Evaluation of a New Glycomacropeptide-Based Protein Substitute in Powdered and Liquid Format in Patients with PKU.

(1) Background: Good adherence to a Phe-restricted diet supplemented with an adequate amount of a protein substitute (PS) is important for good clinical outcomes in PKU. Glycomacropeptide (cGMP)-PSs are innovative, palatable alternatives to amino acid-based PSs (AA-PS). This study aimed to evaluate a new cGMP-PS in liquid and powder formats in PKU. (2) Methods: Children and adults with PKU recruited from eight centres were prescribed at least one serving/day of cGMP-PS for 7-28 days. Adherence, acceptability, and gastrointestinal tolerance were recorded at baseline and the end of the intervention. The blood Phe levels reported as part of routine care during the intervention were recorded. (3) Results: In total, 23 patients (powder group, n = 13; liquid group, n = 10) completed the study. The majority assessed the products to be palatable (77% of powder group; 100% of liquid group) and well tolerated; the adherence to the product prescription was good. A total of 14 patients provided blood Phe results during the intervention, which were within the target therapeutic range for most patients (n = 11) at baseline and during the intervention. (4) Conclusions: These new cGMP-PSs were well accepted and tolerated, and their use did not adversely affect blood Phe control.

Evaluation of a New 'Mix-In' Style Glycomacropeptide-Based Protein Substitute for Food and Drinks in Patients with Phenylketonuria and Tyrosinemia.

(1) Background: Poor palatability, large volume, and lack of variety of some liquid and powdered protein substitutes (PSs) for patients with phenylketonuria (PKU) and tyrosinemia (TYR) can result in poor adherence. This study aimed to evaluate a new unflavoured, powdered GMP-based PS designed to be mixed into drinks, foods, or with other PSs, in patients with PKU and TYR. (2) Methods: Paediatric and adult community-based patients were recruited from eight metabolic centres and prescribed ≥1 sachet/day (10 g protein equivalent (PE)) of the Mix-In-style PS over 28 days. Adherence, palatability, GI tolerance, and metabolic control were recorded at baseline and follow-up. Patients who completed at least 7 days of intervention were included in the final analysis. (3) Results: Eighteen patients (3-45 years, nine males) with PKU (n = 12) and TYR (n = 6) used the Mix-In-style PS for ≥7 days (mean 26.4 days (SD 4.6), range 11-28 days) alongside their previous PS, with a mean intake of 16.7 g (SD 7.7) PE/day. Adherence was 86% (SD 25), and GI tolerance was stable, with n = 14 experiencing no/no new symptoms and n = 3 showing improved symptoms compared to baseline. Overall palatability was rated satisfactory by 78% of patients, who successfully used the Mix-In-style PS in various foods and drinks, including smoothies, squash, and milk alternatives, as a top-up to meet their protein needs. There was no concern regarding safety/metabolic control during the intervention. (4) Conclusions: The 'Mix-In'-style PS was well adhered to, accepted, and tolerated. Collectively, these data show that providing a flexible, convenient, and novel format of PS can help with adherence and meet patients' protein needs.

Structure and activity of botulinum neurotoxin X.

Botulinum neurotoxins (BoNTs) are the most potent toxins known and are used to treat an increasing number of medical disorders. All BoNTs are naturally co-expressed with a protective partner protein (NTNH) with which they form a 300 kDa complex, to resist acidic and proteolytic attack from the digestive tract. We have previously identified a new botulinum neurotoxin serotype, BoNT/X, that has unique and therapeutically attractive properties. We present the cryo-EM structure of the BoNT/X-NTNH/X complex at 3.1 Å resolution. Unexpectedly, the BoNT/X complex is stable and protease resistant at both neutral and acidic pH and disassembles only in alkaline conditions. Using the stabilizing effect of NTNH, we isolated BoNT/X and showed that it has very low potency both in vitro and in vivo . Given the high catalytic activity and translocation efficacy of BoNT/X, low activity of the full toxin is likely due to the receptor-binding domain, which presents weak ganglioside binding and exposed hydrophobic surfaces.

Suitability and Allocation of Protein-Containing Foods According to Protein Tolerance in PKU: A 2022 UK National Consensus.

