Synthetic protein circuits for programmable control of mammalian cell death.

Natural cell death pathways such as apoptosis and pyroptosis play dual roles: they eliminate harmful cells and modulate the immune system by dampening or stimulating inflammation. Synthetic protein circuits capable of triggering specific death programs in target cells could similarly remove harmful cells while appropriately modulating immune responses. However, cells actively influence their death modes in response to natural signals, making it challenging to control death modes. Here, we introduce naturally inspired "synpoptosis" circuits that proteolytically regulate engineered executioner proteins and mammalian cell death. These circuits direct cell death modes, respond to combinations of protease inputs, and selectively eliminate target cells. Furthermore, synpoptosis circuits can be transmitted intercellularly, offering a foundation for engineering synthetic killer cells that induce desired death programs in target cells without self-destruction. Together, these results lay the groundwork for programmable control of mammalian cell death.

Engineering RNA export for measurement and manipulation of living cells.

A system for programmable export of RNA molecules from living cells would enable both non-destructive monitoring of cell dynamics and engineering of cells capable of delivering executable RNA programs to other cells. We developed genetically encoded cellular RNA exporters, inspired by viruses, that efficiently package and secrete cargo RNA molecules from mammalian cells within protective nanoparticles. Exporting and sequencing RNA barcodes enabled non-destructive monitoring of cell population dynamics with clonal resolution. Further, by incorporating fusogens into the nanoparticles, we demonstrated the delivery, expression, and functional activity of exported mRNA in recipient cells. We term these systems COURIER (controlled output and uptake of RNA for interrogation, expression, and regulation). COURIER enables measurement of cell dynamics and establishes a foundation for hybrid cell and gene therapies based on cell-to-cell delivery of RNA.

Diagnosis and management of patients who present with narrow complex tachycardia in the emergency department.

INTRODUCTION: While narrow complex tachycardia (NCT) is a common presentation to the emergency department (ED), little is known about its incidence in the ED or about emergency physician expertise in its diagnosis and management. We sought to compare cases of NCT due to primary arrhythmias to those with a rapid heart rate secondary to a medical issue, as well as to determine the accuracy of ED physician diagnosis and appropriateness of treatment. METHODS: We conducted a health records review at a large academic hospital ED staffed by 95 physicians and included consecutive adult patients over 7 months (2020-2021) with NCT (heart rate ≥ 130 bpm and QRS < 120 ms). Cases were reviewed for accuracy of ECG diagnosis and for correctness of treatment as per guidelines by an adjudication committee. RESULTS: We identified 310 ED visits (0.8% of all ED visits), mean age 65.1 years, 52.6% female. Primary arrhythmias accounted for 54.8%. ED physicians correctly interpreted 86.6% of ECGs. The most common arrhythmias and accuracy of ED physician ECG interpretation were atrial fibrillation 44.5% (95.1%), sinus tachycardia 24.2% (90.5%), atrial flutter 15.8% (61.5%), and supraventricular tachycardia (SVT) 12.9% (81.6%). Treatments were judged optimal in 96.5% of primary NCT and 99.3% in secondary NCT. Treatments were suboptimal for failure to reduce heart rate < 100 bpm prior to discharge in 2.1% of primary cases and failure to treat underlying cause in 0.7% of secondary cases. CONCLUSION: NCT was found in 0.8% of all ED visits, with more being primary NCT. ED physicians correctly interpreted 86.6% of ECGs but had difficulty differentiating atrial flutter and SVT. They implemented appropriate care in most cases but sometimes failed to adequately control heart rate or to treat the underlying condition, suggesting opportunities to improve care of NCT in the ED.

