results indicated that brimonidine was rapidly absorbed and diffused to the retina and sclera. Meanwhile, it maintained higher posterior tissue concentrations, which can effectively activate the alpha-2 adrenergic receptor. This may provide pharmacokinetic evidence for the inhibition of myopia progression by brimonidine in animal experiments.

Cre-mediated recombination can induce apoptosis in vivo by activating the p53 DNA damage-induced pathway.

Cre-mediated apoptosis has been observed in many contexts in mice expressing Cre-recombinase and can confound the analysis of genetically engineered conditional mutant or transgenic alleles. Several mechanisms have been proposed to explain this phenomenon. We find that the degree of apoptosis induced correlates roughly with the copy number of loxP sites present in the genome and that some level of increased apoptosis accompanies the presence of even only a few loxP sites, as occurs in conditional floxed alleles. Cre-induced apoptosis in this context is completely p53-dependent, suggesting that the apoptosis is stimulated by p53 activation in response to DNA damage incurred during the process of Cre-mediated recombination.

Antiviral Efficacy and Host Innate Immunity Associated with SB 9200 Treatment in the Woodchuck Model of Chronic Hepatitis B.

SB 9200, an oral prodrug of the dinucleotide SB 9000, is being developed for the treatment of chronic hepatitis B virus (HBV) infection and represents a novel class of antivirals. SB 9200 is thought to activate the viral sensor proteins, retinoic acid-inducible gene 1 (RIG-I) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) resulting in interferon (IFN) mediated antiviral immune responses in virus-infected cells. Additionally, the binding of SB 9200 to these sensor proteins could also sterically block the ability of the viral polymerase to access pre-genomic RNA for nucleic acid synthesis. The immune stimulating and direct antiviral properties of SB 9200 were evaluated in woodchucks chronically infected with woodchuck hepatitis virus (WHV) by daily, oral dosing at 15 and 30 mg/kg for 12 weeks. Prolonged treatment resulted in 2.2 and 3.7 log10 reductions in serum WHV DNA and in 0.5 and 1.6 log10 declines in serum WHV surface antigen from pretreatment level with the lower or higher dose of SB 9200, respectively. SB 9200 treatment also resulted in lower hepatic levels of WHV nucleic acids and antigen and reduced liver inflammation. Following treatment cessation, recrudescence of viral replication was observed but with dose-dependent delays in viral relapse. The antiviral effects were associated with dose-dependent and long-lasting induction of IFN-α, IFN-ß and IFN-stimulated genes in blood and liver, which correlated with the prolonged activation of the RIG-I/NOD2 pathway and hepatic presence of elevated RIG-I protein levels. These results suggest that in addition to a direct antiviral activity, SB 9200 induces antiviral immunity during chronic hepadnaviral infection via activation of the viral sensor pathway.

[Modified procedure for prolapse and hemorrhoids to prevent and treat postoperative complications].

OBJECTIVE: To investigate the complications of procedure for prolapse and hemorrhoids (PPH) and their management. METHOD: The clinical data of 147 cases of severe hemorrhoids treated by modified PPH were analyzed in view of the operation method and postoperative complications. RESULT: All the prolapsed hemorrhoids recovered immediately after the operation. The main complications on the first postoperative day were abdominal distention caused by dysuria, pain, and constant sense of defecation urgency, which lasted for 1 to 16 days. Thirty-six cases had intermittent hemafecia 1 to 12 days after operation, improved with expectant treatment. No stoma stenosis, copracrasia or relapse of prolapse occurred during the follow-up for 1 to 37 months. CONCLUSION: All postoperative complications can be prevented, alleviated and cured with modified PPH.

Myofibrotic malformation vessels: unique angiodysplasia toward the progression of hemorrhoidal disease.

