The Effectiveness of a Nurse-led Glaucoma Education on Patient Knowledge and Compliance Motivation Levels: A 1-year Prospective Case Series.

Purpose: To evaluate the impact of a nurse-led glaucoma education program on patient knowledge and compliance levels in an Asian population. Materials and Methods: A 1-year prospective case series involving 69 adult glaucoma patients. Each patient attended a standardized nurse-led glaucoma education session. A questionnaire was administered by a single nurse-clinician and analyzed at three time points (preeducation for baseline, immediately posteducation, and at the 1-year follow-up) to evaluate for associations with patient knowledge and compliance motivation levels. Results: A total of 64 patients were included in the final analysis. Patients with higher educational qualifications or who were employed had better baseline knowledge of glaucoma. Younger patients had higher baseline compliance motivation levels. Immediately posteducation, both median patient knowledge score and compliance motivation levels had a statistically significant increase. Patients on more glaucoma eye drops had greater immediate improvement in confidence in eye drop application. Patients with more positive Humphrey visual field mean deviation values had a greater immediate improvement in confidence in their understanding of glaucoma. A total of 34 patients were readministered the questionnaire at the 1-year time point. Median score for patient knowledge was highest at this point. Employed patients demonstrated better patient knowledge at baseline and at 1-year time point compared to unemployed patients. Unemployed patients experienced a significant improvement in scores from baseline to immediately posteducation, but improvement from immediately posteducation to the 1-year time point was insignificant was insignificant. Conclusion: Our study has examined the effectiveness of a nurse-led glaucoma education program in an Asian population, demonstrating improvement in both patient knowledge and compliance motivation levels up to 1 year after intervention. How to cite this article: Sng JJ, Ang BCH, Soo Hoo WC, et al. The Effectiveness of a Nurse-led Glaucoma Education on Patient Knowledge and Compliance Motivation Levels: A 1-year Prospective Case Series. J Curr Glaucoma Pract 2023;17(3):149-156.

An ATP-independent role for Prp16 in promoting aberrant splicing.

The spliceosome is assembled through a step-wise process of binding and release of its components to and from the pre-mRNA. The remodeling process is facilitated by eight DExD/H-box RNA helicases, some of which have also been implicated in splicing fidelity control. In this study, we unveil a contrasting role for the prototypic splicing proofreader, Prp16, in promoting the utilization of aberrant 5' splice sites and mutated branchpoints. Prp16 is not essential for the branching reaction in wild-type pre-mRNA. However, when a mutation is present at the 5' splice site or if Cwc24 is absent, Prp16 facilitates the reaction and encourages aberrant 5' splice site usage independently of ATP. Prp16 also promotes the utilization of mutated branchpoints while preventing the use of nearby cryptic branch sites. Our study demonstrates that Prp16 can either enhance or impede the utilization of faulty splice sites by stabilizing or destabilizing interactions with other splicing components. Thus, Prp16 exerts dual roles in 5' splice site and branch site selection, via ATP-dependent and ATP-independent activities. Furthermore, we provide evidence that these functions of Prp16 are mediated through the step-one factor Cwc25.

The DExD/H-box protein Prp16 has a well-established role in proofreading the 5' splice site and the branch site of precursor mRNA through ATP hydrolysis to ensure the accuracy of the splicing process. Our research has unveiled an unexpected facet of Prp16's function, as it also promotes aberrant selection of the 5' splice site and the branch site through an ATP-independent activity. Prp16 accomplishes these contrasting functions by interacting with the step-one factor Cwc25. It can stabilize Cwc25 to enhance the branching reaction independently of ATP, or destabilize Cwc25 to inhibit the reaction through its ATPase activity. Prp16 exerts dual roles in splice site selection, employing ATP-dependent and ATP-independent mechanisms to regulate splicing fidelity.

Arresting Spliceosome Intermediates at Various Stages of the Splicing Pathway.

