Whole genome and transcriptome integrated analyses guide clinical care of pediatric poor prognosis cancers.

The role for routine whole genome and transcriptome analysis (WGTA) for poor prognosis pediatric cancers remains undetermined. Here, we characterize somatic mutations, structural rearrangements, copy number variants, gene expression, immuno-profiles and germline cancer predisposition variants in children and adolescents with relapsed, refractory or poor prognosis malignancies who underwent somatic WGTA and matched germline sequencing. Seventy-nine participants with a median age at enrollment of 8.8 y (range 6 months to 21.2 y) are included. Germline pathogenic/likely pathogenic variants are identified in 12% of participants, of which 60% were not known prior. Therapeutically actionable variants are identified by targeted gene report and whole genome in 32% and 62% of participants, respectively, and increase to 96% after integrating transcriptome analyses. Thirty-two molecularly informed therapies are pursued in 28 participants with 54% achieving a clinical benefit rate; objective response or stable disease ≥6 months. Integrated WGTA identifies therapeutically actionable variants in almost all tumors and are directly translatable to clinical care of children with poor prognosis cancers.

A single base pair substitution in zebrafish distinguishes between innate and acute startle behavior regulation.

Behavioral thresholds define the lowest stimulus intensities sufficient to elicit a behavioral response. Establishment of baseline behavioral thresholds during development is critical for proper responses throughout the animal's life. Despite the relevance of such innate thresholds, the molecular mechanisms critical to establishing behavioral thresholds during development are not well understood. The acoustic startle response is a conserved behavior whose threshold is established during development yet is subsequently acutely regulated. We have previously identified a zebrafish mutant line (escapist) that displays a decreased baseline or innate acoustic startle threshold. Here, we identify a single base pair substitution on Chromosome 25 located within the coding sequence of the synaptotagmin 7a (syt7a) gene that is tightly linked to the escapist acoustic hypersensitivity phenotype. By generating animals in which we deleted the syt7a open reading frame, and subsequent complementation testing with the escapist line, we demonstrate that loss of syt7a function is not the cause of the escapist behavioral phenotype. Nonetheless, escapist mutants provide a powerful tool to decipher the overlap between acute and developmental regulation of behavioral thresholds. Extensive behavioral analyses reveal that in escapist mutants the establishment of the innate acoustic startle threshold is impaired, while regulation of its acute threshold remains intact. Moreover, our behavioral analyses reveal a deficit in baseline responses to visual stimuli, but not in the acute regulation of responses to visual stimuli. Together, this work eliminates loss of syt7a as causative for the escapist phenotype and suggests that mechanisms that regulate the establishment of behavioral thresholds in escapist larvae can operate independently from those regulating acute threshold regulation.

A single base pair substitution on Chromosome 25 in zebrafish distinguishes between development and acute regulation of behavioral thresholds.

Behavioral thresholds define the lowest stimulus intensities sufficient to elicit a behavioral response. Establishment of baseline behavioral thresholds during development is critical for proper responses throughout the animal's life. Despite the relevance of such innate thresholds, the molecular mechanisms critical to establishing behavioral thresholds during development are not well understood. The acoustic startle response is a conserved behavior whose threshold is established during development yet is subsequently acutely regulated. We have previously identified a zebrafish mutant line ( escapist ) that displays a decreased baseline or innate acoustic startle threshold. Here, we identify a single base pair substitution on Chromosome 25 located within the coding sequence of the synaptotagmin 7a ( syt7a ) gene that is tightly linked to the escapist acoustic hypersensitivity phenotype. By generating animals in which we deleted the syt7a open reading frame, and subsequent complementation testing with the escapist line, we demonstrate that loss of syt7a function is not the cause of the escapist behavioral phenotype. Nonetheless, escapist mutants provide a powerful tool to decipher the overlap between acute and developmental regulation of behavioral thresholds. Extensive behavioral analyses reveal that in escapist mutants the establishment of the innate acoustic startle threshold is impaired, while regulation of its acute threshold remains intact. Moreover, our behavioral analyses reveal a deficit in baseline responses to visual stimuli, but not in the acute regulation of responses to visual stimuli. Together, this work eliminates loss of syt7a as causative for the escapist phenotype and suggests that mechanisms that regulate the establishment of behavioral thresholds in escapist larvae can operate largely independently from those regulating acute threshold regulation.

