Menstrual fluid endometrial stem/progenitor cell and supernatant protein content: cyclical variation and indicative range.

STUDY QUESTION: Does natural variation exist in the endometrial stem/progenitor cell and protein composition of menstrual fluid across menstrual cycles in women? SUMMARY ANSWER: Limited variation exists in the percentage of some endometrial stem/progenitor cell types and abundance of selected proteins in menstrual fluid within and between a cohort of women. WHAT IS KNOWN ALREADY: Menstrual fluid is a readily available biofluid that can represent the endometrial environment, containing endometrial stem/progenitor cells and protein factors. It is unknown whether there is natural variation in the cellular and protein content across menstrual cycles of individual women, which has significant implications for the use of menstrual fluid in research and clinical applications. STUDY DESIGN, SIZE, DURATION: Menstrual fluid was collected from 11 non-pregnant females with regular menstrual cycles. Participants had not used hormonal medications in the previous 3 months. Participants collected menstrual fluid samples from up to five cycles using a silicone menstrual cup worn on Day 2 of menstrual bleeding. PARTICIPANTS/MATERIALS, SETTING, METHODS: Menstrual fluid samples were centrifuged to separate soluble proteins and cells. Cells were depleted of red blood cells and CD45+ leucocytes. Menstrual fluid-derived endometrial stem/progenitor cells were characterized using multicolour flow cytometry including markers for endometrial stem/progenitor cells N-cadherin (NCAD) and stage-specific embryonic antigen-1 (SSEA-1) (for endometrial epithelial progenitor cells; eEPC), and sushi domain containing-2 (SUSD2) (for endometrial mesenchymal stem cells; eMSC). The clonogenicity of menstrual fluid-derived endometrial cells was assessed using colony forming unit assays. Menstrual fluid supernatant was analyzed using a custom magnetic Luminex assay. MAIN RESULTS AND THE ROLE OF CHANCE: Endometrial stem/progenitor cells are shed in menstrual fluid and demonstrate clonogenic properties. The intraparticipant agreement for SUSD2+ menstrual fluid-derived eMSC (MF-eMSC), SSEA-1+ and NCAD+SSEA-1+ MF-eEPC, and stromal clonogenicity were moderate-good (intraclass correlation; ICC: 0.75, 0.56, 0.54 and 0.52, respectively), indicating limited variability across menstrual cycles. Endometrial inflammatory and repair proteins were detectable in menstrual fluid supernatant, with five of eight (63%) factors demonstrating moderate intraparticipant agreement (secretory leukocyte protein inhibitor (SLPI), lipocalin-2 (NGAL), lactoferrin, follistatin-like 1 (FSTL1), human epididymis protein-4 (HE4); ICC ranges: 0.57-0.69). Interparticipant variation was limited for healthy participants, with the exception of key outliers of which some had self-reported menstrual pathologies. LARGE SCALE DATA: N/A. There are no OMICS or other data sets relevant to this study. LIMITATIONS, REASONS FOR CAUTION: The main limitations to this research relate to the difficulty of obtaining menstrual fluid samples across multiple menstrual cycles in a consistent manner. Several participants could only donate across <3 cycles and the duration of wearing the menstrual cup varied between 4 and 6 h within and between women. Due to the limited sample size used in this study, wider studies involving multiple consecutive menstrual cycles and a larger cohort of women will be required to fully determine the normal range of endometrial stem/progenitor cell and supernatant protein content of menstrual fluid. Possibility for selection bias and true representation of the population of women should also be considered. WIDER IMPLICATIONS OF THE FINDINGS: Menstrual fluid is a reliable source of endometrial stem/progenitor cells and related endometrial proteins with diagnostic potential. The present study indicates that a single menstrual sample may be sufficient in characterizing a variety of cellular and protein parameters across women's menstrual cycles. The results also demonstrate the potential of menstrual fluid for identifying endometrial and menstrual abnormalities in both research and clinical settings as a non-invasive method for assessing endometrial health. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Australian National Health and Medical Research Council to C.E.G. (Senior Research Fellowship 1024298 and Investigator Fellowship 1173882) and to J.E. (project grant 1047756), the Monash IVF Research Foundation to C.E.G. and the Victorian Government's Operational Infrastructure Support Program. K.A.W., M.L.D.-T., S.G.S. and J.E. declare no conflicts of interest. C.E.G. reports grants from NHMRC, during the conduct of the study; grants from EndoFound USA, grants from Ferring Research Innovation, grants from United States Department of Defence, grants from Clue-Utopia Research Foundation, outside the submitted work. CEF reports grants from EndoFound USA, grants from Clue-Utopia Research Foundation, outside the submitted work.