The spliceosome is a dynamic ribonucleoprotein particle and is assembled via sequential binding of five snRNAs and numerous protein factors. To understand the molecular mechanism of the splicing reaction, it is necessary to dissect the spliceosome pathway and isolate spliceosome intermediates in various stages of the pathway for biochemical and structural analysis. Here, we describe protocols for preparing intron-containing transcripts, cell-free splicing extracts, and in vitro splicing reactions, as well as procedures to arrest the spliceosome at different stages of the pathway for characterization of specific splicing complexes from the budding yeast Saccharomyces cerevisiae. Methods for arresting spliceosomes at specific stages include depletion with antibodies against factors required for specific steps of the pathway, use of extracts prepared from temperature-sensitive mutants, use of dominant negative mutants of DExD/H-box proteins, and use of mutant substrates.

Pesticide exposure in New Zealand school-aged children: Urinary concentrations of biomarkers and assessment of determinants.

This study aimed to assess pesticide exposure and its determinants in children aged 5-14 years. Urine samples (n = 953) were collected from 501 participating children living in urban areas (participant n = 300), rural areas but not on a farm (n = 76), and living on a farm (n = 125). The majority provided two samples, one in the high and one in the low spraying season. Information on diet, lifestyle, and demographic factors was collected by questionnaire. Urine was analysed for 20 pesticide biomarkers by GC-MS/MS and LC-MS/MS. Nine analytes were detected in > 80% of samples, including six organophosphate insecticide metabolites (DMP, DMTP, DEP, DETP, TCPy, PNP), two pyrethroid insecticide metabolites (3-PBA, trans-DCCA), and one herbicide (2,4-D). The highest concentration was measured for TCPy (median 13 µg/g creatinine), a metabolite of chlorpyrifos and triclopyr, followed by DMP (11 µg/g) and DMTP (3.7 µg/g). Urine metabolite levels were generally similar or low compared to those reported for other countries, while relatively high for TCPy and pyrethroid metabolites. Living on a farm was associated with higher TCPy levels during the high spray season. Living in rural areas, dog ownership and in-home pest control were associated with higher levels of pyrethroid metabolites. Urinary concentrations of several pesticide metabolites were higher during the low spraying season, possibly due to consumption of imported fruits and vegetables. Organic fruit consumption was not associated with lower urine concentrations, but consumption of organic food other than fruit or vegetables was associated with lower concentrations of TCPy in the high spray season. In conclusion, compared to other countries such as the U.S., New Zealand children had relatively high exposures to chlorpyrifos/triclopyr and pyrethroids. Factors associated with exposure included age, season, area of residence, diet, in-home pest control, and pets.

Evidence for complex dynamics during U2 snRNP selection of the intron branchpoint.

Splicing of pre-mRNA is initiated by binding of U1 to the 5' splice site and of Msl5-Mud2 heterodimer to the branch site (BS). Subsequent binding of U2 displaces Msl5-Mud2 from the BS to form the prespliceosome, a step governing branchpoint selection and hence 3' splice site choice, and linking splicing to myelodysplasia and many cancers in human. Two DEAD-box proteins, Prp5 and Sub2, are required for this step, but neither is stably associated with the pre-mRNA during the reaction. Using BS-mutated ACT1 pre-mRNA, we previously identified a splicing intermediate complex, FIC, which contains U2 and Prp5, but cannot bind the tri-snRNP. We show here that Msl5 remains associated with the upstream cryptic branch site (CBS) in the FIC, with U2 binding a few bases downstream of the BS. U2 mutants that restore U2-BS base pairing enable dissociation of Prp5 and allows splicing to proceed. The CBS is required for splicing rescue by compensatory U2 mutants, and for formation of FIC, demonstrating a role for Msl5 in directing U2 to the BS, and of U2-BS base pairing for release of Prp5 and Msl5-Mud2 to form the prespliceosome. Our results provide insights into how the prespliceosome may form in normal splicing reaction.

Gender differences in respiratory health outcomes among farming cohorts around the globe: findings from the AGRICOH consortium.

