Human retinal ganglion cell neurons generated by synchronous BMP inhibition and transcription factor mediated reprogramming.

In optic neuropathies, including glaucoma, retinal ganglion cells (RGCs) die. Cell transplantation and endogenous regeneration offer strategies for retinal repair, however, developmental programs required for this to succeed are incompletely understood. To address this, we explored cellular reprogramming with transcription factor (TF) regulators of RGC development which were integrated into human pluripotent stem cells (PSCs) as inducible gene cassettes. When the pioneer factor NEUROG2 was combined with RGC-expressed TFs (ATOH7, ISL1, and POU4F2) some conversion was observed and when pre-patterned by BMP inhibition, RGC-like induced neurons (RGC-iNs) were generated with high efficiency in just under a week. These exhibited transcriptional profiles that were reminiscent of RGCs and exhibited electrophysiological properties, including AMPA-mediated synaptic transmission. Additionally, we demonstrated that small molecule inhibitors of DLK/LZK and GCK-IV can block neuronal death in two pharmacological axon injury models. Combining developmental patterning with RGC-specific TFs thus provided valuable insight into strategies for cell replacement and neuroprotection.

Frizzled2 receives WntA signaling during butterfly wing pattern formation.

Butterfly color patterns provide visible and biodiverse phenotypic readouts of the patterning processes. Although the secreted ligand WntA has been shown to instruct the color pattern formation in butterflies, its mode of reception remains elusive. Butterfly genomes encode four homologs of the Frizzled-family of Wnt receptors. Here, we show that CRISPR mosaic knockouts of frizzled2 (fz2) phenocopy the color pattern effects of WntA loss of function in multiple nymphalids. Whereas WntA mosaic clones result in intermediate patterns of reduced size, fz2 clones are cell-autonomous, consistent with a morphogen function. Shifts in expression of WntA and fz2 in WntA crispant pupae show that they are under positive and negative feedback, respectively. Fz1 is required for Wnt-independent planar cell polarity in the wing epithelium. Fz3 and Fz4 show phenotypes consistent with Wnt competitive-antagonist functions in vein formation (Fz3 and Fz4), wing margin specification (Fz3), and color patterning in the Discalis and Marginal Band Systems (Fz4). Overall, these data show that the WntA/Frizzled2 morphogen-receptor pair forms a signaling axis that instructs butterfly color patterning and shed light on the functional diversity of insect Frizzled receptors.

CRISPR mediated transactivation in the human disease vector Aedes aegypti.

As a major insect vector of multiple arboviruses, Aedes aegypti poses a significant global health and economic burden. A number of genetic engineering tools have been exploited to understand its biology with the goal of reducing its impact. For example, current tools have focused on knocking-down RNA transcripts, inducing loss-of-function mutations, or expressing exogenous DNA. However, methods for transactivating endogenous genes have not been developed. To fill this void, here we developed a CRISPR activation (CRISPRa) system in Ae. aegypti to transactivate target gene expression. Gene expression is activated through pairing a catalytically-inactive ('dead') Cas9 (dCas9) with a highly-active tripartite activator, VP64-p65-Rta (VPR) and synthetic guide RNA (sgRNA) complementary to a user defined target-gene promoter region. As a proof of concept, we demonstrate that engineered Ae. aegypti mosquitoes harboring a binary CRISPRa system can be used to effectively overexpress two developmental genes, even-skipped (eve) and hedgehog (hh), resulting in observable morphological phenotypes. We also used this system to overexpress the positive transcriptional regulator of the Toll immune pathway known as AaRel1, which resulted in a significant suppression of dengue virus serotype 2 (DENV2) titers in the mosquito. This system provides a versatile tool for research pathways not previously possible in Ae. aegypti, such as programmed overexpression of endogenous genes, and may aid in gene characterization studies and the development of innovative vector control tools.

Interinstitutional simulation of patients with COVID-19 during a remote acute-care advanced pharmacy practice experience.