BACKGROUND: The etiology and pathogenesis of hemorrhoids is unclear, although hemorrhoids are a worldwide disease in men and women, with peak prevalence at 45-65 years of age. Hemorrhoidal cushions as the anal venous plexi are normal anatomical structures from infancy. This study attempts to reveal the angiodysplasia and other pathological changes in association with different degrees of symptomatic hemorrhoids. MATERIALS AND METHODS: A total of 281 patients with internal hemorrhoids from degree I to IV underwent hemorrhoidectomy. The vascular changes were analyzed by microscopic assessment and software analysis, with Masson's trichrome, CD34, and smooth muscle actin. RESULTS: The hemorrhoidal tissues exhibited abnormal vessels in the mucosae and submucosae that we termed them as myofibrotic malformation vessels (MMVs). MMVs are not ascribed to arteries or veins because they exhibit enlarged and tortuous lumens with smooth muscle dysplasia and fibrotic deposition in the walls without overlying mucosal ulceration. The muscularis mucosae also showed smooth muscle dysplasia and fibrosis, even if it were interrupted by the intruding MMVs. The statistical data indicated that the severity of all the changes correlate positively with the progression of hemorrhoids (P<0.001). Hemorrhoidal patients are prone for reoccurrence even with prolapsing hemorrhoid when compared with the conventional hemorrhoidectomy. Multiple logistic regression analysis showed that MMVs in mucosal propria, mean thickness of mucosal muscularis layer, and fibrotic changes in MMV were independent risk factors for MMVs in hemorrhoidal disease. CONCLUSION: MMVs and muscularis mucosae dysplasia reciprocally contribute to hemorrhoidal exacerbation. The novel findings of this study propose that the characteristic features of MMVs and muscularis mucosae dysplasia of the anorectal tube ultimately cause symptomatic hemorrhoids, which could affect the clinical management of hemorrhoidal disease through the use of surgery to target the malformed vessels.

Extreme hyperopia is the result of null mutations in MFRP, which encodes a Frizzled-related protein.

Nanophthalmos is a rare disorder of eye development characterized by extreme hyperopia (farsightedness), with refractive error in the range of +8.00 to +25.00 diopters. Because the cornea and lens are normal in size and shape, hyperopia occurs because insufficient growth along the visual axis places these lensing components too close to the retina. Nanophthalmic eyes show considerable thickening of both the choroidal vascular bed and scleral coat, which provide nutritive and structural support for the retina. Thickening of these tissues is a general feature of axial hyperopia, whereas the opposite occurs in myopia. We have mapped recessive nanophthalmos to a unique locus at 11q23.3 and identified four independent mutations in MFRP, a gene that is selectively expressed in the eye and encodes a protein with homology to Tolloid proteases and the Wnt-binding domain of the Frizzled transmembrane receptors. This gene is not critical for retinal function, as patients entirely lacking MFRP can still have good refraction-corrected vision, produce clinically normal electro-retinograms, and show only modest anomalies in the dark adaptation of photoreceptors. MFRP appears primarily devoted to regulating axial length of the eye. It remains to be determined whether natural variation in its activity plays a role in common refractive errors.

Circular rapid amplification of cDNA ends for high-throughput extension cloning of partial genes.

The rapid amplification of cDNA ends (RACE) procedure is a widely used PCR-based method to clone the cDNA ends of mRNA transcripts. Current RACE methods often produce a high background of nonspecific PCR products, which can exclude the identification of the target cDNA of interest. We describe here an improved RACE procedure using circular cDNA templates and demonstrate the successful extension cloning of 4406 cDNAs.

Cloning and characterization of a GABAA receptor gamma2 subunit variant.

We have cloned a novel gamma-aminobutyric acid type A (GABAA) receptor gamma2 subunit variant named gamma2XL. gamma2XL contains an alternatively spliced exon, resulting in the addition of 40 amino acids to the N-terminal extracellular domain between Ser171 and Tyr172. We show that gamma2XL failed to localize to the cell surface when it was coexpressed with the alpha2 and beta1 subunits in human embryonic kidney 293 cells. Expression of gamma2XL in 293 cells suppressed GABAA receptor binding in a dose-dependent manner by preventing GABAA receptor cell-surface localization. We also generated a gamma2 mutant with Ser171 and Tyr172 converted to glycine and threonine, respectively. We demonstrate that this mutant has a significantly lower affinity for the alpha2 and beta1 subunits and failed to reach the cell surface when coexpressed with these subunits. Together, our results indicate that Ser171 and Tyr172 in the gamma2 subunit constitute a critical motif. When this motif is disrupted by insertion of the alternative exon, access of the gamma2 subunit to the cell surface is prevented.

PCR isolation and cloning of novel splice variant mRNAs from known drug target genes.

Alternative splicing of pre-mRNAs is an important mechanism for the generation of vertebrate protein diversity. Unfortunately, the contribution of alternative splicing to protein diversity is currently not well understood because many full-length mRNA sequences have yet to be identified. In this report, we describe the use of RT-PCR to identify and clone 279 novel alternatively spliced mRNAs from 114 well-known drug target genes. Our findings demonstrate the existence of many novel alternatively spliced mRNA transcripts and suggest that many more genes undergo functionally significant alternative splicing than previously thought.