Purpose: Respiratory hazards of farming have been identified for centuries, with little focus on gender differences. We used data from the AGRICOH consortium, a collective of prospective cohorts of agricultural workers, to assess respiratory disease prevalence among adults in 18 cohorts representing over 200,000 farmers, farm workers, and their spouses from six continents.Methods: Cohorts collected data between 1992 and 2016 and ranged in size from 200 to >128,000 individuals; 44% of participants were female. Farming practices varied from subsistence farming to large-scale industrial agriculture. All cohorts provided respiratory outcome information for their cohort based on their study definitions. The majority of outcomes were based on self-report using standard respiratory questionnaires; the greatest variability in assessment methods was associated with chronic obstructive pulmonary disease (COPD).Results: For all three respiratory symptoms (cough, phlegm, and wheeze), the median prevalence in men was higher than in women, with the greatest difference for phlegm (17% vs. 10%). For asthma, women had a higher prevalence (7.8% vs 6.5%), with the difference associated with allergic asthma. The relative proportion of allergic asthma varied among cohorts. In two of eight cohorts for women and two of seven cohorts for men, allergic asthma was more common than non-allergic asthma.Conclusions: These findings indicate that respiratory outcomes are common among farmers around the world despite differences in agricultural production. As women in the general population are at higher risk of asthma, exploring gender differences in occupational studies is critical for a deeper understanding of respiratory disease among agricultural workers.

Exploring Pasifika wellbeing: findings from a large cluster randomised controlled trial of a mobile health intervention programme.

AIM: The primary objective of this study was to determine the effect of a mobile health (mHealth) intervention on the wellbeing of Pasifika peoples, and to explore factors associated with Pasifika wellbeing. METHODS: The OL@-OR@ mHealth programme was a co-designed smartphone app. Culturally relevant data was collected to examine holistic health and wellbeing status, at baseline, and at 12 weeks (end of the trial). The concept of wellbeing was examined as part of a two-arm, cluster randomised trial, using only the Pasifika data: 389 (of 726) Pasifika adults were randomised to receive the mHealth intervention, while 405 (of 725) Pasifika adults were randomised to receive a control version of the intervention. Culturally relevant data was collected to examine holistic health and wellbeing status, at baseline, and at 12 weeks (end of the trial). The intervention effects and the association of demographic and behavioural relationships with wellbeing, was examined using logistic regression analyses. RESULTS: Relative to baseline, there were significant differences between the intervention and control groups for the 'family/community' wellbeing, at the end of the 12-week trial. There were no significant differences observed for all other wellbeing domains for both groups. Based on our multivariate regression analyses, education and acculturation (assimilation and marginalisation) were identified as positively strong factors associated to Pasifika 'family and community' wellbeing. CONCLUSION: Our study provides new insights on how Pasifika peoples' characteristics and behaviours align to wellbeing. Our findings point to 'family and community' as being the most important wellbeing factor for Pasifika peoples.

Functional analysis of Cwc24 ZF-domain in 5' splice site selection.

The essential splicing factor Cwc24 contains a zinc-finger (ZF) domain required for its function in splicing. Cwc24 binds over the 5' splice site after the spliceosome is activated, and its binding prior to Prp2-mediated spliceosome remodeling is important for proper interactions of U5 and U6 with the 5' splice site sequence and selection of the 5' splice site. Here, we show that Cwc24 transiently interacts with the 5' splice site in formation of the functional RNA catalytic core during spliceosome remodeling, and the ZF-motif is required for specific interaction of Cwc24 with the 5' splice site. Deletion of the ZF domain or mutation of the conserved ZF residues greatly weakened the association of Cwc24 with the spliceosome, and lowered the affinity and specificity of its interaction with the 5' splice site, resulting in atypical interactions of U5, U6 and Prp8 with the 5' splice site, and aberrant cleavage at the 5' splice site. Our results reveal a crucial role of the Cwc24 ZF-motif for defining 5' splice site selection in the first splicing step.

Investigating differences in dietary patterns among a small cross-sectional study of young and old Pacific peoples in NZ using exploratory factor analysis: a feasibility study.