RéSUMé: INTRODUCTION: Bien que la tachycardie à complexe QRS étroite (narrow complex tachycardia [NCT]) soit une présentation courante au service des urgences (SU), on sait peu de choses sur son incidence dans le SU ou sur l'expertise des médecins urgentistes dans son diagnostic et sa prise en charge. Nous avons cherché à comparer les cas de NCT dus à des arythmies primaires à ceux avec une fréquence cardiaque rapide secondaire à un problème médical, ainsi que pour déterminer l'exactitude du diagnostic de médecin ED et la pertinence du traitement. MéTHODES: Nous avons effectué un examen des dossiers médicaux dans les urgences d'un grand hôpital universitaire où travaillent 95 médecins et avons inclus des patients adultes consécutifs sur 7 mois (2020-2021) présentant une NCT (fréquence cardiaque ≥ 130 bpm et QRS < 120 ms). Les cas ont été examinés par un comité d'adjudication pour vérifier l'exactitude du diagnostic ECG et la justesse du traitement conformément aux directives. RéSULTATS: Nous avons recensé 310 visites aux urgences (0,8 % de toutes les visites aux urgences), l'âge moyen étant de 65,1 ans, 52,6 % de femmes. Les arythmies primaires représentaient 54,8 %. Les médecins urgentistes ont correctement interprété 86,6 % des ECG. Les arythmies les plus fréquentes et la précision de l'interprétation de l'ECG par le médecin de l'urgence étaient la fibrillation auriculaire 44,5 % (95,1 %), la tachycardie sinusale 24,2 % (90,5 %), le flutter auriculaire 15,8 % (61,5 %) et la tachycardie supraventriculaire (TSV) 12,9 % (81,6 %). Les traitements ont été jugés optimaux dans 96,5 % des NCT primaires et 99,3 % des NCT secondaires. Les traitements étaient sous-optimaux en raison de l'incapacité à réduire la fréquence cardiaque < 100 bpm avant la sortie de l'hôpital dans 2,1 % des cas primaires et de l'incapacité à traiter la cause sous-jacente dans 0,7 % des cas secondaires. CONCLUSION: Une NCT a été constatée dans 0,8 % de toutes les visites aux urgences, la plupart étant des NCT primaires. Les médecins des services d'urgence ont interprété correctement 86,6 % des ECG mais ont eu des difficultés à différencier le flutter auriculaire et la TSV. Ils ont mis en œuvre des soins appropriés dans la plupart des cas, mais n'ont parfois pas réussi à contrôler adéquatement la fréquence cardiaque ou à traiter l'affection sous-jacente, ce qui laisse entrevoir des possibilités d'améliorer la prise en charge des NCT aux urgences.

The context-dependent, combinatorial logic of BMP signaling.

Cell-cell communication systems typically comprise families of ligand and receptor variants that function together in combinations. Pathway activation depends on the complex way in which ligands are presented extracellularly and receptors are expressed by the signal-receiving cell. To understand the combinatorial logic of such a system, we systematically measured pairwise bone morphogenetic protein (BMP) ligand interactions in cells with varying receptor expression. Ligands could be classified into equivalence groups based on their profile of positive and negative synergies with other ligands. These groups varied with receptor expression, explaining how ligands can functionally replace each other in one context but not another. Context-dependent combinatorial interactions could be explained by a biochemical model based on the competitive formation of alternative signaling complexes with distinct activities. Together, these results provide insights into the roles of BMP combinations in developmental and therapeutic contexts and establish a framework for analyzing other combinatorial, context-dependent signaling systems.

Ligand-receptor promiscuity enables cellular addressing.

In multicellular organisms, secreted ligands selectively activate, or "address," specific target cell populations to control cell fate decision-making and other processes. Key cell-cell communication pathways use multiple promiscuously interacting ligands and receptors, provoking the question of how addressing specificity can emerge from molecular promiscuity. To investigate this issue, we developed a general mathematical modeling framework based on the bone morphogenetic protein (BMP) pathway architecture. We find that promiscuously interacting ligand-receptor systems allow a small number of ligands, acting in combinations, to address a larger number of individual cell types, defined by their receptor expression profiles. Promiscuous systems outperform seemingly more specific one-to-one signaling architectures in addressing capability. Combinatorial addressing extends to groups of cell types, is robust to receptor expression noise, grows more powerful with increases in the number of receptor variants, and is maximized by specific biochemical parameter relationships. Together, these results identify design principles governing cellular addressing by ligand combinations.

