Significance of wastewater surveillance in detecting the prevalence of SARS-CoV-2 variants and other respiratory viruses in the community - A multi-site evaluation.

Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral genome in wastewater has proven to be useful for tracking the trends of virus prevalence within the community. The surveillance also provides precise and early detection of any new and circulating variants, which aids in response to viral outbreaks. Site-specific monitoring of SARS-CoV-2 variants provides valuable information on the prevalence of new or emerging variants in the community. We sequenced the genomic RNA of viruses present in the wastewater samples and analyzed for the prevalence of SARS-CoV-2 variants as well as other respiratory viruses for a period of one year to account for seasonal variations. The samples were collected from the Reno-Sparks metropolitan area on a weekly basis between November 2021 to November 2022. Samples were analyzed to detect the levels of SARS-CoV-2 genomic copies and variants identification. This study confirmed that wastewater monitoring of SARS-CoV-2 variants can be used for community surveillance and early detection of circulating variants and supports wastewater-based epidemiology (WBE) as a complement to clinical respiratory virus testing as a healthcare response effort. Our study showed the persistence of the SARS-CoV-2 virus throughout the year compared to a seasonal presence of other respiratory viruses, implicating SARS-CoV-2's broad genetic diversity and strength to persist and infect susceptible hosts. Through secondary analysis, we further identified antimicrobial resistance (AMR) genes in the same wastewater samples and found WBE to be a feasible tool for community AMR detection and monitoring.

Insect nephrocyte function is regulated by a store operated calcium entry mechanism controlling endocytosis and Amnionless turnover.

Insect nephrocytes are ultrafiltration cells that remove circulating proteins and exogenous toxins from the haemolymph. Experimental disruption of nephrocyte development or function leads to systemic impairment of insect physiology as evidenced by cardiomyopathy, chronic activation of immune signalling and shortening of lifespan. The genetic and structural basis of the nephrocyte's ultrafiltration mechanism is conserved between arthropods and mammals, making them an attractive model for studying human renal function and systemic clearance mechanisms in general. Although dynamic changes to intracellular calcium are fundamental to the function of many cell types, there are currently no studies of intracellular calcium signalling in nephrocytes. In this work we aimed to characterise calcium signalling in the pericardial nephrocytes of Drosophila melanogaster. To achieve this, a genetically encoded calcium reporter (GCaMP6) was expressed in nephrocytes to monitor intracellular calcium both in vivo within larvae and in vitro within dissected adults. Larval nephrocytes exhibited stochastically timed calcium waves. A calcium signal could be initiated in preparations of adult nephrocytes and abolished by EGTA, or the store operated calcium entry (SOCE) blocker 2-APB, as well as RNAi mediated knockdown of the SOCE genes Stim and Orai. Neither the presence of calcium-free buffer nor EGTA affected the binding of the endocytic cargo albumin to nephrocytes but they did impair the subsequent accumulation of albumin within nephrocytes. Pre-treatment with EGTA, calcium-free buffer or 2-APB led to significantly reduced albumin binding. Knock-down of Stim and Orai was non-lethal, caused an increase to nephrocyte size and reduced albumin binding, reduced the abundance of the endocytic cargo receptor Amnionless and disrupted the localisation of Dumbfounded at the filtration slit diaphragm. These data indicate that pericardial nephrocytes exhibit stochastically timed calcium waves in vivo and that SOCE mediates the localisation of the endocytic co-receptor Amnionless. Identifying the signals both up and downstream of SOCE may highlight mechanisms relevant to the renal and excretory functions of a broad range of species, including humans.

Piezo buffers mechanical stress via modulation of intracellular Ca2+ handling in the Drosophila heart.

