Impaired polyamine metabolism causes behavioral and neuroanatomical defects in a mouse model of Snyder-Robinson syndrome.

Snyder-Robinson syndrome (SRS) is a rare X-linked recessive disorder caused by a mutation in the SMS gene, which encodes spermine synthase, and aberrant polyamine metabolism. SRS is characterized by intellectual disability, thin habitus, seizure, low muscle tone/hypotonia and osteoporosis. Progress towards understanding and treating SRS requires a model that recapitulates human gene variants and disease presentations. Here, we evaluated molecular and neurological presentations in the G56S mouse model, which carries a missense mutation in the Sms gene. The lack of SMS protein in the G56S mice resulted in increased spermidine/spermine ratio, failure to thrive, short stature and reduced bone density. They showed impaired learning capacity, increased anxiety, reduced mobility and heightened fear responses, accompanied by reduced total and regional brain volumes. Furthermore, impaired mitochondrial oxidative phosphorylation was evident in G56S cerebral cortex, G56S fibroblasts and Sms-null hippocampal cells, indicating that SMS may serve as a future therapeutic target. Collectively, our study establishes the suitability of the G56S mice as a preclinical model for SRS and provides a set of molecular and functional outcome measures that can be used to evaluate therapeutic interventions for SRS.

Insulin secretion deficits in a Prader-Willi syndrome ß-cell model are associated with a concerted downregulation of multiple endoplasmic reticulum chaperones.

Prader-Willi syndrome (PWS) is a multisystem disorder with neurobehavioral, metabolic, and hormonal phenotypes, caused by loss of expression of a paternally-expressed imprinted gene cluster. Prior evidence from a PWS mouse model identified abnormal pancreatic islet development with retention of aged insulin and deficient insulin secretion. To determine the collective roles of PWS genes in ß-cell biology, we used genome-editing to generate isogenic, clonal INS-1 insulinoma lines having 3.16 Mb deletions of the silent, maternal- (control) and active, paternal-allele (PWS). PWS ß-cells demonstrated a significant cell autonomous reduction in basal and glucose-stimulated insulin secretion. Further, proteomic analyses revealed reduced levels of cellular and secreted hormones, including all insulin peptides and amylin, concomitant with reduction of at least ten endoplasmic reticulum (ER) chaperones, including GRP78 and GRP94. Critically, differentially expressed genes identified by whole transcriptome studies included reductions in levels of mRNAs encoding these secreted peptides and the group of ER chaperones. In contrast to the dosage compensation previously seen for ER chaperones in Grp78 or Grp94 gene knockouts or knockdown, compensation is precluded by the stress-independent deficiency of ER chaperones in PWS ß-cells. Consistent with reduced ER chaperones levels, PWS INS-1 ß-cells are more sensitive to ER stress, leading to earlier activation of all three arms of the unfolded protein response. Combined, the findings suggest that a chronic shortage of ER chaperones in PWS ß-cells leads to a deficiency of protein folding and/or delay in ER transit of insulin and other cargo. In summary, our results illuminate the pathophysiological basis of pancreatic ß-cell hormone deficits in PWS, with evolutionary implications for the multigenic PWS-domain, and indicate that PWS-imprinted genes coordinate concerted regulation of ER chaperone biosynthesis and ß-cell secretory pathway function.

CRISPRa-induced upregulation of human LAMA1 compensates for LAMA2-deficiency in Merosin-deficient congenital muscular dystrophy.