Introduction: There is little practical guidance about suitable food choices for higher natural protein tolerances in patients with phenylketonuria (PKU). This is particularly important to consider with the introduction of adjunct pharmaceutical treatments that may improve protein tolerance. Aim: To develop a set of guidelines for the introduction of higher protein foods into the diets of patients with PKU who tolerate >10 g/day of protein. Methods: In January 2022, a 26-item food group questionnaire, listing a range of foods containing protein from 5 to >20 g/100 g, was sent to all British Inherited Metabolic Disease Group (BIMDG) dietitians (n = 80; 26 Inherited Metabolic Disease [IMD] centres). They were asked to consider within their IMD dietetic team when they would recommend introducing each of the 26 protein-containing food groups into a patient's diet who tolerated >10 g to 60 g/day of protein. The patient protein tolerance for each food group that received the majority vote from IMD dietetic teams was chosen as its tolerance threshold for introduction. A virtual meeting was held using Delphi methodology in March 2022 to discuss and agree final consensus. Results: Responses were received from dietitians from 22/26 IMD centres (85%) (11 paediatric, 11 adult). For patients tolerating protein ≥15 g/day, the following foods were agreed for inclusion: gluten-free pastas, gluten-free flours, regular bread, cheese spreads, soft cheese, and lentils in brine; for protein tolerance ≥20 g/day: nuts, hard cheeses, regular flours, meat/fish, and plant-based alternative products (containing 5−10 g/100 g protein), regular pasta, seeds, eggs, dried legumes, and yeast extract spreads were added; for protein tolerance ≥30 g/day: meat/fish and plant-based alternative products (containing >10−20 g/100 g protein) were added; and for protein tolerance ≥40 g/day: meat/fish and plant-based alternatives (containing >20 g/100 g protein) were added. Conclusion: This UK consensus by IMD dietitians from 22 UK centres describes for the first time the suitability and allocation of higher protein foods according to individual patient protein tolerance. It provides valuable guidance for health professionals to enable them to standardize practice and give rational advice to patients.

Are women with gestational diabetes being screened for type 2 diabetes following pregnancy? A nationwide retrospective cohort study in Aotearoa New Zealand.

AIM: To estimate the proportion of women with a first episode of gestational diabetes mellitus (GDM) in Aotearoa (New Zealand) who received postpartum screening for type 2 diabetes mellitus (T2DM). METHODS: Data from 941,468 pregnancies occurring between 2005 and 2015 were linked with laboratory, community pharmacy, and hospital discharge data from the Ministry of Health's National Collections to identify a cohort of women who had a first episode of GDM (n = 14,443). Proportions receiving a glycated haemoglobin (HbA1c) test or oral glucose tolerance test (OGTT) during the first year postpartum were estimated overall, and by calendar year, ethnic group, age, deprivation, and region. RESULTS: Overall, 40.9% (95% CI 40.1-41.7%) received an HbA1c test or OGTT within 3 months, 53.3% (52.5-54.1%) within 6 months, and 61.0% (60.2-61.8%) within 12 months postpartum. Screening proportions within 12 months were stable over time. Indigenous Maori were less likely to receive screening within 6 months postpartum (35.0% [33.1-37.0%]) than other ethnic groups, as were younger women and those with higher deprivation. There were marked variations by region (between 15.3% and 67.5%). CONCLUSION: Postpartum T2DM screening was low over the period studied, with substantial ethnic and regional inequities across New Zealand.

Antidepressant dispensing before, during, and after pregnancy in New Zealand, 2005-2014.

BACKGROUND: Depression during pregnancy is associated with a number of negative impacts on maternal and infant health, therefore good control of depression in pregnant women is crucial. There is a lack of population-level information about patterns of antidepressant use during pregnancy in New Zealand. AIM: To describe antidepressant dispensing patterns before, during, and after pregnancy in New Zealand, 2005-2014. MATERIALS AND METHODS: Antidepressant dispensing records from 270 days prior to pregnancy through to 360 days after pregnancy end were linked with 805 990 pregnancies in the New Zealand Pregnancy Cohort. Proportions (and 95% confidence intervals) with at least one dispensing were calculated for the periods before, during, and after pregnancy and compared over time and by maternal characteristics. RESULTS: Dispensing during the first trimester was lower than in the pre-pregnancy and post-pregnancy periods, and dropped further in later trimesters. The proportion of pregnancies during which an antidepressant was dispensed rose from 3.1 to 4.9% over the study years. Around 80% of those with a dispensing received a selective serotonin reuptake inhibitor. Dispensing before, during, and after pregnancy varied by ethnicity, age, smoking status, and body mass index. Among women taking an antidepressant before pregnancy, younger women and those of Maori, Pacific, or Asian ethnicity were less likely to continue therapy during pregnancy. CONCLUSIONS: This study has established a baseline for antidepressant use around pregnancy in New Zealand, documented increasing use over time, and demonstrated that known ethnic differences in antidepressant use are also evident in the pregnant population.