Systematic review on raphide morphotype calcium oxalate crystals in angiosperms.

Calcium oxalate (CaOx) crystals are biominerals present in a wide variety of plants. Formation of these crystals is a biomineralization process occurring in vacuoles within specialized cells called crystal idioblasts. This process is dependent on two key components: deprotonated oxalic acid, and calcium ions (Ca2+), and can result in multiple crystal morphologies. Raphides are needle-like CaOx crystals found in various plant organs and tissues. Though their function is highly debated, they can potentially store calcium, sequester heavy metals, protect against herbivory and possibly programmed cell death. The last review of the taxonomic and anatomical distribution of raphides across the plant kingdom dates back to 1980, in a review by Franceschi and Horner, prompting an updated systematic review of raphides in plants. We conduct a broad literature search to record plant taxa and tissue locations containing raphides. We provide an overview of raphide-forming plant taxa, discussing phylogenetic distribution of raphides at the order level, and report on the specific locations of raphides within plants. Our review reveals raphide occurrence has been studied in 33 orders, 76 families and 1305 species, with raphides presence confirmed in 24 orders, 46 families and 797 species. These taxa represented less than 1 % of known species per family. Leaves are the most prominent raphide-containing primary location in all three major angiosperm clades investigated: Eudicots, Magnoliids, and Monocots. Roots are least reported to contain raphides. The collation of such information lays the groundwork to unveil the genetic origin and evolution of raphides in plants, and highlights targets for future studies of the presence and role of plant raphides.

Decreasing the Frequency of Nursing Assessment for Medically Stable Hospitalized Patients.

PURPOSE/OBJECTIVES: During the COVID-19 pandemic, a large Midwest tertiary care medical center had prolonged hospitalizations due to strained staffing and few options for post-acute care recovery. Patients deemed medically ready for discharge were receiving the same care interventions as all other hospitalized medical-surgical patients. The study objective was to appropriately match care assessment frequency for these patients with their individual needs by reducing the frequency of routine nursing assessments. DESCRIPTION OF THE PROJECT/PROGRAM: This quality improvement initiative reduced the frequency of nursing assessments, including routine monitoring of vital signs, to once daily for medically stable patients whose discharge was delayed. OUTCOME: During the 4-week pilot, 40 hospitalized patients were enrolled; 960 assessments were eliminated, and nurses were able to reallocate approximately 500 hours to other nursing tasks. No adverse outcomes were observed among patients who received once-daily assessment. CONCLUSION: By decreasing nursing assessment frequency for hospitalized patients with discharge delays, nurses appropriately matched care interventions with the patient's needs.

USP7 controls NGN3 stability and pancreatic endocrine lineage development.

Understanding the factors and mechanisms involved in beta-cell development will guide therapeutic efforts to generate fully functional beta cells for diabetes. Neurogenin 3 (NGN3) is the key transcription factor that marks endocrine progenitors and drives beta-cell differentiation. Here we screen for binding partners of NGN3 and identify the deubiquitylating enzyme USP7 as a key regulator of NGN3 stability. Mechanistically, USP7 interacts with, deubiquitinates and stabilizes NGN3. In vivo, conditional knockout of Usp7 in the mouse embryonic pancreas causes a dramatic reduction in islet formation and hyperglycemia in adult mice, due to impaired NGN3-mediated endocrine specification during pancreatic development. Furthermore, pharmacological inhibition of USP7 during endocrine specification in human iPSC models of beta-cell differentiation decreases NGN3 expressing progenitor cell numbers and impairs beta cell differentiation. Thus, the USP7-NGN3 axis is an essential mechanism for driving endocrine development and beta-cell differentiation, which can be therapeutically exploited.