OBJECTIVES: Obesity among Pasifika people living in New Zealand is a serious health problem with prevalence rates more than twice those of the general population (67% vs 33%, respectively). Due to the high risk of developing obesity for this population, we investigated diet quality of Pacific youth and their parents and grandparents. Therefore, we examined the dietary diversity of 30 youth and their parents and grandparents (n=34) to identify whether there are generational differences in dietary patterns and investigate the relationship between acculturation and dietary patterns. METHODS: The study design of the overarching study was cross-sectional. Face-to-face interviews were conducted with Pasifika youth, parents and grandparents to investigate dietary diversity, that included both nutritious and discretionary food items and food groups over a 7 day period. Study setting was located in 2 large urban cities, New Zealand. Exploratory factor analyses were used to calculate food scores (means) from individual food items based on proportions consumed over the week, and weights were applied to calculate a standardised food score. The relationship between the level of acculturation and deprivation with dietary patterns was also assessed. RESULTS: Three distinctive dietary patterns across all participants were identified from our analyses. Healthy diet, processed diet and mixed diet. Mean food scores indicated statistically significant differences between the dietary patterns for older and younger generations. Older generations showed greater diversity in food items consumed, as well as eating primarily a 'healthy diet'. The younger generation was more likely to consume a 'processed diet'. There was significant association between acculturation and deprivation with the distinctive dietary patterns. CONCLUSION: Our investigation highlighted generational differences in consuming a limited range of food items. Identified dietary components may, in part, be explained by specific acculturation modes (assimilation and marginalised) and high socioeconomic deprivation among this particular study population.

Germline CDH1 mutations are a significant contributor to the high frequency of early-onset diffuse gastric cancer cases in New Zealand Maori.

New Zealand Maori have a considerably higher incidence of gastric cancer compared to non-Maori, and are one of the few populations worldwide with a higher prevalence of diffuse-type disease. Pathogenic germline CDH1 mutations are causative of hereditary diffuse gastric cancer, a cancer predisposition syndrome primarily characterised by an extreme lifetime risk of developing diffuse gastric cancer. Pathogenic CDH1 mutations are well described in Maori families in New Zealand. However, the contribution of these mutations to the high incidence of gastric cancer is unknown. We have used next-generation sequencing, Sanger sequencing, and Multiplex Ligation-dependent Probe Amplification to examine germline CDH1 in an unselected series of 94 Maori gastric cancer patients and 200 healthy matched controls. Overall, 18% of all cases, 34% of cases diagnosed with diffuse-type gastric cancer, and 67% of cases diagnosed aged less than 45 years carried pathogenic CDH1 mutations. After adjusting for the effect of screening known HDGC families, we estimate that 6% of all advanced gastric cancers and 13% of all advanced diffuse-type gastric cancers would carry germline CDH1 mutations. Our results demonstrate that germline CDH1 mutations are a significant contributor to the high frequency of diffuse gastric cancer in New Zealand Maori.

Dynamic protein-RNA interactions in mediating splicing catalysis.

The spliceosome is assembled via sequential interactions of pre-mRNA with five small nuclear RNAs and many proteins. Recent determination of cryo-EM structures for several spliceosomal complexes has provided deep insights into interactions between spliceosomal components and structural changes of the spliceosome between steps, but information on how the proteins interact with pre-mRNA to mediate the reaction is scarce. By systematic analysis of proteins interacting with the splice sites (SSs), we have identified many previously unknown interactions of spliceosomal components with the pre-mRNA. Prp8 directly binds over the 5'SS and the branch site (BS) for the first catalytic step, and the 5'SS and 3'SS for the second step. Switching the Prp8 interaction from the BS to the 3'SS requires Slu7, which interacts dynamically with pre-mRNA first, and then interacts stably with the 3'-exon after Prp16-mediated spliceosome remodeling. Our results suggest that Prp8 plays a key role in positioning the 5'SS and 3'SS, facilitated by Slu7 through interactions with Prp8 and substrate RNA to advance exon ligation. We also provide evidence that Prp16 first docks on the intron 3' tail, then translocates in the 3' to 5' direction on remodeling the spliceosome.

Inequities in exposure to occupational risk factors between Maori and non-Maori workers in Aotearoa New Zealand.