Merosin-deficient congenital muscular dystrophy (MDC1A) is an autosomal recessive disorder caused by mutations in the LAMA2 gene, resulting in a defective form of the extracellular matrix protein laminin-α2 (LAMA2). Individuals diagnosed with MDC1A exhibit progressive muscle wasting and declining neuromuscular functions. No treatments for this disorder are currently available. We previously showed that postnatal Lama1 upregulation, achieved through CRISPR activation (CRISPRa), compensates for Lama2 deficiency and prevents neuromuscular pathophysiology in a mouse model of MDC1A. In this study, we assessed the feasibility of upregulating human LAMA1 as a potential therapeutic strategy for individuals with MDC1A, regardless of their mutations. We hypothesized that CRISPRa-mediated upregulation of human LAMA1 would compensate for the lack of LAMA2 and rescue cellular abnormalities in MDC1A fibroblasts. Global transcriptomic and pathway enrichment analyses of fibroblasts collected from individuals carrying pathogenic LAMA2 mutations, compared with healthy controls, indicated higher expression of transcripts encoding proteins that contribute to wound healing, including Transforming Growth Factor-ß (TGF-ß) and Fibroblast Growth Factor (FGF). These findings were supported by wound-healing assays indicating that MDC1A fibroblasts migrated significantly more rapidly than the controls. Subsequently, we treated the MDC1A fibroblasts with SadCas9-2XVP64 and sgRNAs targeting the LAMA1 promoter. We observed robust LAMA1 expression, which was accompanied by significant decreases in cell migration and expression of FGFR2, TGF-ß2, and ACTA2, which are involved in the wound-healing mechanism in MDC1A fibroblasts. Collectively, our data suggest that CRISPRa-mediated LAMA1 upregulation may be a feasible mutation-independent therapeutic approach for MDC1A. This strategy might be adapted to address other neuromuscular diseases and inherited conditions in which strong compensatory mechanisms have been identified.

Impaired polyamine metabolism causes behavioral and neuroanatomical defects in a novel mouse model of Snyder-Robinson Syndrome.

Polyamines (putrescine, spermidine, and spermine) are essential molecules for normal cellular functions and are subject to strict metabolic regulation. Mutations in the gene encoding spermine synthase (SMS) lead to accumulation of spermidine in an X-linked recessive disorder known as Snyder-Robinson syndrome (SRS). Presently, no treatments exist for this rare disease that manifests with a spectrum of symptoms including intellectual disability, developmental delay, thin habitus, and low muscle tone. The development of therapeutic interventions for SRS will require a suitable disease-specific animal model that recapitulates many of the abnormalities observed in patients. Here, we characterize the molecular, behavioral, and neuroanatomical features of a mouse model with a missense mutation in Sms gene that results in a glycine-to-serine substitution at position 56 (G56S) of the SMS protein. Mice harboring this mutation exhibit a complete loss of SMS protein and elevated spermidine/spermine ratio in skeletal muscles and the brain. In addition, the G56S mice demonstrate increased anxiety, impaired learning, and decreased explorative behavior in fear conditioning, Morris water maze, and open field tests, respectively. Furthermore, these mice failed to gain weight over time and exhibit abnormalities in brain structure and bone density. Transcriptomic analysis of the cerebral cortex revealed downregulation of genes associated with mitochondrial oxidative phosphorylation and ribosomal protein synthesis. Our findings also revealed impaired mitochondrial bioenergetics in fibroblasts isolated from the G56S mice, indicating a correlation between these processes in the affected mice. Collectively, our findings establish the first in-depth characterization of an SRS preclinical mouse model that identifies cellular processes that could be targeted for future therapeutic development.

Cross-sectional Census Survey of Patients With Cancer who Received a Pharmacist Consultation in a Pharmacist Led Anti-cancer Clinic.

Pharmacists have a critical consulting role in patients undergoing oral antineoplastic drug therapy to ensure harm minimisation. Studies exploring the benefits of pharmacists in this role are limited. This study evaluated patient perceptions, experiences and overall satisfaction with clinical pharmacist consultations in patients treated with oral antineoplastic drugs. Data on 160 patients initiated on oral antineoplastic drugs between January 2019 and February 2021 were collected retrospectively from an outpatient Comprehensive Cancer Centre of a quaternary hospital in Western Australia (demographics, cancer type, oral antineoplastic drugs prescribed). In addition, patients were mailed a hard copy questionnaire in March 2021 to assess their satisfaction with pharmacist consultations in the pharmacist clinic, using a 5-point Likert scale. The statements included perceptions of the patient's understanding, medication adherence, experiences and overall satisfaction with the clinical pharmacist consultation. There were 76 (47.5%) completed questionnaires returned (52.6% female; average age was 63.2 ± 13.9 years). The majority of patients were satisfied with the service offered by the clinical pharmacist (73/76; 96.1%), perceived that clinical pharmacists provided an important service in outpatient cancer care (71/76; 93.4%) and improved their understanding of the use of oral antineoplastic drugs and side-effect management (48/74; 64.9%). Patients' perceived understanding of their medication regimen and additional health services available improved after pharmacist counselling. The patients also reported overall satisfaction with the service provided by the clinical pharmacist and found it beneficial to their care. The study supports the expanding role of the clinical pharmacist in an outpatient cancer centre.