Engineering an Effective Human SNAP-23 Cleaving Botulinum Neurotoxin A Variant.

Botulinum neurotoxin (BoNT) serotype A inhibits neurotransmitter release by cleaving SNAP-25 and represents an established pharmaceutical for treating medical conditions caused by hyperactivity of cholinergic nerves. Oversecretion from non-neuronal cells is often also the cause of diseases. Notably, excessive release of inflammatory messengers is thought to contribute to diseases such as chronic obstructive pulmonary disease, asthma, diabetes etc. The expansion of its application to these medical conditions is prevented because the major non-neuronal SNAP-25 isoform responsible for exocytosis, SNAP-23, is, in humans, virtually resistant to BoNT/A. Based on previous structural data and mutagenesis studies of SNAP-23 we optimized substrate binding pockets of the enzymatic domain for interaction with SNAP-23. Systematic mutagenesis and rational design yielded the mutations E148Y, K166F, S254A, and G305D, each of which individually increased the activity of LC/A against SNAP-23 between 3- to 23-fold. The assembled quadruple mutant showed approximately 2000-fold increased catalytic activity against human SNAP-23 in in vitro cleavage assays. A comparable increase in activity was recorded for the full-length BoNT/A quadruple mutant tested in cultivated primary neurons transduced with a fluorescently tagged-SNAP-23 encoding gene. Equipped with a suitable targeting domain this quadruple mutant promises to complete successfully tests in cells of the immune system.

Patterns of prescription medicine dispensing before and during pregnancy in New Zealand, 2005-2015.

OBJECTIVE: To describe prescription medicine dispensing before and during pregnancy in New Zealand, 2005-2015. METHODS: Members of the New Zealand Pregnancy Cohort were linked with their dispensing records in a national database of prescription products dispensed from community pharmacies. We identified the proportion of pregnancies during which at least one prescription medicine was dispensed, the number of different medicines used and the most commonly dispensed medicine groups both during pregnancy and in the 270 days before conception. Dispensing during pregnancy was assessed by several maternal characteristics. RESULTS: 874,884 pregnancies were included. Over the study timeframe, the proportion of pregnancies exposed to a non-supplement prescription medicine increased from 38.5% to 67.2%. The mean number of different non-supplement medicines dispensed during pregnancy increased from 2.5 to 3.2. Dispensing during pregnancy was weakly associated with body mass index, smoking status and ethnicity. Pregnancy exposure was highest for Antibacterials (26.0%), Analgesics (16.7%) and Antinausea & Vertigo Agents (11.0%). CONCLUSIONS: From 2005-2015, both the proportion of exposed pregnancies and the number of different medicines dispensed to pregnant women in New Zealand increased.

Prescription medicines with potential for foetal harm: dispensing before and during pregnancy in New Zealand, 2005-2015.

PURPOSE: This study describes dispensing of potentially teratogenic prescription medicines before and during pregnancy in New Zealand over the period 2005-2015. METHODS: Records in a national dispensing database were linked with the members of the New Zealand Pregnancy Cohort to determine the proportion of pregnancies with at least one dispensing of a Category D or X medicine, using the Australian pregnancy risk categorisation system. Exposure was examined from 270 days prior to conception through to the end of pregnancy. Pregnancy outcomes of D/X-exposed pregnancies were reviewed. RESULTS: In the study, 874,884 pregnancies were included. Overall, Category D and X medicines were dispensed during 4.3% and 0.058% of pregnancies, respectively. After excluding misoprostol, X exposure decreased to 0.035%. Generally, dispensing declined through the 270-day pre-pregnancy period and continued to decline throughout pregnancy. Dispensing of X medicines increased over the study timeframe, whereas dispensing of D medicines increased from 2005 to 2011 then declined slightly. Smokers were more likely than non-smokers to have been dispensed a D/X medicine, and compared with European women, Maori and Pacific women were less likely to have been dispensed a D/X medicine. Excluding misoprostol, pregnancies exposed to an X medicine were more likely than D/X-unexposed pregnancies to have ended in termination. CONCLUSION: Dispensing of potentially harmful medicines in pregnancy in New Zealand was low, particularly for Category X medicines. However, exposure did increase over the study timeframe. The inclusion of pregnancies that did not progress past early pregnancy better reflects population-level pregnancy exposure to potentially teratogenic medicines.