BACKGROUND: Health inequities between indigenous and non-indigenous people are well documented. However, the contribution of differential exposure to risk factors in the occupational environment remains unclear. This study assessed differences in the prevalence of self-reported exposure to disease risk factors, including dust and chemicals, physical factors and organisational factors, between Maori and non-Maori workers in New Zealand. METHODS: Potential participants were sampled from the New Zealand electoral rolls and invited to take part in a telephone interview, which included questions about current workplace exposures. Logistic regression, accounting for differences in age, socioeconomic status and occupational distribution between Maori and non-Maori, was used to assess differences in exposures. RESULTS: In total, 2344 Maori and 2710 non-Maori participants were included in the analyses. Maori had greater exposure to occupational risk factors than non-Maori. For dust and chemical exposures, the main differences related to Maori working in occupations where these exposures are more common. However, even within the same job, Maori were more likely to be exposed to physical factors such as heavy lifting and loud noise, and organisational factors such as carrying out repetitive tasks and working to tight deadlines compared with non-Maori. CONCLUSIONS: This is one of the first studies internationally to compare occupational risk factors between indigenous and non-indigenous people. These findings suggest that the contribution of the occupational environment to health inequities between Maori and non-Maori has been underestimated and that work tasks may be unequally distributed according to ethnicity.

Cwc23 is a component of the NTR complex and functions to stabilize Ntr1 and facilitate disassembly of spliceosome intermediates.

Cwc23 is a member of the J protein family, and has been shown to interact with Ntr1, a scaffold protein that interacts with Ntr2 and Prp43 to form the NTR complex that mediates spliceosome disassembly. We show that Cwc23 is also an intrinsic component of the NTR complex, and that it interacts with the carboxyl terminus of Ntr1. Metabolic depletion of Cwc23 concurrently depleted Ntr1 and Ntr2, suggesting a role for Cwc23 in stabilizing these two proteins. Ntr1, Ntr2 and Cwc23 are stoichiometrically balanced, and form a stable heterotrimer. Depletion of Cwc23 from splicing extracts using antibodies resulted in depletion of all three proteins and accumulation of intron-lariat in the splicing reaction. Cwc23 is not required for disassembly of intron-lariat spliceosome (ILS), but facilitates disassembly of spliceosome intermediates after the actions of Prp2 and Prp16 by stabilizing the association of Ntr1 with the spliceosome. Cwc23 has a more limited effect on the association of Ntr1 with the ILS. Our data suggest that Cwc23 is important for maintaining the levels of Ntr1 and Ntr2, and that it also plays a regulatory role in targeting spliceosome intermediates for disassembly.

Morbidity in New Zealand pesticide producers exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

OBJECTIVES: To conduct a cross-sectional morbidity survey among 245 former employees of a pesticide production plant exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in New Zealand. METHODS: Demographic factors and health information were collected in face-to-face interviews. TCDD, lipids, thyroid hormones, glucose and immunoglobulin G (IgG) were determined in non-fasting blood. For 111 participants, a neurological examination was conducted. Associations between health outcomes and working in a TCDD exposed job (prevalence 49%) and serum TCDD concentration≥10pg/g lipid (18%) were assessed using logistic regression whilst controlling for age, gender, smoking, body mass index and ethnicity. RESULTS: Diabetes was more common in those who had worked in TCDD exposed jobs (OR 4.0, 95%CI 1.0-15.4) and in those with serum TCDD ≥10pg/g (OR 3.1, 95%CI 0.9-10.7). Non-fasting glucose levels >6.6mmol/l were more common in those with TCDD exposed jobs (OR 3.6, 95%CI 1.0-12.9), as were serum free thyroxine 4<12.8pmol/l (OR 4.5, 95%CI 1.4-14.4), triglycerides >1.7mmol/l (OR 2.5, 95%CI 1.1-5.7) and high density lipoprotein cholesterol (HDL) <1mmol/l (OR 4.0, 95%CI 1.2-13.2). IgG was negatively associated with TCDD (linear regression p=0.05). The neurological examination revealed a higher frequency of abnormal reflexes in those with serum TCDD ≥10pg/g (OR 4.8, 95%CI 1.1-21.0). CONCLUSIONS: In this occupationally exposed population, TCDD was associated with an increased risk of diabetes and a range of subclinical responses in multiple systems (peripheral nervous system, immune system, thyroid hormones and lipid metabolism), several decades after last exposure. These results need to be interpreted with caution due to the small study size and the cross-sectional nature of the study.