Depression symptoms in adolescents with autism spectrum disorder: A contextual approach to mental health comorbidities.

INTRODUCTION: Although research on mental health comorbidities in autism spectrum disorder (ASD) has increased in recent years, little has been done to evaluate potential individual × environment interactions associated with these comorbidities. The current study explored whether ASD-related characteristics (social-communication impairment) and environmental factors (peer and family contexts) had additive or interactive effects on the depression symptoms of youth with ASD. METHOD: In a cross-sectional sample of adolescents with ASD (N = 176; 13-17 years old; 72.7% male), primary caregivers and adolescents responded to a series of surveys online pertaining to adolescents' mental health (Revised Child Anxiety and Depression Scale), family functioning (Self-Report of Family Inventory), and experiences of peer victimization (Peer Experiences Questionnaire-Revised). RESULTS: There were statistically significant interactions between social-communication skills and the environment in both family (△R2 = 0.02) and peer (△R2 = 0.02) contexts. For youth with better social-communication skills, there was a positive association between peer victimization and depression symptoms and a negative association between family competence and depression symptoms. CONCLUSION: Findings support social-push interactive models in which better social-communication skills are associated with fewer depression symptoms in the context of less-stressful peer and family environments, highlight the utility of ecologically informed approaches to the mental health of youth with ASD, and suggest several areas for future study.

A porcine model of phenylketonuria generated by CRISPR/Cas9 genome editing.

Phenylalanine hydroxylase-deficient (PAH-deficient) phenylketonuria (PKU) results in systemic hyperphenylalaninemia, leading to neurotoxicity with severe developmental disabilities. Dietary phenylalanine (Phe) restriction prevents the most deleterious effects of hyperphenylalaninemia, but adherence to diet is poor in adult and adolescent patients, resulting in characteristic neurobehavioral phenotypes. Thus, an urgent need exists for new treatments. Additionally, rodent models of PKU do not adequately reflect neurocognitive phenotypes, and thus there is a need for improved animal models. To this end, we have developed PAH-null pigs. After selection of optimal CRISPR/Cas9 genome-editing reagents by using an in vitro cell model, zygote injection of 2 sgRNAs and Cas9 mRNA demonstrated deletions in preimplantation embryos, with embryo transfer to a surrogate leading to 2 founder animals. One pig was heterozygous for a PAH exon 6 deletion allele, while the other was compound heterozygous for deletions of exon 6 and of exons 6-7. The affected pig exhibited hyperphenylalaninemia (2000-5000 µM) that was treatable by dietary Phe restriction, consistent with classical PKU, along with juvenile growth retardation, hypopigmentation, ventriculomegaly, and decreased brain gray matter volume. In conclusion, we have established a large-animal preclinical model of PKU to investigate pathophysiology and to assess new therapeutic interventions.

Operational research to inform a sub-national surveillance intervention for malaria elimination in Solomon Islands.

BACKGROUND: Successful reduction of malaria transmission to very low levels has made Isabel Province, Solomon Islands, a target for early elimination by 2014. High malaria transmission in neighbouring provinces and the potential for local asymptomatic infections to cause malaria resurgence highlights the need for sub-national tailoring of surveillance interventions. This study contributes to a situational analysis of malaria in Isabel Province to inform an appropriate surveillance intervention. METHODS: A mixed method study was carried out in Isabel Province in late 2009 and early 2010. The quantitative component was a population-based prevalence survey of 8,554 people from 129 villages, which were selected using a spatially stratified sampling approach to achieve uniform geographical coverage of populated areas. Diagnosis was initially based on Giemsa-stained blood slides followed by molecular analysis using polymerase chain reaction (PCR). Local perceptions and practices related to management of fever and treatment-seeking that would impact a surveillance intervention were also explored using qualitative research methods. RESULTS: Approximately 33% (8,554/26,221) of the population of Isabel Province participated in the survey. Only one subject was found to be infected with Plasmodium falciparum (Pf) (96 parasites/µL) using Giemsa-stained blood films, giving a prevalence of 0.01%. PCR analysis detected a further 13 cases, giving an estimated malaria prevalence of 0.51%. There was a wide geographical distribution of infected subjects. None reported having travelled outside Isabel Province in the previous three months suggesting low-level indigenous malaria transmission. The qualitative findings provide warning signs that the current community vigilance approach to surveillance will not be sufficient to achieve elimination. In addition, fever severity is being used by individuals as an indicator for malaria and a trigger for timely treatment-seeking and case reporting. In light of the finding of a low prevalence of parasitaemia, the current surveillance system may not be able to detect and prevent malaria resurgence. CONCLUSION: An adaption to the malERA surveillance framework is proposed and recommendations made for a tailored provincial-level surveillance intervention, which will be essential to achieve elimination, and to maintain this status while the rest of the country catches up.

