A brief peek at the cyclotron in our microscope.

The influence of low energy bremsstrahlung emission on the performance of electron spectrometers and monochromators is investigated. Despite the occurrence of multi-photon events, the effect of the main azimuthal (organ pipe) mode is likely to be negligible. Potentially more serious is a new radial mode not considered in the classical theory but revealed in the quantum mechanics picture. The progress of the finely focused wave at the spectrometer entrance slit is described by a coherent wave packet of many oscillator states. It is shielded from disruption by a relatively much longer half-life. Cavity effects causing additional suppression of bremsstrahlung emission are briefly discussed.

Risk and Quality in Brachytherapy From a Technical Perspective.

AIMS: To provide an overview of the history of incidents in brachytherapy and to describe the pillars in place to ensure that medical physicists deliver high-quality brachytherapy. MATERIALS AND METHODS: A review of the literature was carried out to identify reported incidents in brachytherapy, together with an evaluation of the structures and processes in place to ensure that medical physicists deliver high-quality brachytherapy. In particular, the role of education and training, the use of process and technical quality assurance and the role of international guidelines are discussed. RESULTS: There are many human factors in brachytherapy procedures that introduce additional risks into the process. Most of the reported incidents in the literature are related to human factors. Brachytherapy-related education and training initiatives are in place at the societal and departmental level for medical physicists. Additionally, medical physicists have developed process and technical quality assurance procedures, together with international guidelines and protocols. Education and training initiatives, together with quality assurance procedures and international guidelines may reduce the risk of human factors in brachytherapy. CONCLUSION: Through application of the three pillars (education and training; process control and technical quality assurance; international guidelines), medical physicists will continue to minimise risk and deliver high-quality brachytherapy treatments.

Reproductive and metabolic adaptation to multistressor training in women.

Hypothalamic-pituitary-gonadal (HPG) axis suppression in exercising women can be caused by low energy availability (EA), but the impact of a real-world, multistressor training environment on reproductive and metabolic function is unknown. This study aimed to characterize reproductive and metabolic adaptation in women undertaking basic military training. A prospective cohort study in women undertaking 11-month initial military training (n = 47) was carried out. Dynamic low-dose 1-h gonadotrophin-releasing hormone (GnRH) tests were completed after 0 and 7 mo of training. Urine progesterone was sampled weekly throughout. Body composition (dual X-ray absorptiometry), fasting insulin resistance (homeostatic modeling assessment 2, HOMA2), leptin, sex steroids, anti-Müllerian hormone (AMH), and inhibin B were measured after 0, 7, and 11 mo with an additional assessment of body composition at 3 mo. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) responses were suppressed after 7 mo (both P < 0.001). Among noncontraceptive users (n = 20), 65% had regular (23-35 days) cycles preenrollment, falling to 24% by 7 mo of training. Of women in whom urine progesterone was measured (n = 24), 87% of cycles showed no evidence of ovulation. There was little change in AMH, LH, and estradiol, although inhibin B and FSH increased (P < 0.05). Fat mass fluctuated during training but at month 11 was unchanged from baseline. Fat-free mass did not change. Visceral adiposity, HOMA2, and leptin increased (all P < 0.001). HPG axis suppression with anovulation occurred in response to training without evidence of low EA. Increased insulin resistance may have contributed to the observed pituitary and ovarian dysfunction. Our findings are likely to represent an adaptive response of reproductive function to the multistressor nature of military training.NEW & NOTEWORTHY We characterized reproductive endocrine adaptation to prolonged arduous multistressor training in women. We identified marked suppression of hypothalamic-pituitary-gonadal (HPG) axis function during training but found no evidence of low energy availability despite high energy requirements. Our findings suggest a complex interplay of psychological and environmental stressors with suppression of the HPG axis via activation of the hypothalamic-pituitary adrenal (HPA) axis. The neuroendocrine impact of nonexercise stressors on the HPG axis during arduous training should be considered.

The safety of paediatric surgery between COVID-19 surges: an observational study.