Throughout its lifetime the heart is buffeted continuously by dynamic mechanical forces resulting from contraction of the heart muscle itself and fluctuations in haemodynamic load and pressure. These forces are in flux on a beat-by-beat basis, resulting from changes in posture, physical activity or emotional state, and over longer timescales due to altered physiology (e.g. pregnancy) or as a consequence of ageing or disease (e.g. hypertension). It has been known for over a century of the heart's ability to sense differences in haemodynamic load and adjust contractile force accordingly (Frank, Z. biology, 1895, 32, 370-447; Anrep, J. Physiol., 1912, 45 (5), 307-317; Patterson and Starling, J. Physiol., 1914, 48 (5), 357-79; Starling, The law of the heart (Linacre Lecture, given at Cambridge, 1915), 1918). These adaptive behaviours are important for cardiovascular homeostasis, but the mechanism(s) underpinning them are incompletely understood. Here we present evidence that the mechanically-activated ion channel, Piezo, is an important component of the Drosophila heart's ability to adapt to mechanical force. We find Piezo is a sarcoplasmic reticulum (SR)-resident channel and is part of a mechanism that regulates Ca2+ handling in cardiomyocytes in response to mechanical stress. Our data support a simple model in which Drosophila Piezo transduces mechanical force such as stretch into a Ca2+ signal, originating from the SR, that modulates cardiomyocyte contraction. We show that Piezo mutant hearts fail to buffer mechanical stress, have altered Ca2+ handling, become prone to arrhythmias and undergo pathological remodelling.

Detecting SARS-CoV-2 variants in wastewater and their correlation with circulating variants in the communities.

Detection of SARS-CoV-2 viral load in wastewater has been highly informative in estimating the approximate number of infected individuals in the surrounding communities. Recent developments in wastewater monitoring to determine community prevalence of COVID-19 further extends into identifying SARS-CoV-2 variants, including those being monitored for having enhanced transmissibility. We sequenced genomic RNA derived from wastewater to determine the variants of coronaviruses circulating in the communities. Wastewater samples were collected from Truckee Meadows Water Reclamation Facility (TMWRF) from November 2020 to June 2021. SARS-CoV-2 variants resulting from wastewater were compared with the variants detected in infected individuals' clinical specimens (nasal/nasopharyngeal swabs) during the same period and found conclusively in agreement. Therefore, wastewater monitoring for SARS-CoV-2 variants in the community is a feasible strategy as a complementary tool to clinical specimen testing in the latter's absence.

Detecting SARS-CoV-2 Variants in Wastewater and Their Correlation With Circulating Variants in the Communities.

Detection of SARS-CoV-2 viral load in wastewater has been highly informative in estimating the approximate number of infected individuals in the surrounding communities. Recent developments in wastewater monitoring to determine community prevalence of COVID-19 further extends into identifying SARS-CoV-2 variants, including those being monitored for having enhanced transmissibility. We sequenced genomic RNA derived from wastewater to determine the variants of coronaviruses circulating in the communities. Wastewater samples were collected from Truckee Meadows Water Reclamation Facility (TMWRF) from November 2021 to June 2021 were analyzed for SARS-CoV-2 variants and were compared with the variants detected in the clinical specimens (nasal/nasopharyngeal swabs) of infected individuals during the same period. The comparison was found to be conclusively in agreement. Therefore, wastewater monitoring for SARS-CoV-2 variants in the community is a feasible strategy both as a complementary tool to clinical specimen testing and in the latter's absence.

Exploring the relevance of NUP93 variants in steroid-resistant nephrotic syndrome using next generation sequencing and a fly kidney model.

BACKGROUND: Variants in genes encoding nuclear pore complex (NPC) proteins are a newly identified cause of paediatric steroid-resistant nephrotic syndrome (SRNS). Recent reports describing NUP93 variants suggest these could be a significant cause of paediatric onset SRNS. We report NUP93 cases in the UK and demonstrate in vivo functional effects of Nup93 depletion in a fly (Drosophila melanogaster) nephrocyte model. METHODS: Three hundred thirty-seven paediatric SRNS patients from the National cohort of patients with Nephrotic Syndrome (NephroS) were whole exome and/or whole genome sequenced. Patients were screened for over 70 genes known to be associated with Nephrotic Syndrome (NS). D. melanogaster Nup93 knockdown was achieved by RNA interference using nephrocyte-restricted drivers. RESULTS: Six novel homozygous and compound heterozygous NUP93 variants were detected in 3 sporadic and 2 familial paediatric onset SRNS characterised histologically by focal segmental glomerulosclerosis (FSGS) and progressing to kidney failure by 12 months from clinical diagnosis. Silencing of the two orthologs of human NUP93 expressed in D. melanogaster, Nup93-1, and Nup93-2 resulted in significant signal reduction of up to 82% in adult pericardial nephrocytes with concomitant disruption of NPC protein expression. Additionally, nephrocyte morphology was highly abnormal in Nup93-1 and Nup93-2 silenced flies surviving to adulthood. CONCLUSION: We expand the spectrum of NUP93 variants detected in paediatric onset SRNS and demonstrate its incidence within a national cohort. Silencing of either D. melanogaster Nup93 ortholog caused a severe nephrocyte phenotype, signaling an important role for the nucleoporin complex in podocyte biology. A higher resolution version of the Graphical abstract is available as Supplementary information.