Influence of phosphate on the transport properties of lead in sand.

Temporal moment analysis was used to examine the transport of lead species in sand columns. The influence of sodium phosphate (PO(4(aq))) and hydroxyapatite (HA) on lead transport was also evaluated. Transport properties of lead microparticles (diameter>0.45 µm) were a function of electrophoretic mobility: those particles with electrophoretic mobility less than -1 × 10(-8)m(2)/Vs exhibited significantly lower dimensionless first temporal moment (θ) and second temporal moment (σ(θ)(2)). The forms of lead investigated in this work had a tendency to move in sand over a wide pH range. Although the PO(4(aq)) amendment substantially reduced lead mass recoveries in the sand column effluent, lead microparticles were formed that had a tendency to move rapidly and with minimal dispersion when compared with controls. Treatments with HA provided limited reduction in lead mass recovery and minimal changes in lead transport properties. A colloid stability model was used to predict attachment of lead particles in sand.

A large proportion of asymptomatic Plasmodium infections with low and sub-microscopic parasite densities in the low transmission setting of Temotu Province, Solomon Islands: challenges for malaria diagnostics in an elimination setting.

BACKGROUND: Many countries are scaling up malaria interventions towards elimination. This transition changes demands on malaria diagnostics from diagnosing ill patients to detecting parasites in all carriers including asymptomatic infections and infections with low parasite densities. Detection methods suitable to local malaria epidemiology must be selected prior to transitioning a malaria control programme to elimination. A baseline malaria survey conducted in Temotu Province, Solomon Islands in late 2008, as the first step in a provincial malaria elimination programme, provided malaria epidemiology data and an opportunity to assess how well different diagnostic methods performed in this setting. METHODS: During the survey, 9,491 blood samples were collected and examined by microscopy for Plasmodium species and density, with a subset also examined by polymerase chain reaction (PCR) and rapid diagnostic tests (RDTs). The performances of these diagnostic methods were compared. RESULTS: A total of 256 samples were positive by microscopy, giving a point prevalence of 2.7%. The species distribution was 17.5% Plasmodium falciparum and 82.4% Plasmodium vivax. In this low transmission setting, only 17.8% of the P. falciparum and 2.9% of P. vivax infected subjects were febrile (≥ 38°C) at the time of the survey. A significant proportion of infections detected by microscopy, 40% and 65.6% for P. falciparum and P. vivax respectively, had parasite density below 100/µL. There was an age correlation for the proportion of parasite density below 100/µL for P. vivax infections, but not for P. falciparum infections. PCR detected substantially more infections than microscopy (point prevalence of 8.71%), indicating a large number of subjects had sub-microscopic parasitemia. The concordance between PCR and microscopy in detecting single species was greater for P. vivax (135/162) compared to P. falciparum (36/118). The malaria RDT detected the 12 microscopy and PCR positive P. falciparum, but failed to detect 12/13 microscopy and PCR positive P. vivax infections. CONCLUSION: Asymptomatic malaria infections and infections with low and sub-microscopic parasite densities are highly prevalent in Temotu province where malaria transmission is low. This presents a challenge for elimination since the large proportion of the parasite reservoir will not be detected by standard active and passive case detection. Therefore effective mass screening and treatment campaigns will most likely need more sensitive assays such as a field deployable molecular based assay.

Application of the ICF to referral delay in total joint arthroplasty.