The impact of the World Health Organization Health Promoting Schools framework approach on diet and physical activity behaviours of adolescents in secondary schools: a systematic review.

OBJECTIVE: To examine the effectiveness of interventions using the World Health Organization Health Promoting Schools (HPSs) framework approach in increasing physical activity (PA) and improving the diet of 11-18-year-olds. STUDY DESIGN: A systematic review guided by the National Health Services Centre for Reviews and Dissemination framework and reported in accordance with the Preferred Reporting Items for Systematic reviews and Meta-Analyses. METHODS: Nine databases and trial registries were searched from 2013 to 2018 for cluster randomised controlled trials involving adolescents' aged 11-18 years. We also included relevant studies from a 2014 Cochrane Review of HPS approach on health behaviours. Data were extracted from included studies and assessed for quality. RESULTS: Twelve eligible studies were identified from seven countries. The studies varied in outcome measures, sample size, quality and duration of intervention and follow-up. Only four of the included studies were of high to moderate quality. We found some evidence of effectiveness for physical activity only interventions and limited evidence of effectiveness for nutrition only and combined PA and nutrition interventions. CONCLUSIONS: There were no discernible patterns across the studies to suggest effective mechanisms for the HPS approach. The family/community component was poorly developed and superficially reported in all studies. Future research should seek to understand how best to work in partnership with secondary schools, to foster and sustain a healthy eating and physical activity culture, which aligns with their core aims. More attention should be paid to the restriction of unhealthy foods in the school environment.

How accessible and acceptable are current GP referral mechanisms for IAPT for low-income patients? Lay and primary care perspectives.

Background: Improving Access to Psychological Therapies (IAPT) constitutes a key element of England's national mental health strategy. Accessing IAPT usually requires patients to self-refer on the advice of their GP. Little is known about how GPs perceive and communicate IAPT services with patients from low-income communities, nor how the notion of self-referral is understood and responded to by such patients.Aims: This paper examines how IAPT referrals are made by GPs and how these referrals are perceived and acted on by patients from low-income backgroundsMethod: Findings are drawn from in-depth interviews with low-income patients experiencing mental distress (n = 80); interviews with GPs (n = 10); secondary analysis of video-recorded GP-patient consultations for mental health (n = 26).Results: GPs generally supported self-referral, perceiving it an important initial step towards patient recovery. Most patients however, perceived self-referral as an obstacle to accessing IAPT, and felt their mental health needs were being undermined. The way that IAPT was discussed and the pathway for referral appears to affect uptake of these services.Conclusions: A number of factors deter low-income patients from self-referring for IAPT. Understanding these issues is necessary in enabling the development of more effective referral and support mechanisms within primary care.

The NCS-LSD cohort study: a description of the methods and analyses used to assess the long-term effectiveness of enzyme replacement therapy and substrate reduction therapy in patients with lysosomal storage disorders.

Lysosomal storage disorders (LSDs) comprise more than 50 extremely rare, inherited metabolic diseases resulting from a deficiency of specific lysosomal enzymes required for normal macromolecular metabolism. The National Collaborative Study for Lysosomal Storage Disorders (NCS-LSD), was a longitudinal cohort study which collected prospective and retrospective clinical data, and patient-reported data from adults and children with a confirmed diagnosis of Gaucher disease, Fabry disease, mucopolysaccharidosis type I (MPS I), mucopolysaccharidosis type II (MPS II), Pompe disease and Niemann Pick disease type C (NPC) in the UK. The study aimed to determine the natural history of these conditions and estimate the effectiveness and cost of therapies. Clinical outcomes were chosen to reflect disease progression. Age- and gender-adjusted treatment effects were estimated using generalised linear mixed models. Treated patients contributed data before and during treatment while untreated patients contributed natural history data. A total of 711 adults and children were recruited to this study from the seven LSD treatment centres in England. Data was collected from 2008 to 2011. This paper describes the methods used to collect and analyse clinical data for this study. The clinical findings are reported separately in a series of condition-specific articles in this issue.