Integration of cooperative and opposing molecular programs drives learning-associated behavioral plasticity.

Habituation is a foundational learning process critical for animals to adapt their behavior to changes in their sensory environment. Although habituation is considered a simple form of learning, the identification of a multitude of molecular pathways including several neurotransmitter systems that regulate this process suggests an unexpected level of complexity. How the vertebrate brain integrates these various pathways to accomplish habituation learning, whether they act independently or intersect with one another, and whether they act via divergent or overlapping neural circuits has remained unclear. To address these questions, we combined pharmacogenetic pathway analysis with unbiased whole-brain activity mapping using the larval zebrafish. Based on our findings, we propose five distinct molecular modules for the regulation of habituation learning and identify a set of molecularly defined brain regions associated with four of the five modules. Moreover, we find that in module 1 the palmitoyltransferase Hip14 cooperates with dopamine and NMDA signaling to drive habituation, while in module 3 the adaptor protein complex subunit Ap2s1 drives habituation by antagonizing dopamine signaling, revealing two distinct and opposing roles for dopaminergic neuromodulation in the regulation of behavioral plasticity. Combined, our results define a core set of distinct modules that we propose act in concert to regulate habituation-associated plasticity, and provide compelling evidence that even seemingly simple learning behaviors in a compact vertebrate brain are regulated by a complex and overlapping set of molecular mechanisms.

Tibial Supra-tubercular Deflexion Osteotomy in the Management of Failed Anterior Cruciate Ligament Reconstruction: A Surgical Technique.

Excessive posterior tibial slope (PTS) is a recognized risk factor for failure of anterior cruciate ligament reconstruction (ACLR) and should be considered when planning a revision ACLR. A tibial supra-tubercular deflexion osteotomy can correct excessive PTS with simultaneous or staged ACLR. There are only a handful of technical descriptions offering insight on the respective authors' approach at reducing PTS, all of which vary greatly in their methods. The authors describe a surgical technique using a proximal tibial supra-tubercular deflexion osteotomy in patients with persistent knee instability, a history of at least one failed ACLR, and a PTS greater than 12°. This surgery is not recommended in patients with significant genu recurvatum (>10°), significant varus, or severe tibiofemoral osteoarthritis.

cacna2d3, a voltage-gated calcium channel subunit, functions in vertebrate habituation learning and the startle sensitivity threshold.

BACKGROUND: The ability to filter sensory information into relevant versus irrelevant stimuli is a fundamental, conserved property of the central nervous system and is accomplished in part through habituation learning. Synaptic plasticity that underlies habituation learning has been described at the cellular level, yet the genetic regulators of this plasticity remain poorly understood, as do circuits that mediate sensory filtering. METHODS: To identify genes critical for plasticity, a forward genetic screen for zebrafish genes that mediate habituation learning was performed, which identified a mutant allele, doryp177, that caused reduced habituation of the acoustic startle response. In this study, we combine whole-genome sequencing with behavioral analyses to characterize and identify the gene affected in doryp177 mutants. RESULTS: Whole-genome sequencing identified the calcium voltage-gated channel auxiliary subunit alpha-2/delta-3 (cacna2d3) as a candidate gene affected in doryp177 mutants. Behavioral characterization of larvae homozygous for two additional, independently derived mutant alleles of cacna2d3, together with failure of these alleles to complement doryp177, confirmed a critical role for cacna2d3 in habituation learning. Notably, detailed analyses of the acoustic response in mutant larvae also revealed increased startle sensitivity to acoustic stimuli, suggesting a broader role for cacna2d3 in controlling innate response thresholds to acoustic stimuli. CONCLUSIONS: Taken together, our data demonstrate a critical role for cacna2d3 in sensory filtering, a process that is disrupted in human CNS disorders, e.g. ADHD, schizophrenia, and autism.