BACKGROUND: The factors that affect referral for total joint arthroplasty (TJA) have been widely studied. Implicit in this work is the assumption that patient health status should determine priority for surgery. However, specification of patient health status lacks a strong theoretical framework. This study employs the WHO model of health outcomes, the International Classification of Functioning, Disability and Health (ICF), to examine patient health factors in the referral process for TJA. METHODS: Within 8 weeks prior to TJA, 260 patients electing for primary TJA completed a questionnaire which measured the ICF (impairment, activity limitations and participation restrictions) and four types of delay in their journey from initial consultation with their primary care physician to surgery. RESULTS: Impairment did not affect any stage of the referral process. In contrast, patients who had experienced a delay of 26 weeks or less between referral to a surgeon and being placed on the waiting list for surgery reported greater activity limitations and participation restrictions than patients who had waited more than 26 weeks. Further, patients who reported having wanted surgery for more than 52 weeks reported greater participation restrictions than patients who had wanted surgery for less than 52 weeks. CONCLUSIONS: The ICF identifies three health outcomes, two of which (activity limitations and participation restrictions) are related to delay in the referral process for TJA. The ICF is a useful theoretical framework for the study of factors that influence prioritisation for surgery. The level of functional and social disability appears to inform prioritisation for TJA by consultant orthopaedic surgeons.

Relative adrenal insufficiency in severe congestive heart failure with preserved systolic function: a case report.

Relative adrenal insufficiency in critically ill patients is an important syndrome in septic shock. The insufficient stress response of the hypothalamic-pituitary-adrenal axis in acute illness contributes to hemodynamic instability. Treatment of this state in septic shock improves patient outcomes. In this report, we describe the case of a patient with severe diastolic dysfunction who presented in cardiogenic shock associated with relative adrenal insufficiency and had a complete recovery with corticosteroid replacement. Alteration of the hypothalamic-pituitary-adrenal axis may be more prevalent than suspected in end-stage heart failure, and the diagnosis and treatment of this syndrome may ultimately improve outcomes in a subgroup of heart failure patients.

Using voice-recognition technology to eliminate cardiac cycle segmentation in automated heart sound diagnosis.

Advanced digital signal processing has the potential to revolutionize the stethoscope through the use of mathematical algorithms to interpret heart sound acoustic information. In this study, a novel classification algorithm that does not require cardiac cycle segmentation was used for identifying differences between normal and diseased heart sounds. The heart sound signals were not separated into systole and diastole. A recordable electronic stethoscope was used to record the heart sounds of 163 echocardiogram patients. Mel-cepstrum and Principal Components Analysis were applied to the 60 recorded heart sounds and decision spaces were developed. The algorithm was tested using 100 novel patients. The specificity of the algorithm is 72.4% and the sensitivity is 63.4%.

Structure and ultrastructure of the silk glands and spinneret of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae).

This study provides comprehensive documentation of silk production in the pest moth Helicoverpa armigera from gland secretion to extrusion of silk thread. The structure of the silk glands, accessory structures and extrusion apparatus are reported. The general schema of the paired silk glands follows that found for Lepidoptera. Morphology of the duct, silk press, muscle attachments and spigot are presented as a three-dimensional reconstruction and the cuticular crescent-shaped profile of the silk press is demonstrated in both open and closed forms with attendant muscle blocks, allowing advances in our knowledge of how the silk press functions to regulate the extrusion of silk. Growth of the spigot across instars is documented showing a distinctive developmental pattern for this extrusion device. Its shape and structure are related to use and load-bearing activity.

Development of protoporphyrinogen oxidase as an efficient selection marker for Agrobacterium tumefaciens-mediated transformation of maize.

In this article, we report the isolation of plant protoporphyrinogen oxidase (PPO) genes and the isolation of herbicide-tolerant mutants. Subsequently, an Arabidopsis double mutant (Y426M + S305L) was used to develop a selectable marker system for Agrobacterium tumefaciens-mediated transformation of maize (Zea mays) and to obtain multiple events tolerant to the PPO family of herbicides. Maize transformants were produced via butafenacil selection using a flexible light regime to increase selection pressure. Butafenacil selection per se did not change transgene copy number distribution relative to other selectable marker systems, but the most tolerant events identified in the greenhouse were more likely to contain multiple copies of the introduced mutant PPO gene. To date, more than 2,500 independent transgenic maize events have been produced using butafenacil selection. The high frequency of A. tumefaciens-mediated transformation via PPO selection enabled us to obtain single-copy transgenic maize lines tolerant to field levels of butafenacil.