Long-term effectiveness of enzyme replacement therapy in adults with Gaucher disease: results from the NCS-LSD cohort study.

OBJECTIVES: To determine the effectiveness of enzyme replacement therapies (ERT) for adults with Gaucher disease (GD). DESIGN: A longitudinal, multi-centre cohort study, including prospective and retrospective clinical data. Age- and gender-adjusted treatment effects were estimated using generalised linear mixed models. Treated patients contributed data before and during treatment. Untreated patients contributed natural history data. PARTICIPANTS: Consenting adults (N = 150, aged 16 to 83 years) with a diagnosis of GD who attended a specialist treatment centre in England. At recruitment, 131 patients were receiving ERT (mean treatment duration, 10.8 years; range 0-18 years). OUTCOME MEASURES: Clinical outcomes chosen to reflect disease progression, included platelet count; haemoglobin; absence/presence of bone pain; spleen and liver volumes and AST levels. RESULTS: One hundred and fifty adults were recruited. Duration of ERT was associated with statistically significant improvements in platelet count (p < 0.001), haemoglobin (p < 0.001), liver and spleen volumes (p < 0.001) and AST levels (p = 0.02). CONCLUSIONS: These data provide further evidence of the long-term effectiveness of ERT in adults with GD.

Long-term effectiveness of enzyme replacement therapy in children with Gaucher disease: results from the NCS-LSD cohort study.

OBJECTIVES: To determine the effectiveness of enzyme replacement therapies (ERT) for children with Gaucher disease (GD). DESIGN: A longitudinal cohort study including prospective and retrospective clinical data. Age- and gender-adjusted treatment effects were estimated using generalised linear mixed models. Children on treatment contributed data before and during treatment. Children not on treatment contributed natural history data. PARTICIPANTS: Consenting children (N = 25, aged 1.1 to 15.6 years) with a diagnosis of GD (14 with GD1 and 11 with GD3) who attended a specialist treatment centre in England. At recruitment, 24 patients were receiving ERT (mean treatment duration, 5.57 years; range 0-13.7 years). OUTCOME MEASURES: Clinical outcomes chosen to reflect disease progression, included platelet count; haemoglobin and absence/presence of bone pain. RESULTS: Duration of ERT was associated with statistically significant improvements in platelet count (p < 0.001), haemoglobin (p < 0.001), and reported bone pain (p = 0.02). The magnitude of effect on haematological parameters was greater in children with GD3 than in those with GD1. CONCLUSIONS: These data provide further evidence of the long-term effectiveness of ERT in children with GD.

Effectiveness of enzyme replacement therapy in adults with late-onset Pompe disease: results from the NCS-LSD cohort study.

OBJECTIVES: To determine the effectiveness of enzyme replacement therapy (ERT) for adults with late-onset Pompe disease. DESIGN: A longitudinal cohort study including prospective and retrospective clinical outcome data. Age- and gender-adjusted treatment effects were estimated using generalised linear mixed models. Treated patients contributed data before and during treatment. Untreated patients contributed natural history data. PARTICIPANTS: Consenting adults (N = 62) with a diagnosis of late-onset Pompe disease who attended a specialist treatment centre in England. This cohort represented 83 % of all patients in the UK with a confirmed diagnosis of this rare condition. At study entry, all but three patients were receiving ERT (range of treatment duration, 0 to 3.1 years). OUTCOME MEASURES: Percent predicted forced vital capacity (%FVC); ventilation dependency; mobility; 6 min walk test (6MWT); muscle strength and body mass index (BMI). RESULTS: An association was found between time on ERT and significant increases in the distance walked in the 6MWT (p < 0.001) and muscle strength scores (p < 0.001). Improvements in both these measures were seen over the first 2 years of treatment with ERT. No statistically significant relationship was found between time on ERT and respiratory function or in BMI. CONCLUSIONS: These data provide some further evidence of the effectiveness of ERT in adults with late-onset Pompe disease. SYNOPSIS: The results of this longitudinal cohort study of 62 adults with late-onset Pompe disease, provide further evidence on the effectiveness of ERT in this rare condition.