Risk of stomach cancer in Aotearoa/New Zealand: A Maori population based case-control study.

Maori, the indigenous people of New Zealand, experience disproportionate rates of stomach cancer, compared to non-Maori. The overall aim of the study was to better understand the reasons for the considerable excess of stomach cancer in Maori and to identify priorities for prevention. Maori stomach cancer cases from the New Zealand Cancer Registry between 1 February 2009 and 31 October 2013 and Maori controls, randomly selected from the New Zealand electoral roll were matched by 5-year age bands to cases. Logistic regression was used to estimate odd ratios (OR) and 95% confidence intervals (CI) between exposures and stomach cancer risk. Post-stratification weighting of controls was used to account for differential non-response by deprivation category. The study comprised 165 cases and 480 controls. Nearly half (47.9%) of cases were of the diffuse subtype. There were differences in the distribution of risk factors between cases and controls. Of interest were the strong relationships seen with increased stomach risk and having >2 people sharing a bedroom in childhood (OR 3.30, 95%CI 1.95-5.59), testing for H pylori (OR 12.17, 95%CI 6.15-24.08), being an ex-smoker (OR 2.26, 95%CI 1.44-3.54) and exposure to environmental tobacco smoke in adulthood (OR 3.29, 95%CI 1.94-5.59). Some results were attenuated following post-stratification weighting. This is the first national study of stomach cancer in any indigenous population and the first Maori-only population-based study of stomach cancer undertaken in New Zealand. We emphasize caution in interpreting the findings given the possibility of selection bias. Population-level strategies to reduce the incidence of stomach cancer in Maori include expanding measures to screen and treat those infected with H pylori and a continued policy focus on reducing tobacco consumption and uptake.

New Zealanders working non-standard hours also have greater exposure to other workplace hazards.

Exposure to workplace hazards, such as dust, solvents, and fumes, has the potential to adversely affect the health of people. However, the effects of workplace hazards on health may differ when exposure occurs at different times in the circadian cycle, and among people who work longer hours or who do not obtain adequate sleep. The aim of the present study was to document exposures to workplace hazards across a national sample of New Zealanders, comparing people who work a standard 08:00 -17:00 h Monday-to-Friday working week (Std hours) and those who do not (N-Std hours). New Zealanders (n = 10 000) aged 20-64 yrs were randomly selected from the Electoral Roll to take part in a nationwide survey of workplace exposures. Telephone interviews were conducted between 2004 and 2006, using a six-part questionnaire addressing demographics, detailed information on the current or most recent job (including exposures to a range of workplace hazards), sleep, sleepiness, and health status. N-Std hours were categorised on the basis of: being required to start work prior to 07:00 h or finish work after 21:00 h and/or; having a regular on-call commitment (at least once per week) and/or; working rotating shifts and/or; working night shift(s) in the last month. The response rate was 37% (n = 3003), with 22.2% of participants (n = 656) categorised as working N-Std hours. Industry sectors with the highest numbers of participants working N-Std hours were manufacturing, health and community services, and agriculture, fishing, and forestry. Response rate was 37% (n = 3003) with 22.2% (n = 656) categorised as working N-Std hours. Participants working N-Std hours were more likely to be exposed to all identified hazards, including multiple hazards (OR = 2.45, 95% CI = 2.01-3.0) compared to those working Std hours. Participants working N-Std hours were also more likely to report 'never/rarely' getting enough sleep (OR = 1.38, 95% CI = 1.15-1.65), 'never/rarely' waking refreshed (OR = 1.23, 95% CI = 1.04-1.47), and excessive sleepiness (OR = 1.77, 95% CI = 1.29-2.42). New Zealanders working N-Std hours are more likely to be exposed to hazards in the workplace, to be exposed to multiple hazards, and to report inadequate sleep and excessive sleepiness than their colleagues working a standard 08:00-17:00 h Monday-to-Friday working week. More research is needed on the effects of exposure to hazardous substances outside the usual waking day, on the effects of exposure to multiple hazards, and on the combination of hazard exposure and sleep restriction as a result of shift work.

A central role of Cwc25 in spliceosome dynamics during the catalytic phase of pre-mRNA splicing.