Genomic surveillance of Nevada patients revealed prevalence of unique SARS-CoV-2 variants bearing mutations in the RdRp gene.

Patients with signs of COVID-19 were tested through diagnostic RT-PCR for SARS-CoV-2 using RNA extracted from the nasopharyngeal/nasal swabs. To determine the variants of SARS-CoV-2 circulating in the state of Nevada, specimens from 200 COVID-19 patients were sequenced through our robust sequencing platform, which enabled sequencing of SARS-CoV-2 from specimens with even very low viral loads, without the need of culture-based amplification. High genome coverage allowed the identification of single and multi-nucleotide variants in SARS-CoV-2 in the community and their phylogenetic relationships with other variants present during the same period of the outbreak. We report the occurrence of a novel mutation at 323aa (314aa of orf1b) of nsp12 (RNA-dependent RNA polymerase) changed to phenylalanine (F) from proline (P), in the first reported isolate of SARS-CoV-2, Wuhan-Hu-1. This 323F variant was present at a very high frequency in Northern Nevada. Structural modeling determined this mutation in the interface domain, which is important for the association of accessory proteins required for the polymerase. In conclusion, we report the introduction of specific SARS-CoV-2 variants at very high frequency in distinct geographic locations, which is important for understanding the evolution and circulation of SARS-CoV-2 variants of public health importance, while it circulates in humans.

Genomic evidence for reinfection with SARS-CoV-2: a case study.

BACKGROUND: The degree of protective immunity conferred by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently unknown. As such, the possibility of reinfection with SARS-CoV-2 is not well understood. We describe an investigation of two instances of SARS-CoV-2 infection in the same individual. METHODS: A 25-year-old man who was a resident of Washoe County in the US state of Nevada presented to health authorities on two occasions with symptoms of viral infection, once at a community testing event in April, 2020, and a second time to primary care then hospital at the end of May and beginning of June, 2020. Nasopharyngeal swabs were obtained from the patient at each presentation and twice during follow-up. Nucleic acid amplification testing was done to confirm SARS-CoV-2 infection. We did next-generation sequencing of SARS-CoV-2 extracted from nasopharyngeal swabs. Sequence data were assessed by two different bioinformatic methodologies. A short tandem repeat marker was used for fragment analysis to confirm that samples from both infections came from the same individual. FINDINGS: The patient had two positive tests for SARS-CoV-2, the first on April 18, 2020, and the second on June 5, 2020, separated by two negative tests done during follow-up in May, 2020. Genomic analysis of SARS-CoV-2 showed genetically significant differences between each variant associated with each instance of infection. The second infection was symptomatically more severe than the first. INTERPRETATION: Genetic discordance of the two SARS-CoV-2 specimens was greater than could be accounted for by short-term in vivo evolution. These findings suggest that the patient was infected by SARS-CoV-2 on two separate occasions by a genetically distinct virus. Thus, previous exposure to SARS-CoV-2 might not guarantee total immunity in all cases. All individuals, whether previously diagnosed with COVID-19 or not, should take identical precautions to avoid infection with SARS-CoV-2. The implications of reinfections could be relevant for vaccine development and application. FUNDING: Nevada IDEA Network of Biomedical Research, and the National Institute of General Medical Sciences (National Institutes of Health).

Characteristics of viral specimens collected from asymptomatic and fatal cases of COVID-19.