Long-term effectiveness of enzyme replacement therapy in Fabry disease: results from the NCS-LSD cohort study.

OBJECTIVES: To determine the effectiveness of enzyme replacement therapy (ERT) for adults and children with Fabry disease. DESIGN: Cohort study including prospective and retrospective clinical data. Age- and gender-adjusted treatment effects were estimated using generalised linear mixed models. Treated patients contributed data before and during treatment and untreated patients contributed natural history data. PARTICIPANTS: Consenting adults (N = 289) and children (N = 22) with a confirmed diagnosis of Fabry disease attending a specialist Lysosomal Storage Disorder treatment centre in England. At recruitment 211 adults and seven children were on ERT (range of treatment duration, 0 to 9.7 and 0 to 4.2 years respectively). OUTCOME MEASURES: Clinical outcomes chosen to reflect disease progression included left ventricular mass index (LVMI); proteinuria; estimated glomerular filtration rate (eGFR); pain; hearing and transient ischaemic attacks (TIA)/stroke. RESULTS: We found evidence of a statistically significant association between time on ERT and a small linear decrease in LVMI (p = 0.01); a reduction in the risk of proteinuria after adjusting for angiotensin-converting enzyme inhibitors and angiotensin receptor blockers (p < 0.001) and a small increase in eGFR in men and women without pre-treatment proteinuria (p = 0.01 and p < 0.001 respectively). The same analyses in children provided no statistically significant results. No associations between time on ERT and pain, risk of needing a hearing aid, or risk of stroke or TIAs, were found. CONCLUSIONS: These data provide some further evidence on the long-term effectiveness of ERT in adults with Fabry disease, but evidence of effectiveness could not be demonstrated in children.

A search for new back contacts for CdTe solar cells.

There is widespread interest in reaching the practical efficiency of cadmium telluride (CdTe) thin-film solar cells, which suffer from open-circuit voltage loss due to high surface recombination velocity and Schottky barrier at the back contact. Here, we focus on back contacts in the superstrate configuration with the goal of finding new materials that can provide improved passivation, electron reflection, and hole transport properties compared to the commonly used material, ZnTe. We performed a computational search among 229 binary and ternary tetrahedrally bonded structures using first-principles methods and transport models to evaluate critical material design criteria, including phase stability, electronic structure, hole transport, band alignments, and p-type dopability. Through this search, we have identified several candidate materials and their alloys (AlAs, AgAlTe2, ZnGeP2, ZnSiAs2, and CuAlTe2) that exhibit promising properties for back contacts. We hope that these new material recommendations and associated guidelines will inspire new directions in hole transport layer design for CdTe solar cells.

Scalable ultrafast epitaxy of large-grain and single-crystal II-VI semiconductors.

A general problem for semiconductor applications is that very slow deposition on expensive single-crystal substrates yields high crystalline quality with excellent electro-optical properties, but at prohibitive costs and throughput for many applications. In contrast, rapid deposition on inexpensive substrates or nanocrystalline films yields low costs, but comparatively inferior crystallinity, carrier transport, and recombination. Here, we present methods to deposit single-crystal material at rates 2-3 orders of magnitude faster than state-of-the-art epitaxy with low-cost methods without compromising crystalline or electro-optical quality. For example, single-crystal CdTe and CdZnTe films that would take several days to grow by molecular-beam epitaxy are deposited in 8 minutes by close-spaced sublimation, yet retain the same crystalline quality measured by X-ray diffraction rocking curves. The fast deposition is coupled with effective n- and p-type in-situ doping by In, P, and As. The epitaxy can be extended to nanocrystalline substrates. For example, we recrystallize thin CdTe films on glass to deposit large grains with low defect density. The results provide new research paths for photovoltaics, detectors, infrared imaging, flexible electronics, and other applications.

SnO2-Catalyzed Oxidation in High-Efficiency CdTe Solar Cells.