Diagnosis and management of wide complex tachycardia in the emergency department.

INTRODUCTION: While wide complex tachycardia (WCT) is potentially lethal, little is known about its incidence in the ED or about expertise of ED physicians in diagnosing and treating it. We sought to compare WCT ED cases that were primary arrhythmias versus those with rapid heart rate secondary to medical issues, as well as the accuracy of ED diagnosis and appropriateness of treatment. METHODS: We conducted a health records review at a large academic hospital ED staffed by 95 physicians and included consecutive patients over 28 months (2018-2020) with WCT (heart rate ≥ 120 bpm and QRS ≥ 120 ms). Cases were adjudicated for the accuracy of ECG diagnosis versus the cardiology read and for correctness of treatment as per guidelines by two ED physicians and one cardiologist. RESULTS: We identified 306 eligible cases (0.2% of all ED visits): mean age 73.9 years, male 66.0%, admitted 53.3%, died in ED 2.3%. Primary arrhythmias and secondary tachycardias were each 50.0% (95% CI 44.4-55.6%). ED physicians correctly interpreted 81.2% of ECGs. The most common presenting arrythmias and % correct were: atrial fibrillation 42.7% (95.0%), atrial flutter 22.2% (63.5%), sinus tachycardia 12.0% (78.6%), and supraventricular tachycardia (SVT) 11.1% (68.0%). Treatments were judged optimal in 84.3% of primary WCT and 86.9% in secondary WCT. Treatments were suboptimal for: inappropriate drug (3.9% for primary versus 1.3% for secondary), failure to reduce heart rate < 100 prior to discharge home (9.1% for primary versus 34.4% for secondary), and not treating the underlying cause in 5.9% of secondary WCT. CONCLUSIONS: WCT cases were evenly split between primary arrhythmias and secondary cases. ED physicians interpreted the ECG correctly in 81% but over-called atrial flutter and SVT. They implemented appropriate care in most cases but sometimes failed to adequately control heart rate or to treat the underlying condition, suggesting opportunities to improve care of WCT in the ED.

RéSUMé: INTRODUCTION: Bien que la tachycardie à complexe large (wide complex tachycardia WCT) soit potentiellement mortelle, on sait peu de choses sur son incidence aux urgences ou sur l'expertise des médecins des urgences en matière de diagnostic et de traitement. Nous avons cherché à comparer les cas WCT aux urgences qui étaient des arythmies primaires par rapport à ceux avec une fréquence cardiaque rapide secondaire à des problèmes médicaux, ainsi que la précision du diagnostic des urgences et la pertinence du traitement. MéTHODES: Nous avons effectué un examen des dossiers médicaux dans les urgences d'un grand hôpital universitaire où travaillent 95 médecins et avons inclus des patients consécutifs sur 28 mois (2018-2020) présentant un WCT (fréquence cardiaque ≥120 bpm et QRS ≥120 ms). Les cas ont été jugés par deux médecins des urgences et un cardiologue pour l'exactitude du diagnostic ECG par rapport à la lecture cardiologique et pour l'exactitude du traitement conformément aux directives. RéSULTATS: Nous avons identifié 306 cas éligibles (0,2 % de toutes les visites aux urgences) : âge moyen 73,9 ans, hommes 66,0 %, admis 53,3 %, décédés aux urgences 2,3 %. Les arythmies primaires et les tachycardies secondaires étaient chacune de 50,0 % (IC 95 % 44,4-55,6 %). Les médecins des urgences ont interprété correctement 81,2 % des ECG. Les arythmies les plus fréquentes et les pourcentages d'interprétation correcte étaient les suivants : fibrillation auriculaire 42,7 % (95,0 %), flutter auriculaire 22,2 % (63,5 %), tachycardie sinusale 12,0 % (78,6 %) et tachycardie supraventriculaire (TSV) 11,1 % (68,0 %). Les traitements ont été jugés optimaux dans 84,3 % des WCT primaires et 86,9 % des WCT secondaires. Les traitements étaient sous-optimaux pour les raisons suivantes : médicament inapproprié (3,9 % pour le primaire contre 1,3 % pour le secondaire), incapacité à réduire la fréquence <100 avant le retour à domicile (9,1 % pour le primaire contre 34,4 % pour le secondaire) et absence de traitement de la cause sous-jacente dans 5,9 % du WCT secondaire. CONCLUSIONS: Les cas de WCT étaient répartis uniformément entre les arythmies primaires et les cas secondaires. Les médecins du service d'urgence ont correctement interprété l'ECG dans 81 % des cas, mais ont surévalué le flutter auriculaire et la TSV. Ils ont mis en œuvre des soins appropriés dans la plupart des cas, mais n'ont pas toujours réussi à contrôler adéquatement la fréquence cardiaque ou à traiter la maladie sous-jacente, ce qui laisse entrevoir des possibilités d'améliorer les soins de WCT au service d'urgence.