Despite the ongoing coronavirus disease 2019 (COVID-19) pandemic, elective paediatric surgery must continue safely through the first, second and subsequent waves of disease. This study presents outcome data from a children's hospital in north-west England, the region with the highest prevalence of COVID-19 in England. Children and young people undergoing elective surgery isolated within their household for 14 days, then presented for real-time reverse transcriptase polymerase chain reaction testing for severe acute respiratory syndrome coronavirus disease-2 (SARS-CoV-2) within 72 h of their procedure (or rapid testing within 24 h in high-risk cases), and completed a screening questionnaire on admission. Planned surgery resumed on 26 May 2020; in the four subsequent weeks, there were 197 patients for emergency and 501 for elective procedures. A total of 488 out of 501 (97.4%) elective admissions proceeded, representing a 2.6% COVID-19-related cancellation rate. There was no difference in the incidence of SARS-CoV-2 among children and young people who had or had not isolated for 14 days (p > 0.99). One out of 685 (0.1%) children who had surgery re-presented to the hospital with symptoms potentially consistent with SARS-CoV-2 within 14 days of surgery. Outcomes were similar to those in the same time period in 2019 for length of stay (p = 1.0); unplanned critical care admissions (p = 0.59); and 14-day hospital re-admission (p = 0.17). However, the current cohort were younger (p = 0.037); of increased complexity (p < 0.001) and underwent more complex surgery (p < 0.001). The combined use of household self-isolation, testing and screening questionnaires has allowed the re-initiation of elective paediatric surgery at high volume while maintaining pre-COVID-19 outcomes in children and young people undergoing surgery. This may provide a model for addressing the ongoing challenges posed by COVID-19, as well as future pandemics.

BrachyView: initial preclinical results for a real-time in-body HDR PBT source tracking system with simultaneous TRUS image fusion.

A prototype in-body gamma camera system with integrated trans-rectal ultrasound (TRUS) and associated real-time image acquisition and analysis software was developed for intraoperative source tracking in high dose rate (HDR) brachytherapy. The accuracy and temporal resolution of the system was validated experimentally using a deformable tissue-equivalent prostate gel phantom and a full clinical HDR treatment plan. The BrachyView system was able to measure 78% of the 200 source positions with an accuracy of better than 1 mm. A minimum acquisition time of 0.28 s/frame was required to achieve this accuracy, restricting dwell times to a minimum of 0.3 s. Additionally, the performance of the BrachyView-TRUS fusion probe for mapping the spatial location of the tracked source within the prostate volume was evaluated. A global coordinate system was defined by scanning the phantom with the probe in situ using a CT scanner, and was subsequently used for co-registration of the BrachyView and TRUS fields of view (FoVs). TRUS imaging was used to segment the prostate volume and reconstruct it into a three-dimensional (3D) image. Fusion of the estimated source locations with the 3D prostate image was performed using integrated 3D visualisation software. HDR BrachyView is demonstrated to be a valuable tool for intraoperative source tracking in HDR brachytherapy, capable of resolving source dwell locations relative to the prostate anatomy when combined with TRUS.

Continued skirmishing on the wave-particle frontier.

Historical developments in electron microscopy where understanding lies at the interface between the particle and wave pictures of the electron are reviewed. Electron optics has remained mainly particle-based but imaging is now substantially dependent on wave methods which are now pushing it to new heights. Spectroscopy in the low loss region has used Fermi's classical electron model which can also describe the main effects of Johnson noise. It is confirmed that the RMS Johnson noise scattering of an aloof beam electron is proportional to its wavelength and to the square root of the temperature. If the conductivity σ is high, it is independent of σ. In the performance of magnetic spectrometers, the angular deflections due to the walls may be less significant than that generated in the slits. The angular deflections due to the many bremmstrahlung events in the spectrometer seem to be negligible but the influence of Johnson noise on them is beyond the scope of the classical Fermi theory and will need to be addressed by the quantum mechanical spontaneous wave theory.

Urinary specific gravity as an alternative for the normalisation of endocrine metabolite concentrations in giant panda (Ailuropoda melanoleuca) reproductive monitoring.