Interfaces at the front of superstrate CdTe-based solar cells are critical to carrier transport, recombination, and device performance, yet determination of the chemical structure of these nanoscale regions has remained elusive. This is partly due to changes that occur at the front interfaces during high temperature growth and substantive changes occurring during postdeposition processing. In addition, these buried interfaces are extremely difficult to access in a way that preserves chemical information. In this work, we use a recently developed thermomechanical cleaving technique paired with X-ray photoelectron spectroscopy to probe oxidation states at the SnO2 interface of CdTe solar cells. We show that the tin oxide front electrode promotes the formation of nanometer-scale oxides of tellurium and sulfur. Most oxidation occurs during CdCl2/O2 activation. Surprisingly, we show that relatively low-temperature anneals (180-260 °C) used to diffuse and activate copper acceptors in a doping/back contact process also cause significant changes in oxidation at the front of the cell, providing a heretofore missing aspect of how back contact processes can modify device transport, recombination, and performance. Device performance is shown to correlate with the extent of tellurium and sulfur oxidation within this nanometer-scale region. Mechanisms responsible for these beneficial effects are proposed.

Factors Associated With High-Voltage Impedance and Subcutaneous Implantable Defibrillator Ventricular Fibrillation Conversion Success.

BACKGROUND: The ability to predict defibrillation efficacy at the time of subcutaneous implantable cardioverter-defibrillator implantation without the need to induce ventricular fibrillation might eliminate the need for defibrillation testing. The purpose of this study was to determine the association of high-voltage impedance and system implant position on ventricular fibrillation conversion success with a submaximal 65-J shock. METHODS: In the subcutaneous implantable cardioverter-defibrillator IDE study (Investigational Device Exemption), a successful conversion test required 2 consecutive ventricular fibrillation conversions at 65 J in either shock vector. Chest radiographs were obtained after implantation. Patients with imaging and impedance data were included. Suboptimal device position was defined as an inferior electrode or pulse generator or electrode coil depth >3 mm anterior to the sternum. Absence of suboptimal positional parameters was defined as appropriate position. Conversion success rate was calculated among all 65-J tests. RESULTS: Of 314 patients who underwent subcutaneous implantable cardioverter-defibrillator implantation, 282 patients were included in this analysis. There were 637 inductions to test defibrillation at 65 J. Sixty-two conversion failures (9.7%) occurred in 42 (14.9%) patients. Lower body mass index and lower shock impedance were associated with higher conversion success rate, whereas white race was associated with lower conversion success rate. Suboptimal position was more common in obese patients. Inferior electrode and greater distance between the lead and sternum were associated with a higher impedance. When appropriate system position was achieved, conversion failure was not associated with high body mass index. CONCLUSIONS: Subcutaneous implantable cardioverter-defibrillator shock efficacy is associated with system position and high-voltage system impedance. A high impedance is associated with inferiorly placed pulse generator and electrode system, inadequate coil depth, and a lower rate of defibrillator success. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov . Unique identifier: NCT01064076.

Overcoming Carrier Concentration Limits in Polycrystalline CdTe Thin Films with In Situ Doping.

Thin film materials for photovoltaics such as cadmium telluride (CdTe), copper-indium diselenide-based chalcopyrites (CIGS), and lead iodide-based perovskites offer the potential of lower solar module capital costs and improved performance to microcrystalline silicon. However, for decades understanding and controlling hole and electron concentration in these polycrystalline films has been extremely challenging and limiting. Ionic bonding between constituent atoms often leads to tenacious intrinsic compensating defect chemistries that are difficult to control. Device modeling indicates that increasing CdTe hole density while retaining carrier lifetimes of several nanoseconds can increase solar cell efficiency to 25%. This paper describes in-situ Sb, As, and P doping and post-growth annealing that increases hole density from historic 1014 limits to 1016-1017 cm-3 levels without compromising lifetime in thin polycrystalline CdTe films, which opens paths to advance solar performance and achieve costs below conventional electricity sources.

Evaluation of the risks of chemotherapy in dogs with thrombocytopenia.