Publisher Correction: In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription.

Molecular barcoding technologies that uniquely identify single cells are hampered by limitations in barcode measurement. Readout by sequencing does not preserve the spatial organization of cells in tissues, whereas imaging methods preserve spatial structure but are less sensitive to barcode sequence. Here we introduce a system for image-based readout of short (20-base-pair) DNA barcodes. In this system, called Zombie, phage RNA polymerases transcribe engineered barcodes in fixed cells. The resulting RNA is subsequently detected by fluorescent in situ hybridization. Using competing match and mismatch probes, Zombie can accurately discriminate single-nucleotide differences in the barcodes. This method allows in situ readout of dense combinatorial barcode libraries and single-base mutations produced by CRISPR base editors without requiring barcode expression in live cells. Zombie functions across diverse contexts, including cell culture, chick embryos and adult mouse brain tissue. The ability to sensitively read out compact and diverse DNA barcodes by imaging will facilitate a broad range of barcoding and genomic recording strategies.

Primary Sertoli Cell Cultures From Adult Mice Have Different Properties Compared With Those Derived From 20-Day-Old Animals.

Cultures of Sertoli cells isolated from 20-day-old mice are widely used in research as substitutes for adult Sertoli cell cultures. This practice is based on the fact that Sertoli cells cease to proliferate and become mature in vivo by 16 to 20 days after birth. However, it is important to verify whether cultured Sertoli cells derived from 20-day-old mice do not proliferate ex vivo and whether they have the same properties as cultured adult Sertoli cells. Herein we described an isolation/culture method of Sertoli cells from 10-week-old adult mice with > 90% purity. Properties of these cultured adult Sertoli cells were then compared with those of cultured Sertoli cells derived from 20-day-old mice (also > 90% purity). By cell counting, bromo-2-deoxyuridine incorporation, and metaphase plate detection, we demonstrated that only adult Sertoli cells did not proliferate throughout 12 culture days. In contrast, Sertoli cells derived from 20-day-old mice still proliferated until Day 10 in culture. The morphology and profiles of intracellular lipidomics and spent medium proteomics of the 2 cultures were also different. Cultured adult Sertoli cells were larger in size and contained higher levels of triacylglycerols, cholesteryl esters, and seminolipid, and the proteins in their spent medium were mainly engaged in cellular metabolism. In contrast, proteins involved in cell division, including anti-Mullerian hormone, cell division cycle protein 42 (CDC42), and collagen isoforms, were at higher levels in Sertoli cell cultures derived from 20-day-old mice. Therefore, cultured Sertoli cells derived from 10-week-old mice, rather than those from 20-day-old animals, should be used for studies on properties of adult Sertoli cells.

Toward a Synergistic Operating Model for Westmead Research Hub Biobanks: A Questionnaire Study.