Reproductive monitoring for captive breeding in giant pandas is based on behavioural observation and non-invasive hormone analysis. In urine, interpretation of results requires normalisation due to an animal's changing hydration. Correction of urinary concentrations based on creatinine is the gold standard. In this study, a largely unexplored, easy-to-perform normalisation technique, based on urinary specific gravity (USpG), was examined and compared to creatinine. To this extent, six cycles from two female pandas (SB741(1) and SB569(5)) were monitored through urine analysis for oestrogen, progesterone, ceruloplasmin and 13,14-dihydro-15-keto-PGF2a (PGFM). The Pearson's correlation between creatinine and USpG was high (r = 0.805-0.894; p < 0.01), indicative for a similar performance of both normalisation methods. However, generally lower values were observed during pro-oestrus and primary (progesterone) rise. This could be associated with huge shifts in appetite, monitored by faecal output (kg) with an averaged > 50% decrease during oestrus and >50% increase during primary progesterone rise. In parallel, respectively highest and lowest creatinine and USpG levels, were measured, with creatinine obviously more affected as a result of linkage with muscle tissue metabolism affected by reproductive hormones. As a consequence, metabolite levels were significantly different between both corrected datasets with significantly higher oestrogen peak levels during oestrus ranging from 2.13-86.93 and 31.61-306.45 ng/mL (USpG correction) versus 2.33-31.20 and 36.36-249.05 ng/mL Cr (creatinine correction) for SB569 and SB741 respectively, and significant lower progesterone levels during primary progesterone rise ranging from 0.35-3.21 and 0.85-6.80 ng/mL (USpG correction) versus 0.52-10.31 and 2.10-272.74 ng/mL Cr (creatinine correction) for SB569 and SB741 respectively. Consequently, USpG correction rendered unbiased profiles, less subject to variation and metabolic artefacts and therefore allowed a more straightforward identification of peak oestrogen and onset of secondary progesterone rise, being potentially advantageous for future studies unravelling key giant panda reproductive events, including (delayed) implantation. The alternative application of USpG as a normalisation factor was further supported by its easy application and environmental and technical robustness.

New instrumentation and cutting edge research.

Stimulated by Ondrej Krivanek's contributions, the complex interaction between research and innovations in the instrumentation for electron microscopy is discussed. Specific attention is given to aberration correction and to spectroscopy in both the valence region and at the energies of localised phonons or bond vibrations. Current thinking about projection imaging and dielectric excitation theory may be challenged. More significantly in the new fields of investigation opened up to them, electron microscopists will need to build closer relations, particularly with the photonics and scanning tunnelling microscopy communities. Further improvements in instrumentation could usefully be directed towards imaging and spectroscopy at higher scattering angles as well as the incorporation of other facilities such as photon stimulation and secondary electron imaging.

Placental hormone profiles as predictors of preterm birth in twin pregnancy: A prospective cohort study.

OBJECTIVE: The objective of the study was to analyse placental hormone profiles in twin pregnancies to determine if they could be used to predict preterm birth. STUDY DESIGN: Progesterone, estradiol, estriol and corticotropin-releasing hormone were measured using competitive immunoassay and radioimmunoassay in serum and saliva samples of 98 women with twin pregnancies,at 3 or more gestational timepoints. Hormone profiles throughout gestation were compared between very preterm (<34 weeks; n = 8), preterm (<37 weeks; n = 40) and term (37+ weeks; n = 50) deliveries. RESULTS: No significant differences were found between preterm and term deliveries in either absolute hormone concentrations or ratios. Estimated hormone concentrations and ratios at 26 weeks did not appear to predict preterm delivery. Salivary and serum hormone concentrations were generally poorly correlated. CONCLUSION: Our results suggest that serial progesterone, estradiol, estriol and corticotropin-releasing hormone measurements in saliva and serum are not robust biomarkers for preterm birth in twin pregnancies.

The evaluation of a 2D diode array in "magic phantom" for use in high dose rate brachytherapy pretreatment quality assurance.