GREM1 is required to maintain cellular heterogeneity in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) shows pronounced epithelial and mesenchymal cancer cell populations1-4. Cellular heterogeneity in PDAC is an important feature in disease subtype specification3-5, but how distinct PDAC subpopulations interact, and the molecular mechanisms that underlie PDAC cell fate decisions, are incompletely understood. Here we identify the BMP inhibitor GREM16,7 as a key regulator of cellular heterogeneity in pancreatic cancer in human and mouse. Grem1 inactivation in established PDAC in mice resulted in a direct conversion of epithelial into mesenchymal PDAC cells within days, suggesting that persistent GREM1 activity is required to maintain the epithelial PDAC subpopulations. By contrast, Grem1 overexpression caused an almost complete 'epithelialization' of highly mesenchymal PDAC, indicating that high GREM1 activity is sufficient to revert the mesenchymal fate of PDAC cells. Mechanistically, Grem1 was highly expressed in mesenchymal PDAC cells and inhibited the expression of the epithelial-mesenchymal transition transcription factors Snai1 (also known as Snail) and Snai2 (also known as Slug) in the epithelial cell compartment, therefore restricting epithelial-mesenchymal plasticity. Thus, constant suppression of BMP activity is essential to maintain epithelial PDAC cells, indicating that the maintenance of the cellular heterogeneity of pancreatic cancer requires continuous paracrine signalling elicited by a single soluble factor.

Dynamic plastid and mitochondrial genomes in Chaetopeltidales (Chlorophyceae) and characterization of a new chlorophyte taxon.

PREMISE: Chaetopeltidales is a poorly characterized order in the Chlorophyceae, with only two plastid and no mitochondrial genomes published. Here we describe a new taxon in Chaetopeltidales, Gormaniella terricola gen. et sp. nov. and characterize both of its organellar genomes. METHODS: Gormaniella terricola was inadvertently isolated from a surface-sterilized hornwort thallus. Light microscopy was used to characterize its vegetative morphology. Organellar genomes were assembled, annotated, and analyzed using a variety of software packages. RESULTS: The mitochondrial genome (66,927 bp) represents the first complete mitochondrial genome published for Chaetopeltidales. The chloroplast genome, measuring 428,981 bp, is one of the largest plastid genomes published to date and shares this large size and an incredible number of short, dispersed repeats with the other sequenced chloroplast genomes in Chaetopeltidales. Despite these shared features, the chloroplast genomes of Chaetopeltidales appear to be highly rearranged when compared to one another, with numerous inversions, translocations, and duplications, suggesting a particularly dynamic chloroplast genome. Both the chloroplast and mitochondrial genomes of G. terricola contain a number of mobile group I and group II introns, which appear to have invaded separately. Three of the introns within the mitochondrial genome encode homing endonucleases that are phylogenetically nested within those found in fungi, rather than algae, suggesting a possible case of horizontal gene transfer. CONCLUSIONS: These results help to shed light on a poorly understood group of algae and their unusual organellar genomes, raising additional questions about the unique patterns of genome evolution within Chaetopeltidales.

Zebrafish behavior as a gateway to nervous system assembly and plasticity.

Nervous system assembly relies on a diversity of cellular processes ranging from dramatic tissue reorganization to local, subcellular changes all driven by precise molecular programs. Combined, these processes culminate in an animal's ability to plan and execute behaviors. Animal behavior can, therefore, serve as a functional readout of nervous system development. Benefitting from an expansive and growing set of molecular and imaging tools paired with an ever-growing number of assays of diverse behaviors, the zebrafish system has emerged as an outstanding platform at the intersection of nervous system assembly, plasticity and behavior. Here, we summarize recent advancements in the field, including how developing neural circuits are refined to shape complex behaviors and plasticity.

A scoping review of bryophyte microbiota: diverse microbial communities in small plant packages.