Nutritional and Metabolic Characteristics of UK Adult Phenylketonuria Patients with Varying Dietary Adherence.

The nutritional and metabolic characteristics of adult phenylketonuria (PKU) patients in the UK with varying dietary adherence is unknown. In other countries, nutritional and metabolic abnormalities have been reported in nonadherent patients compared to adherent counterparts. A pooled analysis of primary baseline data from two UK multi-centre studies was therefore performed to establish whether this is true from a UK perspective. Adult PKU patients who had provided 3-day food records and amino acid blood samples were included and grouped according to dietary adherence (adherent; n = 16 vs. nonadherent; n = 14). Nonadherent patients consumed greater amounts of natural protein compared to adherent patients (61.6 ± 30.7 vs. 18.3 ± 7.7 g/day; q < 0.001). In contrast, the contribution of protein substitutes to total protein intake was lower in nonadherent compared to adherent patients (3.9 ± 9.2 g/day vs. 58.6 ± 10.2 g/day; q < 0.001). Intakes of iron, zinc, vitamin D3, magnesium, calcium, selenium, iodine, vitamin C, vitamin A and copper were significantly lower in nonadherent compared to adherent patients and were below UK Reference Nutrient Intakes. Similarly, intakes of thiamin, riboflavin, niacin, vitamin B6 and phosphorus were significantly lower in nonadherent compared to adherent patients but met the UK Reference Nutrient Intakes. Phenylalanine concentrations in nonadherent patients were significantly higher than adherent patients (861 ± 348 vs. 464 ± 196 µmol/L; q=0.040) and fell outside of European treatment target ranges. This study shows the nutritional and metabolic consequences of deviation from phenylalanine restriction and intake of PKU protein substitutes in nonadherent adult PKU patients. Collectively, these data further underlie the importance of life-long adherence to the PKU diet.

A comparison of biological activity of commercially available purified native botulinum neurotoxin serotypes A1 to F1 in vitro, ex vivo, and in vivo.

Botulinum neurotoxin (BoNT) is a major therapeutic agent. Of seven native BoNT serotypes (A to G), only A and B are currently used in the clinic. Here we compared the potency of commercially available purified native serotypes A1 to F1 across in vitro, ex vivo, and in vivo assays. BoNT potency in vitro was assessed in rat primary cells (target protein cleavage and neurotransmitter release assays) in supraspinal, spinal, and sensory systems. BoNT potency ex vivo was measured in the mouse phrenic nerve hemidiaphragm (PNHD) assay, measuring muscle contractility. In vivo, BoNT-induced muscle relaxation in mice and rats was assessed in the Digit Abduction Score (DAS) test, while effects on body weight (BW) gain were used to assess tolerability. In all assays, all BoNT serotypes were potent toxins, except serotype D1 in vivo which failed to produce significant muscle flaccidity in mice and rats. In rats, all serotypes were well-tolerated, whereas in mice, reductions in BW were detected at high doses. Serotype A1 was the most potent serotype across in vitro, ex vivo, and in vivo assays. The rank order of potency of the serotypes revealed differences among assays. For example, species-specificity was seen for serotype B1, and to a lesser extent for serotype C1. Serotypes F1 and C1, not currently in the clinic, showed preference for sensory over motor models and therefore could be considered for development in conditions involving the somatosensory system.

Generation of a pregnancy cohort for medicine utilisation and medicine safety studies in New Zealand.