Standardization and sustainability are ideals within the biobanking world, and the demand for high-quality well-annotated specimens is growing just as rapidly as the ever-increasing precision and throughput of today's high-tech scientific methods. In the state of New South Wales (NSW) in Australia, the state government has allocated significant funding toward this requirement in recent years, with the launch of the NSW Health Statewide Biobank in central Sydney in 2017, and the introduction of the voluntary NSW Biobank Certification Program, and Consent Toolkit. For new and established biobanks, the influence of these new resources has been twofold: first they have provided valuable guidance for moving toward standardized practices and raising the bar for biobanking quality standards; second, they have brought to the forefront the challenges of sustainability and transitioning to a certification standard of biobanking. In Westmead, ∼20 km from Sydney's central business district, the Westmead Research Hub has responded to these challenges with a collaborative biobanking project initiated in 2015. As the site of almost 30 individual biobanks, and to inform a pilot project of central biobank services, a questionnaire was developed and administered to all of the biobanks. This article reports on the results from the questionnaire and the rationale for subsequent initiation of a core biobanking facility.

Properties, metabolism and roles of sulfogalactosylglycerolipid in male reproduction.

Sulfogalactosylglycerolipid (SGG, aka seminolipid) is selectively synthesized in high amounts in mammalian testicular germ cells (TGCs). SGG is an ordered lipid and directly involved in cell adhesion. SGG is indispensable for spermatogenesis, a process that greatly depends on interaction between Sertoli cells and TGCs. Spermatogenesis is disrupted in mice null for Cgt and Cst, encoding two enzymes essential for SGG biosynthesis. Sperm surface SGG also plays roles in fertilization. All of these results indicate the significance of SGG in male reproduction. SGG homeostasis is also important in male fertility. Approximately 50% of TGCs become apoptotic and phagocytosed by Sertoli cells. SGG in apoptotic remnants needs to be degraded by Sertoli lysosomal enzymes to the lipid backbone. Failure in this event leads to a lysosomal storage disorder and sub-functionality of Sertoli cells, including their support for TGC development, and consequently subfertility. Significantly, both biosynthesis and degradation pathways of the galactosylsulfate head group of SGG are the same as those of sulfogalactosylceramide (SGC), a structurally related sulfoglycolipid important for brain functions. If subfertility in males with gene mutations in SGG/SGC metabolism pathways manifests prior to neurological disorder, sperm SGG levels might be used as a reporting/predicting index of the neurological status.

Combinatorial Signal Perception in the BMP Pathway.

The bone morphogenetic protein (BMP) signaling pathway comprises multiple ligands and receptors that interact promiscuously with one another and typically appear in combinations. This feature is often explained in terms of redundancy and regulatory flexibility, but it has remained unclear what signal-processing capabilities it provides. Here, we show that the BMP pathway processes multi-ligand inputs using a specific repertoire of computations, including ratiometric sensing, balance detection, and imbalance detection. These computations operate on the relative levels of different ligands and can arise directly from competitive receptor-ligand interactions. Furthermore, cells can select different computations to perform on the same ligand combination through expression of alternative sets of receptor variants. These results provide a direct signal-processing role for promiscuous receptor-ligand interactions and establish operational principles for quantitatively controlling cells with BMP ligands. Similar principles could apply to other promiscuous signaling pathways.

Grey matter abnormalities in children and adolescents with functional neurological symptom disorder.

OBJECTIVE: Functional neurological symptom disorder refers to the presence of neurological symptoms not explained by neurological disease. Although this disorder is presumed to reflect abnormal function of the brain, recent studies in adults show neuroanatomical abnormalities in brain structure. These structural brain abnormalities have been presumed to reflect long-term adaptations to the disorder, and it is unknown whether child and adolescent patients, with illness that is typically of shorter duration, show similar deficits or have normal brain structure. METHOD: High-resolution, three-dimensional T1-weighted magnetic resonance images (MRIs) were acquired in 25 patients (aged 10-18 years) and 24 healthy controls. Structure was quantified in terms of grey matter volume using voxel-based morphometry. Post hoc, we examined whether regions of structural difference related to a measure of motor readiness to emotional signals and to clinical measures of illness duration, illness severity, and anxiety/depression. RESULTS: Patients showed greater volumes in the left supplementary motor area (SMA) and right superior temporal gyrus (STG) and dorsomedial prefrontal cortex (DMPFC) (corrected p < 0.05). Previous studies of adult patients have also reported alterations of the SMA. Greater SMA volumes correlated with faster reaction times in identifying emotions but not with clinical measures. CONCLUSIONS: The SMA, STG, and DMPFC are known to be involved in the perception of emotion and the modulation of motor responses. These larger volumes may reflect the early expression of an experience-dependent plasticity process associated with increased vigilance to others' emotional states and enhanced motor readiness to organize self-protectively in the context of the long-standing relational stress that is characteristic of this disorder.