Introduction: Faculty and staff from Duquesne University and the University of Pittsburgh Schools of Pharmacy created a simulation activity focused on the care of critically ill patients with coronavirus disease 2019 (COVID-19). Students on remote, short-term-care advanced pharmacy practice experiences (APPE) rotations from both universities worked in comingled teams and completed two educational electronic health record reviews, complex simulation sessions, and debriefs. Individually, students completed two educational electronic health record reviews and verbal patient presentations before and after the simulation sessions. Objectives: Evaluate the effects of a simulation activity during a remote short-term-care APPE on student confidence and knowledge surrounding the care of a critically ill patient with COVID-19. Methods: Student knowledge surrounding COVID-19 short-term-care treatment principles was assessed through pre-/postcase-based multiple-choice examinations and an intermittent clinical examination (ICE). Student confidence and perceptions were gathered through anonymous pre-/postsurveys. The written examination and patient presentation recordings were compared from baseline to the final assessment using the Wilcoxon signed-rank test. Results: In total, 92 students participated in the activity. There was a statistically significant improvement from baseline to the final assessment (preassessment median [interquartile range (IQR)]: 55.3% [50%-60.5%]; postassessment median [IQR]: 68.4 [60.5%-73.7%]; P < .001) on the written examination. ICE total scores improved from baseline (preassessment median [range]: 33 [28-36] vs postassessment median [range]: 36.5 [29.5-43.52]; P = .004) as well as the objective (P < .001), plan (P < .001), and monitoring (P < .001) subdomain scores. Student confidence reported on surveys improved from baseline in all domains. Conclusion: Remote simulation sessions improve student knowledge and confidence and provide an opportunity for students to experience caring for patients with COVID-19 in a safe environment. Collaboration between schools of pharmacy can be successfully employed to leverage resources and expertise to expand opportunities for students.

Cortex cis-regulatory switches establish scale colour identity and pattern diversity in Heliconius.

In Heliconius butterflies, wing colour pattern diversity and scale types are controlled by a few genes of large effect that regulate colour pattern switches between morphs and species across a large mimetic radiation. One of these genes, cortex, has been repeatedly associated with colour pattern evolution in butterflies. Here we carried out CRISPR knockouts in multiple Heliconius species and show that cortex is a major determinant of scale cell identity. Chromatin accessibility profiling and introgression scans identified cis-regulatory regions associated with discrete phenotypic switches. CRISPR perturbation of these regions in black hindwing genotypes recreated a yellow bar, revealing their spatially limited activity. In the H. melpomene/timareta lineage, the candidate CRE from yellow-barred phenotype morphs is interrupted by a transposable element, suggesting that cis-regulatory structural variation underlies these mimetic adaptations. Our work shows that cortex functionally controls scale colour fate and that its cis-regulatory regions control a phenotypic switch in a modular and pattern-specific fashion.

Heliconius butterflies have bright patterns on their wings that tell potential predators that they are toxic. As a result, predators learn to avoid eating them. Over time, unrelated species of butterflies have evolved similar patterns to avoid predation through a process known as Müllerian mimicry. Worldwide, there are over 180,000 species of butterflies and moths, most of which have different wing patterns. How do genes create this pattern diversity? And do butterflies use similar genes to create similar wing patterns? One of the genes involved in creating wing patterns is called cortex. This gene has a large region of DNA around it that does not code for proteins, but instead, controls whether cortex is on or off in different parts of the wing. Changes in this non-coding region can act like switches, turning regions of the wing into different colours and creating complex patterns, but it is unclear how these switches have evolved. Butterfly wings get their colour from tiny structures called scales, which each have their own unique set of pigments. In Heliconius butterflies, there are three types of scales: yellow/white scales, black scales, and red/orange/brown scales. Livraghi et al. used a DNA editing technique called CRISPR to find out whether the cortex gene affects scale type. First, Livraghi et al. confirmed that deleting cortex turned black and red scales yellow. Next, they used the same technique to manipulate the non-coding DNA around the cortex gene to see the effect on the wing pattern. This manipulation turned a black-winged butterfly into a butterfly with a yellow wing band, a pattern that occurs naturally in Heliconius butterflies. The next step was to find the mutation responsible for the appearance of yellow wing bands in nature. It turns out that a bit of extra genetic code, derived from so-called 'jumping genes', had inserted itself into the non-coding DNA around the cortex gene, 'flipping' the switch and leading to the appearance of the yellow scales. Genetic information contains the instructions to generate shape and form in most organisms. These instructions evolve over millions of years, creating everything from bacteria to blue whales. Butterfly wings are visual evidence of evolution, but the way their genes create new patterns isn't specific to butterflies. Understanding wing patterns can help researchers to learn how genetic switches control diversity across other species too.