PURPOSE: The aim of this study was to use national health databases to assemble a pregnancy cohort for undertaking medicine utilisation and safety studies in New Zealand. METHOD: Pregnancies conceived between January 2005 and March 2015 were identified in the National Maternity Collection, the National Minimum Dataset, the Mortality Collection, and the Laboratory Claims Collection. Pregnancy start and end dates were calculated and used in conjunction with the National Health Index number to merge the records from the four collections to create the New Zealand Pregnancy Cohort. Records of live born and stillborn infants identified in the National Maternity Collection and the Mortality Collection that were linkable with a cohort member formed the baby cohort. RESULTS: The cohort consists of 941 468 pregnancies to 491 272 women. One-third of the pregnancies, predominantly early pregnancy losses and terminations, were not found in the National Maternity Collection. Records of 632 090 live born or stillborn infants are linked with 623 099 pregnancies. CONCLUSIONS: The New Zealand Pregnancy Cohort is a comprehensive collection of virtually all pregnancies which ended in a live or stillbirth and many, though not all, which ended as early pregnancy losses or terminations in New Zealand over the past decade, and better represents the pregnant population than a cohort generated from the National Maternity Collection alone would do. This cohort will be valuable for investigating patterns of medicine use during pregnancy in New Zealand and developing a fuller understanding of potential impacts of foetal exposure in early pregnancy.

Botulinum neurotoxin A and an engineered derivate targeted secretion inhibitor (TSI) A enter cells via different vesicular compartments.

Botulinum neurotoxins (BoNTs) are highly potent multi-domain proteins, responsible for botulism in animals and humans. The modular structural organization of BoNTs has led to the development of novel engineered bio-therapeutic proteins called targeted secretion inhibitors (TSIs). We report here that botulinum neurotoxin A (BoNT/A) and a TSI/A in which the neuronal binding domain of BoNT/A has been substituted by an epidermal growth factor (EGF) ligand, named EGFR-targeted TSI/A, exploit different routes to gain entry in the same in vitro neuroblastoma cell system, SiMa cells. We found that the EGF ligand conferred the affinity to the EGFR-targeted TSI/A at the EGF receptor when compared to an untargeted TSI/A and also the ability to internalize into the cells and cleave its cytosolic target protein SNAP-25. Using high content analysis we found that both BoNT/A and the EGFR-targeted TSI/A enter the cell in a concentration-dependent manner and in compartments which are able to translocate the proteins into the cytosol within 4 h. The EGFR-targeted TSI/A internalized into a compartment which gave a punctate staining pattern by immunofluorescence and partially overlapped with structures positive for the early endosomal marker EAA1; whereas BoNT/A did not internalize into a punctate compartment but did so in an acidifying compartment consistent with local synaptic vesicle recycling. These findings show that the BoNT/A translocation domain, common to both BoNT/A and the EGFR-targeted TSI/A, is a versatile tool for cytosolic delivery from distinct intracellular vesicular compartments.

Impact of community based peer support in type 2 diabetes: a cluster randomised controlled trial of individual and/or group approaches.

BACKGROUND: Diabetes peer support, where one person with diabetes helps guide and support others, has been proposed as a way to improve diabetes management. We have tested whether different diabetes peer support strategies can improve metabolic and/or psychological outcomes. METHODS: People with type 2 diabetes (n = 1,299) were invited to participate as either 'peer' or 'peer support facilitator' (PSF) in a 2x2 factorial randomised cluster controlled trial across rural communities (130 clusters) in England. Peer support was delivered over 8-12 months by trained PSFs, supported by monthly meetings with a diabetes educator. Primary end point was HbA1c. Secondary outcomes included quality of life, diabetes distress, blood pressure, waist, total cholesterol and weight. Outcome assessors and investigators were masked to arm allocation. Main factors were 1:1 or group intervention. Analysis was by intention-to-treat adjusting for baseline. RESULTS: The 4 arms were well matched (Group n = 330, 1:1(individual) n = 325, combined n = 322, control n = 322); 1035 (79·7%) completed the mid-point postal questionnaire and 1064 (81·9%) had a final HbA1c. A limitation was that although 92.6% PSFs and peers were in telephone contact, only 61.4% of intervention participants attended a face to face session. Mean baseline HbA1c was 57 mmol/mol (7·4%), with no significant change across arms. Follow up systolic blood pressure was 2·3 mm Hg (0.6 to 4.0) lower among those allocated group peer-support and 3·0 mm Hg (1.1 to 5.0) lower if the group support was attended at least once. There was no impact on other outcomes by intention to treat or significant differences between arms in self-reported adherence or medication. CONCLUSIONS: Group diabetes peer support over 8-12 months was associated with a small improvement in blood pressure but no other significant outcomes. Long term benefits should be investigated. TRIAL REGISTRATION: ISRCTN.com ISRCTN6696362166963621.