Thrombocytopenia is commonly encountered in veterinary oncology. Currently, there are no standard guidelines regarding the administration of chemotherapy to the patients with thrombocytopenia. This observational epidemiological cohort study aimed to determine whether thrombocytopenic dogs were at increased risk of gastrointestinal adverse effects (vomiting, diarrhoea, inappetence) or haemorrhage following administration of standard doses of chemotherapy. The adverse effects following 77 prospectively identified episodes of thrombocytopenia (platelet count, <200 000 µL-1 ) were compared with the adverse effects experienced in a retrospective cohort (platelet count >200 000 µL-1 ), and evaluated by statistical analysis. Overall, there was no statistically significant difference in the incidence of gastrointestinal adverse effects or haemorrhage between thrombocytopenic and control dogs. The control group of dogs with lymphoma were statistically more likely to experience vomiting as an adverse effect of chemotherapy (P = 0.028). The results presented here showed no evidence for an increased risk of gastrointestinal adverse effects or haemorrhage in thrombocytopenic dogs after receiving standard doses of chemotherapy.

The impact of active stakeholder involvement on recruitment, retention and engagement of schools, children and their families in the cluster randomised controlled trial of the Healthy Lifestyles Programme (HeLP): a school-based intervention to prevent obesity.

BACKGROUND: Recruitment and retention of participants is crucial for statistical power and internal and external validity and participant engagement is essential for behaviour change. However, many school-based interventions focus on programme content rather than the building of supportive relationships with all participants and tend to employ specific standalone strategies, such as incentives, to improve retention. We believe that actively involving stakeholders in both intervention and trial design improves recruitment and retention and increases the chances of creating an effective intervention. METHODS: The Healthy Lifestyles Programme, HeLP (an obesity prevention programme for children 9-10 years old) was developed using intervention mapping and involved extensive stakeholder involvement in both the design of the trial and the intervention to ensure that: (i) delivery methods were suitably engaging, (ii) deliverers had the necessary skills and qualities to build relationships and (iii) the intervention dovetailed with the National Curriculum. HeLP was a year-long intervention consisting of 4 multi-component phases using a range of delivery methods. We recruited 1324 children from 32 schools from the South West of England to a cluster-randomised controlled trial to determine the effectiveness of HeLP in preventing obesity. The primary outcome was change in body mass index standard deviation score (BMI SDS) at 24 months post randomisation. Secondary outcomes included additional anthropometric and behavioural (physical activity and diet) measures at 18 and 24 months. RESULTS: Anthropometric and behavioural measures were taken in 99%, 96% and 94% of children at baseline, 18 and 24 months, respectively, with no differential follow up between the control and intervention groups at each time point. All children participated in the programme and 92% of children and 77% of parents across the socio-economic spectrum were considered to have actively engaged with HeLP. CONCLUSIONS: We attribute our excellent retention and engagement results to the high level of stakeholder involvement in both trial and intervention design, the building of relationships using appropriate personnel and creative delivery methods that are accessible to children and their families across the social spectrum. TRIAL REGISTRATION: International Standard Randomised Controlled Trials Register, ISRCTN15811706 . Registered on 1 May 2012.

Furosemide for prevention of cyclophosphamide-associated sterile haemorrhagic cystitis in dogs receiving metronomic low-dose oral cyclophosphamide.

Sterile haemorrhagic cystitis (SHC) is a known risk of cyclophosphamide treatment. Diuresis using furosemide is effective in canines when maximally tolerated dosed cyclophosphamide is administered. This retrospective study aimed to determine whether orally administered furosemide decreased the incidence of SHC. Secondary aims were to identify predisposing factors for SHC. One-hundred and fifteen dogs treated with metronomic cyclophosphamide were analysed retrospectively. Populations were not randomized. 25 dogs (21.7%) developed SHC. Furosemide administration significantly reduced the likelihood of SHC development (P = 0.010, where SHC was diagnosed in 30.3% of dogs administered cyclophosphamide without furosemide, and 10.2% of dogs administered cyclophosphamide with furosemide). Age, gender, breed, bodyweight, number of cyclophosphamide treatments, piroxicam use and previous or pre-existing disease were not found to be associated with SHC development. This study demonstrates furosemide is effective in the prevention of SHC and its use may be considered when implementing metronomic cyclophosphamide therapy.