Synthetic recording and in situ readout of lineage information in single cells.

Reconstructing the lineage relationships and dynamic event histories of individual cells within their native spatial context is a long-standing challenge in biology. Many biological processes of interest occur in optically opaque or physically inaccessible contexts, necessitating approaches other than direct imaging. Here we describe a synthetic system that enables cells to record lineage information and event histories in the genome in a format that can be subsequently read out of single cells in situ. This system, termed memory by engineered mutagenesis with optical in situ readout (MEMOIR), is based on a set of barcoded recording elements termed scratchpads. The state of a given scratchpad can be irreversibly altered by CRISPR/Cas9-based targeted mutagenesis, and later read out in single cells through multiplexed single-molecule RNA fluorescence hybridization (smFISH). Using MEMOIR as a proof of principle, we engineered mouse embryonic stem cells to contain multiple scratchpads and other recording components. In these cells, scratchpads were altered in a progressive and stochastic fashion as the cells proliferated. Analysis of the final states of scratchpads in single cells in situ enabled reconstruction of lineage information from cell colonies. Combining analysis of endogenous gene expression with lineage reconstruction in the same cells further allowed inference of the dynamic rates at which embryonic stem cells switch between two gene expression states. Finally, using simulations, we show how parallel MEMOIR systems operating in the same cell could enable recording and readout of dynamic cellular event histories. MEMOIR thus provides a versatile platform for information recording and in situ, single-cell readout across diverse biological systems.

Inferring Cell-State Transition Dynamics from Lineage Trees and Endpoint Single-Cell Measurements.

As they proliferate, living cells undergo transitions between specific molecularly and developmentally distinct states. Despite the functional centrality of these transitions in multicellular organisms, it has remained challenging to determine which transitions occur and at what rates without perturbations and cell engineering. Here, we introduce kin correlation analysis (KCA) and show that quantitative cell-state transition dynamics can be inferred, without direct observation, from the clustering of cell states on pedigrees (lineage trees). Combining KCA with pedigrees obtained from time-lapse imaging and endpoint single-molecule RNA-fluorescence in situ hybridization (RNA-FISH) measurements of gene expression, we determined the cell-state transition network of mouse embryonic stem (ES) cells. This analysis revealed that mouse ES cells exhibit stochastic and reversible transitions along a linear chain of states ranging from 2C-like to epiblast-like. Our approach is broadly applicable and may be applied to systems with irreversible transitions and non-stationary dynamics, such as in cancer and development.

A pharmacological network for lifespan extension in Caenorhabditis elegans.

One goal of aging research is to find drugs that delay the onset of age-associated disease. Studies in invertebrates, particularly Caenorhabditis elegans, have uncovered numerous genes involved in aging, many conserved in mammals. However, which of these encode proteins suitable for drug targeting is unknown. To investigate this question, we screened a library of compounds with known mammalian pharmacology for compounds that increase C. elegans lifespan. We identified 60 compounds that increase longevity in C. elegans, 33 of which also increased resistance to oxidative stress. Many of these compounds are drugs approved for human use. Enhanced resistance to oxidative stress was associated primarily with compounds that target receptors for biogenic amines, such as dopamine or serotonin. A pharmacological network constructed with these data reveal that lifespan extension and increased stress resistance cluster together in a few pharmacological classes, most involved in intercellular signaling. These studies identify compounds that can now be explored for beneficial effects on aging in mammals, as well as tools that can be used to further investigate the mechanisms underlying aging in C. elegans.

The basement membrane of hair follicle stem cells is a muscle cell niche.