Testing a peer support intervention for people with type 2 diabetes: a pilot for a randomised controlled trial.

BACKGROUND: People with Type 2 diabetes face various psycho-social, self-management and clinical care issues and evidence is mixed whether support from others with diabetes, 'peer support', can help. We now describe a 2 month pilot study of different peer support interventions. METHODS: The intervention was informed by formative evaluation using semi-structured interviews with health professionals, community support groups and observation of diabetes education and support groups. Invitations to participate were mailed from 4 general practices and included a survey of barriers to care. Participants were randomized by practice to receive individual, group, combined (both individual and group) or no peer support. Evaluation included ethnographic observation, semi-structured interviews and questionnaires at baseline and post-intervention. RESULTS: Of 1,101 invited, 15% expressed an interest in participating in the pilot. Sufficient numbers volunteered to become peer supporters, although 50% of these (8/16) withdrew. Those in the pilot were similar to other patients, but were less likely to feel they knew enough about diabetes (60.8% vs 44.6% p = 0.035) and less likely to be happy with the diabetes education/care to date (75.4% vs 55.4% p = 0.013). Key issues identified were the need to recruit peer supporters directly rather than through clinicians, to address participant diabetes educational needs early and the potential for group sessions to have lower participation rates than 1:1 sessions. CONCLUSIONS: Recruitment to a full trial of peer support within the existing study design is feasible with some amendments. Attendance emerged as a key issue needing close monitoring and additional intervention during the trial.

Successful use of acarbose to manage post-prandial glycaemia in two patients with type 1 diabetes on continuous subcutaneous insulin infusion.

Post-prandial hyperglycaemia is a particular problem for some patients with diabetes despite administering continuous subcutaneous insulin infusion (CSII) to deliver insulin flexibly. We describe two cases of patients on CSII with persistent post-prandial hyperglycaemia despite varying insulin doses and timing. Treatment with acarbose improved their glycaemic control.

P-Rex1 and Vav1 cooperate in the regulation of formyl-methionyl-leucyl-phenylalanine-dependent neutrophil responses.

G protein-coupled receptor (GPCR) activation elicits neutrophil responses such as chemotaxis and reactive oxygen species (ROS) formation, which depend on the small G protein Rac and are essential for host defense. P-Rex and Vav are two families of guanine-nucleotide exchange factors (GEFs) for Rac, which are activated through distinct mechanisms but can both control GPCR-dependent neutrophil responses. It is currently unknown whether they play specific roles or whether they can compensate for each other in controlling these responses. In this study, we have assessed the function of neutrophils from mice deficient in P-Rex and/or Vav family GEFs. We found that both the P-Rex and the Vav family are important for LPS priming of ROS formation, whereas particle-induced ROS responses and cell spreading are controlled by the Vav family alone. Surprisingly, fMLF-stimulated ROS formation, adhesion, and chemotaxis were synergistically controlled by P-Rex1 and Vav1. These responses were more severely impaired in neutrophils lacking both P-Rex1 and Vav1 than those lacking the entire P-Rex family, the entire Vav family, or both P-Rex1 and Vav3. P-Rex1/Vav1 (P1V1) double-deficient cells also showed the strongest reduction in fMLF-stimulated activation of Rac1 and Rac2. This reduction in Rac activity may be sufficient to cause the defects observed in fMLF-stimulated P1V1 neutrophil responses. Additionally, Mac-1 surface expression was reduced in P1V1 cells, which might contribute further to defects in responses involving integrins, such as GPCR-stimulated adhesion and chemotaxis. We conclude that P-Rex1 and Vav1 together are the major fMLFR-dependent Dbl family Rac-GEFs in neutrophils and cooperate in the control of fMLF-stimulated neutrophil responses.

Gastroenterology service in a teaching hospital in rural New Zealand, 1991-2003.