PURPOSE: High dose rate (HDR) brachytherapy is a treatment method that is used increasingly worldwide. The development of a sound quality assurance program for the verification of treatment deliveries can be challenging due to the high source activity utilized and the need for precise measurements of dwell positions and times. This paper describes the application of a novel phantom, based on a 2D 11 × 11 diode array detection system, named "magic phantom" (MPh), to accurately measure plan dwell positions and times, compare them directly to the treatment plan, determine errors in treatment delivery, and calculate absorbed dose. METHODS: The magic phantom system was CT scanned and a 20 catheter plan was generated to simulate a nonspecific treatment scenario. This plan was delivered to the MPh and, using a custom developed software suite, the dwell positions and times were measured and compared to the plan. The original plan was also modified, with changes not disclosed to the primary authors, and measured again using the device and software to determine the modifications. A new metric, the "position­time gamma index," was developed to quantify the quality of a treatment delivery when compared to the treatment plan. The MPh was evaluated to determine the minimum measurable dwell time and step size. The incorporation of the TG-43U1 formalism directly into the software allows for dose calculations to be made based on the measured plan. The estimated dose distributions calculated by the software were compared to the treatment plan and to calibrated EBT3 film, using the 2D gamma analysis method. RESULTS: For the original plan, the magic phantom system was capable of measuring all dwell points and dwell times and the majority were found to be within 0.93 mm and 0.25 s, respectively, from the plan. By measuring the altered plan and comparing it to the unmodified treatment plan, the use of the position­time gamma index showed that all modifications made could be readily detected. The MPh was able to measure dwell times down to 0.067 ± 0.001 s and planned dwell positions separated by 1 mm. The dose calculation carried out by the MPh software was found to be in agreement with values calculated by the treatment planning system within 0.75%. Using the 2D gamma index, the dose map of the MPh plane and measured EBT3 were found to have a pass rate of over 95% when compared to the original plan. CONCLUSIONS: The application of this magic phantom quality assurance system to HDR brachytherapy has demonstrated promising ability to perform the verification of treatment plans, based upon the measured dwell positions and times. The introduction of the quantitative position­time gamma index allows for direct comparison of measured parameters against the plan and could be used prior to patient treatment to ensure accurate delivery.

The evaluation of a 2D diode array in "magic phantom" for use in high dose rate brachytherapy pretreatment quality assurance.

PURPOSE: High dose rate (HDR) brachytherapy is a treatment method that is used increasingly worldwide. The development of a sound quality assurance program for the verification of treatment deliveries can be challenging due to the high source activity utilized and the need for precise measurements of dwell positions and times. This paper describes the application of a novel phantom, based on a 2D 11 × 11 diode array detection system, named "magic phantom" (MPh), to accurately measure plan dwell positions and times, compare them directly to the treatment plan, determine errors in treatment delivery, and calculate absorbed dose. METHODS: The magic phantom system was CT scanned and a 20 catheter plan was generated to simulate a nonspecific treatment scenario. This plan was delivered to the MPh and, using a custom developed software suite, the dwell positions and times were measured and compared to the plan. The original plan was also modified, with changes not disclosed to the primary authors, and measured again using the device and software to determine the modifications. A new metric, the "position-time gamma index," was developed to quantify the quality of a treatment delivery when compared to the treatment plan. The MPh was evaluated to determine the minimum measurable dwell time and step size. The incorporation of the TG-43U1 formalism directly into the software allows for dose calculations to be made based on the measured plan. The estimated dose distributions calculated by the software were compared to the treatment plan and to calibrated EBT3 film, using the 2D gamma analysis method. RESULTS: For the original plan, the magic phantom system was capable of measuring all dwell points and dwell times and the majority were found to be within 0.93 mm and 0.25 s, respectively, from the plan. By measuring the altered plan and comparing it to the unmodified treatment plan, the use of the position-time gamma index showed that all modifications made could be readily detected. The MPh was able to measure dwell times down to 0.067 ± 0.001 s and planned dwell positions separated by 1 mm. The dose calculation carried out by the MPh software was found to be in agreement with values calculated by the treatment planning system within 0.75%. Using the 2D gamma index, the dose map of the MPh plane and measured EBT3 were found to have a pass rate of over 95% when compared to the original plan. CONCLUSIONS: The application of this magic phantom quality assurance system to HDR brachytherapy has demonstrated promising ability to perform the verification of treatment plans, based upon the measured dwell positions and times. The introduction of the quantitative position-time gamma index allows for direct comparison of measured parameters against the plan and could be used prior to patient treatment to ensure accurate delivery.

Stimulated excitation electron microscopy and spectroscopy.

Recent advances in instrumentation for electron optics and spectroscopy have prompted exploration of ultra-low excitations such as phonons, bond vibrations and Johnson noise. These can be excited not just with fast electrons but also thermally or by other external sources of radiation. The near-field theory of electron energy loss and gain provides a convenient platform for analysing these processes. Possibilities for selected phonon mapping and imaging are discussed. Effects should certainly be observable in atomic resolution structure imaging but diffraction contrast imaging could perhaps be more informative. Additional exciting prospects to be explored include the transition from phonon excitation to single atom recoil and the boosting of energy loss and gain signals with tuned laser illumination.