Plant health depends not only on the condition of the plant itself but also on its diverse community of microbes, or microbiota. Just like the better-studied angiosperms, bryophytes (mosses, liverworts, and hornworts) harbor diverse communities of bacteria, archaea, fungi, and other microbial eukaryotes. Bryophytes are increasingly recognized as important model systems for understanding plant evolution, development, physiology, and symbiotic interactions. Much of the work on bryophyte microbiota in the past focused on specific symbiont types for each bryophyte group, but more recent studies are taking a broader view acknowledging the coexistence of diverse microbial communities in bryophytes. Therefore, this review integrates studies of bryophyte microbes from both perspectives to provide a holistic view of the existing research for each bryophyte group and on key themes. The systematic search also reveals the taxonomic and geographic biases in this field, including a severe under-representation of the tropics, very few studies on viruses or eukaryotic microbes beyond fungi, and a focus on mycorrhizal fungi studies in liverworts. Such gaps may have led to errors in conclusions about evolutionary patterns in symbiosis. This analysis points to a wealth of future research directions that promise to reveal how the distinct life cycles and physiology of bryophytes interact with their microbiota.

USP25 promotes pathological HIF-1-driven metabolic reprogramming and is a potential therapeutic target in pancreatic cancer.

Deubiquitylating enzymes (DUBs) play an essential role in targeted protein degradation and represent an emerging therapeutic paradigm in cancer. However, their therapeutic potential in pancreatic ductal adenocarcinoma (PDAC) has not been explored. Here, we develop a DUB discovery pipeline, combining activity-based proteomics with a loss-of-function genetic screen in patient-derived PDAC organoids and murine genetic models. This approach identifies USP25 as a master regulator of PDAC growth and maintenance. Genetic and pharmacological USP25 inhibition results in potent growth impairment in PDAC organoids, while normal pancreatic organoids are insensitive, and causes dramatic regression of patient-derived xenografts. Mechanistically, USP25 deubiquitinates and stabilizes the HIF-1α transcription factor. PDAC is characterized by a severely hypoxic microenvironment, and USP25 depletion abrogates HIF-1α transcriptional activity and impairs glycolysis, inducing PDAC cell death in the tumor hypoxic core. Thus, the USP25/HIF-1α axis is an essential mechanism of metabolic reprogramming and survival in PDAC, which can be therapeutically exploited.

Does Physician Gender and Gender Composition of Clinical Teams Affect Guideline Concordance and Patient Outcomes in Out-of-Hospital Cardiac Arrest?

Objective: Prior literature has shown improved outcomes in morbidity and mortality for admitted patients cared for by female physicians. One theory is that female physicians adhere closely to guideline recommendations. We sought to determine whether patients who have out-of-hospital cardiac arrest (OHCA) experience more guideline-concordant postcardiac arrest care and potentially better outcomes based on the gender of their treating physician and gender distribution of the treatment teams. Methods: This study is a retrospective cohort study from the Colorado Cardiac Arrest Registry, local registry of OHCA patients treated at one academic urban tertiary care hospital. We analyzed adult OHCA patients who survived to hospital admission but were comatose. Patient demographic data and arrest characteristics were abstracted for subjects, and the gender of the provider was abstracted from the medical record. Results: Patients were admitted by a female attending in 28.5% of the cohort. The difference in guideline-concordant care between male and female providers was not significant. No statistical difference was found between all-male or mixed gender teams in adherence to guideline-concordant care. No patient was cared for by an all-female team. Neither gender of the admitting physician nor gender of the physician who led the family meeting to discuss prognosis was associated with a survival difference. Conclusions: Prior literature has described differences in outcome based on gender of the treating physician. Our analysis targeted a similar question in a cohort of OHCA patients with survival to hospital admission. We determined that there was no difference in postcardiac arrest guideline concordance and survival to hospital discharge based on treating physician gender. This finding differs from the prior literature and supports the importance of diverse clinical teams in medicine.

A platform for oncogenomic reporting and interpretation.