AIM: To retrospectively collect inpatient and outpatient data and to assess the use of endoscopic procedures during the years 1991, 1997 and 2003 to analyse for trends. METHODS: This retrospective survey was conducted in a University-associated Gastroenterology Unit offering secondary and tertiary health care services for a population of approximately 182,000 people in Southern New Zealand. Data collected included patient contacts (in- and outpatients), gastroscopic and colonoscopic investigations. RESULTS: We observed a significant increase in the absolute numbers of patient contacts over the years (1991: 2308 vs 1997: 2022 vs 2003: 2783, P < 0.0001) with inflammatory bowel disease, other diseases of the colon, anus and rectum and iron studies related disorders decreasing significantly but liver disease and constipation increasing linearly over time. The use of endoscopy services remained relatively stable but colonoscopic investigations for a positive family history of colorectal cancer increased significantly while more gastroscopies were performed for unexplained anaemia. CONCLUSION: The whole spectrum of gastroenterology contacts was studied. A substantial proportion of colonoscopies and outpatient consultations were undertaken to screen for colorectal cancer. This proportion is likely to grow further. Our findings have implications for the recruitment and training of the next generation of gastroenterologists.

P-Rex2 regulates Purkinje cell dendrite morphology and motor coordination.

The small GTPase Rac controls cell morphology, gene expression, and reactive oxygen species formation. Manipulations of Rac activity levels in the cerebellum result in motor coordination defects, but activators of Rac in the cerebellum are unknown. P-Rex family guanine-nucleotide exchange factors activate Rac. We show here that, whereas P-Rex1 expression within the brain is widespread, P-Rex2 is specifically expressed in the Purkinje neurons of the cerebellum. We have generated P-Rex2(-/-) and P-Rex1(-/-)/P-Rex2(-/-) mice, analyzed their Purkinje cell morphology, and assessed their motor functions in behavior tests. The main dendrite is thinned in Purkinje cells of P-Rex2(-/-) pups and dendrite structure appears disordered in Purkinje cells of adult P-Rex2(-/-) and P-Rex1(-/-)/P-Rex2(-/-) mice. P-Rex2(-/-) mice show a mild motor coordination defect that progressively worsens with age and is more pronounced in females than in males. P-Rex1(-/-)/P-Rex2(-/-) mice are ataxic, with reduced basic motor activity and abnormal posture and gait, as well as impaired motor coordination even at a young age. We conclude that P-Rex1 and P-Rex2 are important regulators of Purkinje cell morphology and cerebellar function.

Membrane translocation of P-Rex1 is mediated by G protein betagamma subunits and phosphoinositide 3-kinase.

P-Rex1 is a guanine-nucleotide exchange factor (GEF) for the small GTPase Rac that is directly activated by the betagamma subunits of heterotrimeric G proteins and by the lipid second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)), which is generated by phosphoinositide 3-kinase (PI3K). Gbetagamma subunits and PIP(3) are membrane-bound, whereas the intracellular localization of P-Rex1 in basal cells is cytosolic. Activation of PI3K alone is not sufficient to promote significant membrane translocation of P-Rex1. Here we investigated the subcellular localization of P-Rex1 by fractionation of Sf9 cells co-expressing P-Rex1 with Gbetagamma and/or PI3K. In basal, serum-starved cells, P-Rex1 was mainly cytosolic, but 7% of the total was present in the 117,000 x g membrane fraction. Co-expression of P-Rex1 with either Gbetagamma or PI3K caused only an insignificant increase in P-Rex1 membrane localization, whereas Gbetagamma and PI3K together synergistically caused a robust increase in membrane-localized P-Rex1 to 23% of the total. PI3K-driven P-Rex1 membrane recruitment was wortmannin-sensitive. The use of P-Rex1 mutants showed that the isolated Dbl homology/pleckstrin homology domain tandem of P-Rex1 is sufficient for synergistic Gbetagamma- and PI3K-driven membrane localization; that the enzymatic GEF activity of P-Rex1 is not required for membrane translocation; and that the other domains of P-Rex1 (DEP, PDZ, and IP4P) contribute to keeping the enzyme localized in the cytosol of basal cells. In vitro Rac2-GEF activity assays showed that membrane-derived purified P-Rex1 has a higher basal activity than cytosol-derived P-Rex1, but both can be further activated by PIP(3) and Gbetagamma subunits.