We sought to determine the characteristics of viral specimens associated with fatal cases, asymptomatic cases and non-fatal symptomatic cases of COVID-19. This included the analysis of 1264 specimens found reactive for at least two SARS-CoV-2 specific loci from people screened for infection in Northern Nevada in March-May of 2020. Of these, 30 were specimens from fatal cases, while 23 were from positive, asymptomatic cases. We assessed the relative amounts of SARS-CoV-2 RNA from sample swabs by real-time PCR and use of the threshold crossing value (Ct). Moreover, we compared the amount of human RNase P found on the same swabs. A considerably higher viral load was found to be associated with swabs from cases involving fatality and the difference was found to be strongly statistically significant. Noting this difference, we sought to assess whether any genetic correlation could be found in association with virus from fatal cases using whole genome sequencing. While no common genetic elements were discerned, one branch of epidemiologically linked fatal cases did have two point mutations, which no other of 156 sequenced cases from northern Nevada had. The mutations caused amino acid changes in the 3'-5' exonuclease protein, and the product of the gene, orf8.

Genomic surveillance of Nevada patients revealed prevalence of unique SARS-CoV-2 variants bearing mutations in the RdRp gene.

Patients with signs of COVID-19 were tested with CDC approved diagnostic RT-PCR for SARS-CoV-2 using RNA extracted from nasopharyngeal/nasal swabs. In order to determine the variants of SARS-CoV-2 circulating in the state of Nevada, 200 patient specimens from COVID-19 patients were sequenced through our robust protocol for sequencing SARS-CoV-2 genomes. Our protocol enabled sequencing of SARS-CoV-2 genome directly from the specimens, with even very low viral loads, without the need of culture-based amplification. This allowed the identification of specific nucleotide variants including those coding for D614G and clades defining mutations. These sequences were further analyzed for determining SARS-CoV-2 variants circulating in the state of Nevada and their phylogenetic relationships with other variants present in the united states and the world during the same period of the outbreak. Our study reports the occurrence of a novel variant in the nsp12 (RNA dependent RNA Polymerase) protein at residue 323 (314aa of orf1b) to Phenylalanine (F) from Proline (P), present in the original isolate of SARS-CoV-2 (Wuhan-Hu-1). This 323F variant is found at a very high frequency (46% of the tested specimen) in Northern Nevada. Functional significance of this unique and highly prevalent variant of SARS-CoV-2 with RdRp mutation is currently under investigation but structural modeling showed this 323aa residue in the interface domain of RdRp, which is required for association with accessory proteins. In conclusion, we report the introduction of specific SARS-CoV-2 variants at a very high frequency within a distinct geographic location, which is important for clinical and public health perspectives in understanding the evolution of SARS-CoV-2 while in circulation.

Modeling Podocyte Biology Using Drosophila Nephrocytes.

Vertebrate podocytes are kidney glomerular cells critically required for normal renal filtration. To fulfill their role, podocytes form molecular sieves known as slit diaphragms that contribute to the glomerular filtration barrier. The disruption of podocyte biology or slit diaphragm formation in humans is a precursor to albuminuria, renal failure, and cardiovascular morbidity. Due to genetic and functional similarities, the nephrocytes of Drosophila are increasingly used to model the genetic and metabolic basis of human podocyte biology. They have the advantage that they are a much quicker system to study compared to other murine transgenic models. In this chapter we present methods to modulate and study Drosophila nephrocyte function and diaphragm formation.

Nephrocytes Remove Microbiota-Derived Peptidoglycan from Systemic Circulation to Maintain Immune Homeostasis.

Preventing aberrant immune responses against the microbiota is essential for the health of the host. Microbiota-shed pathogen-associated molecular patterns translocate from the gut lumen into systemic circulation. Here, we examined the role of hemolymph (insect blood) filtration in regulating systemic responses to microbiota-derived peptidoglycan. Drosophila deficient for the transcription factor Klf15 (Klf15NN) are viable but lack nephrocytes-cells structurally and functionally homologous to the glomerular podocytes of the kidney. We found that Klf15NN flies were more resistant to infection than wild-type (WT) counterparts but exhibited a shortened lifespan. This was associated with constitutive Toll pathway activation triggered by excess peptidoglycan circulating in Klf15NN flies. In WT flies, peptidoglycan was removed from systemic circulation by nephrocytes through endocytosis and subsequent lysosomal degradation. Thus, renal filtration of microbiota-derived peptidoglycan maintains immune homeostasis in Drosophila, a function likely conserved in mammals and potentially relevant to the chronic immune activation seen in settings of impaired blood filtration.