A highly-sensitive anti-Müllerian hormone assay improves analysis of ovarian function following chemotherapy for early breast cancer.

AIM: Anti-Müllerian hormone (AMH) shows promise as a biomarker of the ovarian reserve but current assays are insufficiently sensitive to allow assessment of this post-chemotherapy in most women. We have assessed a new highly sensitive AMH assay (Ansh picoAMH) in the evaluation of ovarian activity in women with very low ovarian reserve after chemotherapy. METHODS: A prospective cohort and an independent validation cohort of premenopausal women with early breast cancer (eBC) were recruited at the time of diagnosis (combined n=98), and ovarian reserve markers 2-5 years later following chemotherapy were assessed in relation to menstrual activity. RESULTS: The picoAMH assay had a limit of detection of 7.5 pg/ml. AMH clearly distinguished women with ongoing menses from those with amenorrhoea at 2 years after diagnosis (mean 522 ± 169 versus 8.9 ± 1.3 pg/ml, P<0.0001) with high predictive value for continuing menses or amenorrhoea for the subsequent 3 years. AMH was detectable in more women than using a previous assay (P=0.004). Other markers of the ovarian reserve (follicle-stimulating hormone (FSH), inhibin B) were also of discriminatory value but to lesser extents. This finding was validated in a second, independent cohort of women treated for eBC. CONCLUSION: The 10-fold increased assay sensitivity showed very clear distinction between groups based on ovarian activity with excellent prediction of future menses or amenorrhoea. This will improve assessment of post-chemotherapy ovarian function in women and may aid treatment decisions.

Altered maternal hypothalamic-pituitary-adrenal axis activity in obese pregnancy is associated with macrosomia and prolonged pregnancy.

BACKGROUND: The hypothalamic-pituitary-adrenal (HPA) axis is important for fetal growth and timing of parturition. Maternal obesity is associated with macrosomia (birthweight ⩾4000g) and prolonged pregnancy (⩾41weeks). We aimed to characterise HPA axis hormones in obese pregnancy and to test associations with these pregnancy outcomes. METHOD: Fasting cortisol was measured by radioimmunoassay in venous blood at 16, 28 and 36 weeks of gestation in 286 obese (BMI 44.05±3.98kg/m(2)) and 137 lean (BMI 22.71±1.66kg/m(2)) pregnant women. In subsets (n=20 obese, 20 lean) we measured corticosteroid binding globulin (CBG) and CRH by radioimmunoassay; progesterone, estradiol (E2), estriol (E3) and sex-hormone-binding-globulin (SHBG) by ELISA; and albumin by bromocresol green binding. Free cortisol levels were calculated using Coolen's equation. RESULTS: Cortisol, CBG, calculated free cortisol, CRH, E2, E3, progesterone and SHBG levels rose similarly during pregnancy in obese and lean, but were significantly lower in obese (p<0.05). In obese, lower free cortisol at 16 weeks was associated with higher birthweight (r=-0.46, p<0.05). Cortisol was not associated with labour onset. CRH was significantly lower at 36 weeks in women who delivered at ⩾41weeks and in women with macrosomic babies (p<0.05); and correlated negatively with gestation at delivery in obese (r=-0.557, p<0.05). CONCLUSION: Our findings suggest that decreased HPA axis activity in obese pregnancy may be a mechanism underlying macrosomia and prolonged pregnancy.

Profiling the impact of medium formulation on morphology and functionality of primary hepatocytes in vitro.

The characterization of fully-defined in vitro hepatic culture systems requires testing of functional and morphological variables to obtain the optimal trophic support, particularly for cell therapeutics including bioartificial liver systems (BALs). Using serum-free fully-defined culture medium formulations, we measured synthetic, detoxification and metabolic variables of primary porcine hepatocytes (PPHs)--integrated these datasets using a defined scoring system and correlated this hepatocyte biological activity index (HBAI) with morphological parameters. Hepatic-specific functions exceeded those of both primary human hepatocytes (PHHs) and HepaRG cells, whilst retaining biotransformation potential and in vivo-like ultrastructural morphology, suggesting PPHs as a potential surrogate for PHHs in various biotech applications. The HBAI permits assessment of global functional capacity allowing the rational choice of optimal trophic support for a defined operational task (including BALs, hepatocellular transplantation, and cytochrome P450 (CYP450) drug metabolism studies), mitigates risk associated with sub-optimal culture systems, and reduces time and cost of research and therapeutic applications.