Manual interpretation of variants remains rate limiting in precision oncology. The increasing scale and complexity of molecular data generated from comprehensive sequencing of cancer samples requires advanced interpretative platforms as precision oncology expands beyond individual patients to entire populations. To address this unmet need, we introduce a Platform for Oncogenomic Reporting and Interpretation (PORI), comprising an analytic framework that facilitates the interpretation and reporting of somatic variants in cancer. PORI integrates reporting and graph knowledge base tools combined with support for manual curation at the reporting stage. PORI represents an open-source platform alternative to commercial reporting solutions suitable for comprehensive genomic data sets in precision oncology. We demonstrate the utility of PORI by matching 9,961 pan-cancer genome atlas tumours to the graph knowledge base, calculating therapeutically informative alterations, and making available reports describing select individual samples.

Optimization of magnetic bead-based nucleic acid extraction for SARS-CoV-2 testing using readily available reagents.

The COVID-19 pandemic has highlighted the need for generic reagents and flexible systems in diagnostic testing. Magnetic bead-based nucleic acid extraction protocols using 96-well plates on open liquid handlers are readily amenable to meet this need. Here, one such approach is rigorously optimized to minimize cross-well contamination while maintaining sensitivity.

The use of abaloparatide to treat nonunion following dorsal closing wedge osteotomy for Freiberg's disease: A case report.

A middle-aged woman who was previously a long-distance trail runner presented with chronic right forefoot pain and was diagnosed with Freiberg's disease. She suffered from nonunion after undergoing a dorsal closing wedge osteotomy. The nonunion achieved full osseous union after treatment with abaloparatide. Patient reported outcome measures taken at 19 months postoperatively indicated good to excellent clinical outcomes and satisfaction. The aim of this case is to report on the effectiveness of abaloparatide to treat nonunion and support fracture healing.

Are all pain tolerance tasks the same? Convergent validity of four behavioral pain tolerance tasks, self-reported capability for suicide, and lifetime self-injurious behaviors.

OBJECTIVES: Discrepancies persist regarding the extent to which different pain measures provide similar information and relate to capability for suicide and self-injurious behaviors. This study examined pain threshold, tolerance, and persistence across four modalities (cold, heat, pressure, shock) and assessed associations with self-reported capability for suicide, non-suicidal self-injury (NSSI), and suicide attempts. METHODS: A sample of 211 students who reported lifetime suicidal ideation completed four behavioral pain tasks and self-reported on capability for suicide, NSSI, and self-injurious behaviors. RESULTS: All pain thresholds, tolerances, and persistences were positively correlated across the four tasks. Pain facets were related to self-reported capability for suicide with small effect sizes but generally did not differ across suicide attempt or NSSI histories. CONCLUSIONS: Pain thresholds, tolerances, and persistences demonstrated convergent validity across the four modalities, suggesting that these tasks provide similar information. Although the relation between pain and self-injurious behaviors remains unclear, these tasks can generally be used interchangeably.

Monodopsis and Vischeria Genomes Shed New Light on the Biology of Eustigmatophyte Algae.

Members of eustigmatophyte algae, especially Nannochloropsis and Microchloropsis, have been tapped for biofuel production owing to their exceptionally high lipid content. Although extensive genomic, transcriptomic, and synthetic biology toolkits have been made available for Nannochloropsis and Microchloropsis, very little is known about other eustigmatophytes. Here we present three near-chromosomal and gapless genome assemblies of Monodopsis strains C73 and C141 (60 Mb) and Vischeria strain C74 (106 Mb), which are the sister groups to Nannochloropsis and Microchloropsis in the order Eustigmatales. These genomes contain unusually high percentages of simple repeats, ranging from 12% to 21% of the total assembly size. Unlike Nannochloropsis and Microchloropsis, long interspersed nuclear element repeats are abundant in Monodopsis and Vischeria and might constitute the centromeric regions. We found that both mevalonate and nonmevalonate pathways for terpenoid biosynthesis are present in Monodopsis and Vischeria, which is different from Nannochloropsis and Microchloropsis that have only the latter. Our analysis further revealed extensive spliced leader trans-splicing in Monodopsis and Vischeria at 36-61% of genes. Altogether, the high-quality genomes of Monodopsis and Vischeria not only serve as the much-needed outgroups to advance Nannochloropsis and Microchloropsis research, but also shed new light on the biology and evolution of eustigmatophyte algae.