Structural Variation Detection by Proximity Ligation from Formalin-Fixed, Paraffin-Embedded Tumor Tissue.

The clinical management and therapy of many solid tumor malignancies depends on detection of medically actionable or diagnostically relevant genetic variation. However, a principal challenge for genetic assays from tumors is the fragmented and chemically damaged state of DNA in formalin-fixed, paraffin-embedded (FFPE) samples. From highly fragmented DNA and RNA there is no current technology for generating long-range DNA sequence data as is required to detect genomic structural variation or long-range genotype phasing. We have developed a high-throughput chromosome conformation capture approach for FFPE samples that we call Fix-C, which is similar in concept to Hi-C. Fix-C enables structural variation detection from archival FFPE samples. This method was applied to 15 clinical adenocarcinoma- and sarcoma-positive control specimens spanning a broad range of tumor purities. In this panel, Fix-C analysis achieves a 90% concordance rate with fluorescence in situ hybridization assays, the current clinical gold standard. In addition, novel structural variation undetected by other methods could be identified, and long-range chromatin configuration information recovered from these FFPE samples harboring highly degraded DNA. This powerful approach will enable detailed resolution of global genome rearrangement events during cancer progression from FFPE material and will inform the development of targeted molecular diagnostic assays for patient care.

As time flies by: Investigating cardiac aging in the short-lived Drosophila model.

Aging is associated with a decline in heart function across the tissue, cellular, and molecular levels. The risk of cardiovascular disease grows significantly over time, and as developed countries continue to see an increase in lifespan, the cost of cardiovascular healthcare for the elderly will undoubtedly rise. The molecular basis for cardiac function deterioration with age is multifaceted and not entirely clear, and there is a limit to what investigations can be performed on human subjects or mammalian models. Drosophila melanogaster has emerged as a useful model organism for studying aging in a short timeframe, benefitting from a suite of molecular and genetic tools and displaying highly conserved traits of cardiac senescence. Here, we discuss recent advances in our understanding of cardiac aging and how the fruit fly has aided in these developments.

The Impact of Reactogenicity After the First Dose of Recombinant Zoster Vaccine on the Physical Functioning and Quality of Life of Older Adults: An Open-Label, Phase III Trial.

BACKGROUND: Herpes zoster and its related complications are associated with significant medical burden, which negatively affects quality of life and daily functioning of the patients. The recently licensed recombinant zoster vaccine (RZV) offers high efficacy but is associated with local and systemic reactions. This study assessed the impact of RZV on the quality of life and daily functioning of participants and implications for caregivers. METHODS: Four hundred and one adults aged 50 years or older received single RZV doses at 0 and 2 months in this open-label, single-arm, multicenter study (NCT02979639). Change in mean SF-36 Physical Functioning score following first-dose administration, quality of life, reactogenicity, safety, productivity loss, and health care resource utilization was assessed. The current analysis was performed post-vaccine dose-1; safety follow-up will continue until 1 year post-dose-2. RESULTS: The most common solicited local symptoms were injection-site pain (77.5%), redness (23.0%), and swelling (13.3%); the most frequent solicited systemic reactions were fatigue (33.5%), headache (28.3%), and myalgia (26.8%). Grade 3 reactogenicity occurred in 9.5% of participants and was associated with a transient clinically important decrease in SF-36 Physical Functioning score (affecting activities such as walking, carrying groceries, climbing stairs) on Days 1 and 2 post-first vaccination. No clinically meaningful reductions in mean SF-36 Physical Functioning scale scores from pre- to post-RZV dose-1 were observed (mean +1.9 points, primary end point), and no overall quality-adjusted-life-year loss was recorded post-dose-1. Five participants reported lost workdays; caregiver workload was not increased. CONCLUSIONS: Overall, the physical functioning and quality of life of older adults were not affected by a first RZV dose. The observed reactogenicity was consistent with previous studies.

Drosophila pericardial nephrocyte ultrastructure changes during ageing.