3D Lifetime Tomography Reveals How CdCl2 Improves Recombination Throughout CdTe Solar Cells.

Using two-photon tomography, carrier lifetimes are mapped in polycrystalline CdTe photovoltaic devices. These 3D maps probe subsurface carrier dynamics that are inaccessible with traditional optical techniques. They reveal that CdCl2 treatment of CdTe solar cells suppresses nonradiative recombination and enhances carrier lifetimes throughout the film with substantial improvements particularly near subsurface grain boundaries and the critical buried p-n junction.

Both ankyrin and band 4.1 are required to restrict the rotational mobility of band 3 in the human erythrocyte membrane.

A population of band 3 proteins in the human erythrocyte membrane is known to have restricted rotational mobility due to interaction with cytoskeletal proteins. We have further investigated the cause of this restriction by measuring the effects on band 3 rotational mobility of rebinding ankyrin and band 4.1 to ghosts stripped of these proteins as well as spectrin and actin. Rebinding either ankyrin or 4.1 alone has no detectable effect on band 3 mobility. Rebinding both these proteins together does, however, reimpose a restriction on band 3 rotation. The effect on band 3 rotational mobility of rebinding ankyrin and 4.1 are similar irrespective of whether or not band 4.2 is removed from the membrane. We suggest that ankyrin and 4.1 together promote the formation of slowly rotating clusters of band 3.

The P23T cataract mutation causes loss of solubility of folded gammaD-crystallin.

Mutations in the human gammaD-crystallin gene have been linked to several types of congenital cataracts. In particular, the Pro23 to Thr (P23T) mutation of human gammaD crystallin has been linked to cerulean, lamellar, coralliform, and fasciculiform congenital cataracts. We have expressed and purified wild-type human gammaD, P23T, and the Pro23 to Ser23 (P23S) mutant. Our measurements show that P23T is significantly less soluble than wild-type human gammaD, with P23S having an intermediate solubility. Using synchrotron radiation circular dichroism spectroscopy, we have determined that the P23T mutant has a slightly increased content of beta-sheet, which may be attributed to the extension of an edge beta-strand due to the substitution of Pro23 with a residue able to form hydrogen bonds. Neither of the point mutations appears to have reduced the thermal stability of the protein significantly, nor its resistance to guanidine hydrochloride-induced unfolding. These results suggest that insolubility, rather than loss of stability, is the primary basis for P23T congenital cataracts.

Thyroid status and nitric oxide in rat arterial vessels.

Thyroid disease has profound effects on cardiovascular function. Hypo- and hyperthyroidism, for example, are associated with reduced and increased maximal endothelium-dependent vasodilation respectively. We therefore hypothesized that the capacity for vascular nitric oxide (NO) formation is decreased in hypothyroidism and increased in hyperthyroidism. To test this hypothesis, rats were made hypothyroid (HYPO) with propylthiouracil or hyperthyroid (HYPER) with triiodothyronine over 3-4 months. Compared with euthyroid control rats (EUT), HYPO exhibited blunted growth and lower citrate synthase activity in the soleus muscle; HYPER exhibited left ventricular hypertrophy and higher citrate synthase activity in the soleus muscle (P<0.05 for all effects). The capacity for NO formation was determined in aortic extracts by formation of [3H]L-citrulline from [3H]L-arginine, i.e. NO synthase (NOS) activity. Thyroid status modulated NOS activity (EUT, 36.8 +/- 5.5 fmol/h per mg protein; HYPO, 26.0 +/- 7.9; HYPER, 64.6 +/- 12.7; P<0.05, HYPER vs HYPO). Expression of endothelial and neural isoforms of NOS was modulated by thyroid status in a parallel fashion. Capacity for responding to NO was also determined via measuring cGMP concentration in aortae incubated with sodium nitroprusside. Stimulated cGMP formation was also modulated by thyroid status (EUT, 73.0 +/- 20.2 pmol/mg protein; HYPO, 152.4 +/- 48.7; HYPER, 10.4 +/- 2.6; P<0.05, HYPER vs HYPO). These data indicate that thyroid status alters capacities for both formation of and responding to NO. The former finding may contribute to previous findings concerning vascular function in thyroid disease states.