Splicing of precursor mRNA occurs via two consecutive steps of transesterification reaction; both require ATP and several proteins. Despite the energy requirement in the catalytic phase, incubation of the purified spliceosome under proper ionic conditions can elicit competitive reversible transesterification, debranching, and spliced-exon-reopening reactions without the necessity for ATP or other factors, suggesting that small changes in the conformational state of the spliceosome can lead to disparate chemical consequences for the substrate. We show here that Cwc25 plays a central role in modulating the conformational state of the catalytic spliceosome during normal splicing reactions. Cwc25 binds tightly to the spliceosome after the reaction and is then removed from the spliceosome, which normally requires DExD/H-box protein Prp16 and ATP hydrolysis, to allow the occurrence of the second reaction. When deprived of Cwc25, the purified first-step spliceosome catalyzes both forward and reverse splicing reactions under normal splicing conditions without requiring energy. Both reactions are inhibited when Cwc25 is added back, presumably due to the stabilization of first-step conformation. Prp16 is dispensable for the second reaction when splicing is carried out under conditions that destabilize Cwc25. We also show that the purified precatalytic spliceosome can catalyze two steps of the reaction at a low efficiency without requiring Cwc25, Slu7, or Prp18 when incubated under proper conditions. Our study reveals conformational modulation of the spliceosome by Cwc25 and Prp16 in stabilization and destabilization of first-step conformation, respectively, to facilitate the splicing process.

Role of Cwc24 in the First Catalytic Step of Splicing and Fidelity of 5' Splice Site Selection.

Cwc24 is an essential splicing factor but only transiently associates with the spliceosome, with an unknown function. The protein contains a RING finger and a zinc finger domain in the carboxyl terminus. The human ortholog of Cwc24, RNF113A, has been associated with the disorder trichothiodystrophy. Here, we show that the zinc finger domain is essential for Cwc24 function, while the RING finger domain is dispensable. Cwc24 binds to the spliceosome after the Prp19-associated complex and is released upon Prp2 action. Cwc24 is not required for Prp2-mediated remodeling of the spliceosome, but the spliceosome becomes inactive if remodeling occurs before the addition of Cwc24. Cwc24 binds directly to pre-mRNA at the 5' splice site, spanning the splice junction. In the absence of Cwc24, U5 and U6 modes of interaction with the 5' splice site are altered, and splicing is very inefficient, with aberrant cleavage at the 5' splice site. Our data suggest roles for Cwc24 in orchestrating organization of the spliceosome into an active configuration prior to Prp2-mediated spliceosome remodeling and in promoting specific interaction of U5 and U6 with the 5' splice site for fidelity of 5' splice site selection.

Sex ratio of the offspring of New Zealand phenoxy herbicide producers exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

OBJECTIVES: Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has inconsistently been associated with a decreased sex ratio of the offspring (number of male births divided by total births). We conducted a study among men and women who were employed in a New Zealand phenoxy herbicide production plant between 1969 and 1984, to study their offspring sex ratio in relation to their back-calculated TCDD serum concentrations determined in 2007/2008. METHODS: A total of 127 men and 21 women reported that 355 children were conceived after starting employment at the plant. The association between their lipid-standardised TCDD serum concentrations back-calculated to the time of their offspring's birth and the probability of a male birth was estimated through logistic regression, adjusting for the age of the exposed parent at birth, current body mass index and smoking. RESULTS: The overall sex ratio was 0.55 (197 boys, 158 girls). For fathers with serum TCDD concentrations ≥20 pg/g lipid at time of birth, the sex ratio was 0.47 (OR 0.49; 95% CI 0.30 to 0.79). The probability of a male birth decreased with higher paternal serum TCDD at time of birth (<4; 4-20; 20-100; ≥100 pg/g lipid), with ORs of 1.00 (reference); 1.00 (95% CI 0.50 to 2.02); 0.52 (95% CI 0.29 to 0.92); 0.45 (95% CI 0.23 to 0.89), p trend 0.007. For exposed mothers, the sex ratio was not reduced. CONCLUSIONS: This study indicates that paternal serum TCDD concentrations in excess of an estimated 20 pg/g lipid at time of conception are associated with a reduced sex ratio.