Here we show that a labyrinth channel compartment and slit diaphragms, which are the histological structures enabling insect nephrocytes ultrafiltration, are established during embryogenesis first by the garland nephrocytes (GCNs). The later pericardial nephrocytes, which represent the majority of functional nephrocytes in larvae and adults, lack these characteristic features at the embryonic stage. During larval development, a subpopulation of the pericardial cells survives and matures into functional nephrocytes (PCNs) displaying a fully differentiated slit diaphragm and a labyrinth channel compartment. Likely the embryonic pericardial cells have primary functions other than ultrafiltration (e.g. in production and secretion of ECM constituents). We also show, for the first time, that PCNs in the adult fly undergo dramatic histological degeneration upon ageing. The slit diaphragms disappear, the labyrinth channel system degenerates and the lysosomal compartment becomes highly enriched with electron-dense material. When using nephrocytes as a model for genetic screening purposes or to investigate the specific role of genes involved in endocytosis, histological changes occurring upon ageing need to be taken into account when interpreting structural data.

The impact of SPARC on age-related cardiac dysfunction and fibrosis in Drosophila.

Tissue fibrosis, an accumulation of extracellular matrix proteins such as collagen, accompanies cardiac ageing in humans and this is linked to an increased risk of cardiac failure. The mechanisms driving age-related tissue fibrosis and cardiac dysfunction are unclear, yet clinically important. Drosophila is amenable to the study of cardiac ageing as well as collagen deposition; however it is unclear whether collagen accumulates in the ageing Drosophila heart. This work examined collagen deposition and cardiac function in ageing Drosophila, in the context of reduced expression of collagen-interacting protein SPARC (Secreted Protein Acidic and Rich in Cysteine) an evolutionarily conserved protein linked with fibrosis. Heart function was measured using high frame rate videomicroscopy. Collagen deposition was monitored using a fluorescently-tagged collagen IV reporter (encoded by the Viking gene) and staining of the cardiac collagen, Pericardin. The Drosophila heart accumulated collagen IV and Pericardin as flies aged. Associated with this was a decline in cardiac function. SPARC heterozygous flies lived longer than controls and showed little to no age-related cardiac dysfunction. As flies of both genotypes aged, cardiac levels of collagen IV (Viking) and Pericardin increased similarly. Over-expression of SPARC caused cardiomyopathy and increased Pericardin deposition. The findings demonstrate that, like humans, the Drosophila heart develops a fibrosis-like phenotype as it ages. Although having no gross impact on collagen accumulation, reduced SPARC expression extended Drosophila lifespan and cardiac health span. It is proposed that cardiac fibrosis in humans may develop due to the activation of conserved mechanisms and that SPARC may mediate cardiac ageing by mechanisms more subtle than gross accumulation of collagen.

Chronic Disease and Self-Injection: Ethnographic Investigations into the Patient Experience During Treatment.

INTRODUCTION: Drug administration by self-injection provides an option to treat chronic inflammatory diseases such as rheumatoid arthritis (RA) and Crohn's disease (CD). However, a negative self-injection experience for patients may reduce patient adherence to the recommended treatment regimen. In this study, a holistic approach was used to identify common themes along the treatment pathway and at self-injection that, if changed, could improve patient experience and treatment outcomes. METHODS: Two ethnographic studies were conducted: Field Insights CODE (FI[CODE]) examined the treatment pathway within the context of the experience of living with RA or CD, and Injection Mission 2020 (IM2020) focused on the moment of self-injection. FI(CODE) used an open ethnographic approach to interview 62 patients and 10 healthcare professionals (HCPs) from the US and UK. IM2020 included a review of over 50 injection device design information sources from the sponsor, and interviews with 9 patients, 8 HCPs, and 5 medical device designers from the US, UK, Canada, and Japan. RESULTS: FI(CODE) identified suboptimal treatment practices along the treatment pathway in four key areas: treatment team communication, treatment choice, patient empowerment, and treatment delivery. Patients with more treatment options and greater disease understanding were less likely to struggle with the treatment process. IM2020 demonstrated that five related components influenced the self-injection experience: delivery process, emotional state, social perception, educational level, and ritualization of the self-injection process. CONCLUSION: These analyses highlight several potential areas for improvement, including aligning the device more to patients' needs to improve treatment adherence, better accessibility to educational resources to increase patient disease understanding, and guidance to empower patients to develop an optimal personalized self-injection ritual. FUNDING: UCB Pharma.