Near field and exit wave computations for electron microscopy.

The partial wave phase shift formalism of atomic scattering is applied to compute exit wave functions for isolated Au and Si atoms under both plane wave and focused probe illumination. Connections between the far field and near field (exit) waves are clarified. This approach treats the Coulomb singularity properly though at 100 keV large numbers of phase shifts are required. In principle any form of incident wave can be handled so it may provide a means for testing traditional scattering theories used in electron microscopy. By applying the analysis to an atom embedded in a constant potential rather than free space, exit spheres of radius half the interatomic spacing can be used.

Ischemic preconditioning in the liver is independent of regulatory T cell activity.

Ischemic preconditioning (IPC) protects organs from ischemia reperfusion injury (IRI) through unknown mechanisms. Effector T cell populations have been implicated in the pathogenesis of IRI, and T regulatory cells (Treg) have become a putative therapeutic target, with suggested involvement in IPC. We explored the role of Treg in hepatic IRI and IPC in detail. IPC significantly reduced injury following ischemia reperfusion insults. Treg were mobilized rapidly to the circulation and liver after IRI, but IPC did not further increase Treg numbers, nor was it associated with modulation of circulating pro-inflammatory chemokine or cytokine profiles. We used two techniques to deplete Treg from mice prior to IRI. Neither Treg depleted FoxP3.LuciDTR mice, nor wildtyoe mice depleted of Tregs with PC61, were more susceptible to IRI compared with controls. Despite successful enrichment of Treg in the liver, by adoptive transfer of both iTreg and nTreg or by in vivo expansion of Treg with IL-2/anti-IL-2 complexes, no protection against IRI was observed.We have explored the role of Treg in IRI and IPC using a variety of techniques to deplete and enrich them within both the liver and systemically. This work represents an important negative finding that Treg are not implicated in IPC and are unlikely to have translational potential in hepatic IRI.

Mechanisms of decoherence in electron microscopy.

The understanding and where possible the minimisation of decoherence mechanisms in electron microscopy were first studied in plasmon loss, diffraction contrast images but are of even more acute relevance in high resolution TEM phase contrast imaging and electron holography. With the development of phase retrieval techniques they merit further attention particularly when their effect cannot be eliminated by currently available energy filters. The roles of electronic excitation, thermal diffuse scattering, transition radiation and bremsstrahlung are examined here not only in the specimen but also in the electron optical column. Terahertz-range aloof beam electronic excitation appears to account satisfactorily for recent observations of decoherence in electron holography. An apparent low frequency divergence can emerge for the calculated classical bremsstrahlung event probability but can be ignored for photon wavelengths exceeding the required coherence distance or path lengths in the equipment. Most bremsstrahlung event probabilities are negligibly important except possibly in large-angle bending magnets or mandolin systems. A more reliable procedure for subtracting thermal diffuse scattering from diffraction pattern intensities is proposed.

Functional characterisation of the H365Y mutation of the 21-hydroxylase gene in congenital adrenal hyperplasia.

The study subject was a 13 day-old boy admitted to hospital, with weight loss since birth. He presented with the vomiting and hypotension that are classical features of congenital adrenal hyperplasia (CAH). The most common type of CAH is an autosomal recessive disorder caused by mutations in the 21-hydroxylase (CYP21A2) gene. To examine the CYP21A2 gene, gene-specific PCR was carried out, followed by sequencing. The baby was shown to be a compound heterozygote H365Y/R356W for two CYP21A2 gene mutations each inherited from a different parent. One of the mutations has not previously been functionally characterised. The mutations were reconstructed in an expression plasmid and characterised in vitro after transient transfection into human embryonic kidney (HEK293T) and hepatoblastoma (C3A) cell lines followed by measurement of enzyme activity. The CYP21A2 H365Y mutant exhibited minimal 21-hydroxylase activity to convert 17-hydroxyprogesterone to 11-deoxycortisol or progesterone to 11-deoxycorticosterone. Western immunoblotting indicated that the H365Y enzyme was produced in more variable amounts than wild type; in particular, the H365Y mutant protein may be unstable and/or subject to a more rapid degradation by the human proteosome as well as catalytically inefficient. The double mutant genotype with a severe mutation on each allele is compatible with the clinical presentation.