The hair follicle bulge in the epidermis associates with the arrector pili muscle (APM) that is responsible for piloerection ("goosebumps"). We show that stem cells in the bulge deposit nephronectin into the underlying basement membrane, thus regulating the adhesion of mesenchymal cells expressing the nephronectin receptor, α8ß1 integrin, to the bulge. Nephronectin induces α8 integrin-positive mesenchymal cells to upregulate smooth muscle markers. In nephronectin knockout mice, fewer arrector pili muscles form in the skin, and they attach to the follicle above the bulge, where there is compensatory upregulation of the nephronectin family member EGFL6. Deletion of α8 integrin also abolishes selective APM anchorage to the bulge. Nephronectin is a Wnt target; epidermal ß-catenin activation upregulates epidermal nephronectin and dermal α8 integrin expression. Thus, bulge stem cells, via nephronectin expression, create a smooth muscle cell niche and act as tendon cells for the APM. Our results reveal a functional role for basement membrane heterogeneity in tissue patterning. PAPERCLIP:

Simple sequence repeats in Helicobacter canadensis and their role in phase variable expression and C-terminal sequence switching.

BACKGROUND: Helicobacter canadensis is an emerging human pathogen and zoonotic agent. The genome of H. canadensis was sequenced previously and determined to contain 29 annotated coding regions associated with homopolymeric tracts. RESULTS: Twenty-one of the repeat-associated coding regions were determined to be potentially transcriptionally or translationally phase variable. In each case the homopolymeric tract was within the predicted promoter region or at the 5' end of the coding region, respectively. However, eight coding sequences were identified with simple sequence repeats toward the 3' end of the open reading frame. In these cases, the repeat tract would be too far into the coding region to be mediating translational phase variation. All of the 29 coding region-associated homopolymeric tracts display variability in tract length in the sequencing read data. CONCLUSIONS: Twenty-nine coding regions have been identified in the genome sequence of Helicobacter canadensis strain NCTC13241 that show variations in homopolymeric tract length in the bacterial population, indicative of phase variation. Five of these are potentially associated with promoter regions, which would lead to transcriptional phase variation. Translational phase variation usually switches expression of a gene ON and OFF due to the repeat region being located sufficiently close to the initiation codon for the resulting frame-shift to lead to a premature termination codon and stop the translation of the protein. Sixteen of the 29 coding regions have homopolymeric tracts characteristic of translational phase variation. For eight coding sequences with repeats located later in the reading frame, changes in the repeat tract length would alter the protein sequence at the C-terminus but not stop the expression of the protein. This mechanism of C-terminal phase variation has implications for stochastic switching of protein sequence in bacterial species that already undergo transcriptional and translational phase variation.

The ECM protein nephronectin promotes kidney development via integrin alpha8beta1-mediated stimulation of Gdnf expression.

Development of the metanephric kidney crucially depends on proper interactions between cells and the surrounding extracellular matrix. For example, we showed previously that in the absence of alpha8beta1 integrin, invasion by the ureteric bud into the metanephric mesenchyme is inhibited, resulting in renal agenesis. Here we present genetic evidence that the extracellular matrix protein nephronectin is an essential ligand that engages alpha8beta1 integrin during early kidney development. We show that embryos lacking a functional nephronectin gene frequently display kidney agenesis or hypoplasia, which can be traced to a delay in the invasion of the metanephric mesenchyme by the ureteric bud at an early stage of kidney development. Significantly, we detected no defects in extracellular matrix organization in the nascent kidneys of the nephronectin mutants. Instead, we found that Gdnf expression was dramatically reduced in both nephronectin- and alpha8 integrin-null mutants specifically in the metanephric mesenchyme at the time of ureteric bud invasion. We show that this reduction is sufficient to explain the agenesis and hypoplasia observed in both mutants. Interestingly, the reduction in Gdnf expression is transient, and its resumption presumably enables the nephronectin-deficient ureteric buds to invade the metanephric mesenchyme and begin branching. Our results thus place nephronectin and alpha8beta1 integrin in a pathway that regulates Gdnf expression and is essential for kidney development.