Some medicines used to treat long-term conditions, such as rheumatoid arthritis or Crohn's disease, are injected under the skin. Often, patients can choose to inject medicines themselves (self-injection). This must be done correctly for the medicines to work properly. But, the training surrounding self-injection is uneven and often cannot address the fundamental problems facing all self-injecting patients.What healthcare improvements could help patients self-inject successfully? To find out, we interviewed people living with rheumatoid arthritis or Crohn's disease, while others were doctors, nurses, and people who design injection devices.We found four common problems in the overall healthcare that patients received:1.There were communication problems between different healthcare professionals and between healthcare professionals and patients, for example about treatment options or goals.2.Each level in the healthcare system (e.g., the nurse, doctor, hospital board, health insurance company) made decisions that limited how many treatment options were presented to patients for consideration.3.Patients were not empowered, as they felt they lacked personal input, information, and control in treatment decisions.4.Healthcare professionals focused on disease treatment but not patient experience; they did not fully explain how to perform injections (delivery), leaving patients to figure it out by trial and error. In addition, five factors were identified that affected patients' experiences of self-injection:1.Process of injection: minimal one-on-one instruction for self-injection left some patients anxious and more prone to mistakes.2.Emotions: some patients were better than others at 'overriding' emotions (e.g., fear) when self-injecting.3.Views on injections: there was negative social stigma around injections, but patients had greater trust in more technological, modern devices.4.Education: doctors often failed to explain how to manage fear and anxiety.5.Developing a ritual: patients with a ritualized routine for when, where, and how to self-inject were more confident. If doctors and nurses can support patients by providing a greater choice of treatments and injection devices, and teaching more about self-injection, this could improve patients' experiences and allow medications to work better. Healthcare professionals should help patients to develop their own, optimal routine for self-injection.

SPARC-Dependent Cardiomyopathy in Drosophila.

BACKGROUND: The Drosophila heart is an important model for studying the genetics underpinning mammalian cardiac function. The system comprises contractile cardiomyocytes, adjacent to which are pairs of highly endocytic pericardial nephrocytes that modulate cardiac function by uncharacterized mechanisms. Identifying these mechanisms and the molecules involved is important because they may be relevant to human cardiac physiology. METHODS AND RESULTS: This work aimed to identify circulating cardiomodulatory factors of potential relevance to humans using the Drosophila nephrocyte-cardiomyocyte system. A Kruppel-like factor 15 (dKlf15) loss-of-function strategy was used to ablate nephrocytes and then heart function and the hemolymph proteome were analyzed. Ablation of nephrocytes led to a severe cardiomyopathy characterized by a lengthening of diastolic interval. Rendering adult nephrocytes dysfunctional by disrupting their endocytic function or temporally conditional knockdown of dKlf15 led to a similar cardiomyopathy. Proteomics revealed that nephrocytes regulate the circulating levels of many secreted proteins, the most notable of which was the evolutionarily conserved matricellular protein Secreted Protein Acidic and Rich in Cysteine (SPARC), a protein involved in mammalian cardiac function. Finally, reducing SPARC gene dosage ameliorated the cardiomyopathy that developed in the absence of nephrocytes. CONCLUSIONS: The data implicate SPARC in the noncell autonomous control of cardiac function in Drosophila and suggest that modulation of SPARC gene expression may ameliorate cardiac dysfunction in humans.

Chromosome-scale shotgun assembly using an in vitro method for long-range linkage.

Long-range and highly accurate de novo assembly from short-read data is one of the most pressing challenges in genomics. Recently, it has been shown that read pairs generated by proximity ligation of DNA in chromatin of living tissue can address this problem, dramatically increasing the scaffold contiguity of assemblies. Here, we describe a simpler approach ("Chicago") based on in vitro reconstituted chromatin. We generated two Chicago data sets with human DNA and developed a statistical model and a new software pipeline ("HiRise") that can identify poor quality joins and produce accurate, long-range sequence scaffolds. We used these to construct a highly accurate de novo assembly and scaffolding of a human genome with scaffold N50 of 20 Mbp. We also demonstrated the utility of Chicago for improving existing assemblies by reassembling and scaffolding the genome of the American alligator. With a single library and one lane of Illumina HiSeq sequencing, we increased the scaffold N50 of the American alligator from 508 kbp to 10 Mbp.