Engineered reproductively isolated species drive reversible population replacement.

Engineered reproductive species barriers are useful for impeding gene flow and driving desirable genes into wild populations in a reversible threshold-dependent manner. However, methods to generate synthetic barriers are lacking in advanced eukaryotes. Here, to overcome this challenge, we engineer SPECIES (Synthetic Postzygotic barriers Exploiting CRISPR-based Incompatibilities for Engineering Species), an engineered genetic incompatibility approach, to generate postzygotic reproductive barriers. Using this approach, we create multiple reproductively isolated SPECIES and demonstrate their reproductive isolation and threshold-dependent gene drive capabilities in D. melanogaster. Given the near-universal functionality of CRISPR tools, this approach should be portable to many species, including insect disease vectors in which confinable gene drives could be of great practical utility.

Improving the safety of shoulder arthroscopy in the beach chair position: a prospective randomized trial investigating the effect of compression stockings on cerebral desaturation events in obese patients.

BACKGROUND: Devastating transient and permanent postoperative neurocognitive complications in previously healthy, low-risk patients have been observed after elective shoulder arthroscopy in the beach chair position (BCP). Continuous monitoring of cerebral oxygen saturation has been recommended to identify cerebral desaturation events (CDEs) and improve patient safety. However, the relatively high cost and limited availability of monitoring may not be cost-effective. More cost-effective and available measures, including the use of thigh-high compression stockings (CS), have been investigated. However, efficacy data of CS usage is limited, especially for obese patients, who have been shown to be at increased risk for CDEs. The purpose of this was study was to determine if the intraoperative addition of thigh-high compression stockings decreases the incidence, frequency, and magnitude of CDEs in obese patients undergoing shoulder arthroscopy in the BCP. METHODS: Thirty-three patients in the treatment group wore both thigh-high compression stockings (CS) and sequential compression devices (SCDs), and the remaining 33 patients in the control group wore SCDs alone. Cerebral oximetry was monitored during surgery using near-infrared spectroscopy. RESULTS: The incidence of CDEs was equal between groups, with 9 patients (27%) in each experiencing desaturation events. The median number of CDEs per patient was 3 for the control group and 1 for patients wearing CS (P = .29). There was no difference between groups in terms of median time from induction of anesthesia to onset of CDE (P = .79), median time from upright positioning to onset of CDE (P = .60), mean CDE duration per patient (P = .22), and median cumulative CDE duration (P = .19). The median maximal desaturation from baseline was also not different between groups: 27.6% in the control group and 24.3% in the treatment group (P = .35). CONCLUSION: The combination of thigh-high CS and SCDs did not decrease the incidence, frequency, or magnitude of CDEs in patients undergoing shoulder arthroscopy in the BCP. Twenty-seven percent of patients undergoing shoulder arthroscopy in the BCP demonstrated CDEs with or without the use of CS. Therefore, further research is required to identify cost-effective, minimally invasive, and universally available methods of decreasing the incidence of CDEs during this common surgical procedure.

A transposable element insertion is associated with an alternative life history strategy.

Tradeoffs affect resource allocation during development and result in fitness consequences that drive the evolution of life history strategies. Yet despite their importance, we know little about the mechanisms underlying life history tradeoffs. Many species of Colias butterflies exhibit an alternative life history strategy (ALHS) where females divert resources from wing pigment synthesis to reproductive and somatic development. Due to this reallocation, a wing color polymorphism is associated with the ALHS: either yellow/orange or white. Here we map the locus associated with this ALHS in Colias crocea to a transposable element insertion located downstream of the Colias homolog of BarH-1, a homeobox transcription factor. Using CRISPR/Cas9 gene editing, antibody staining, and electron microscopy we find white-specific expression of BarH-1 suppresses the formation of pigment granules in wing scales and gives rise to white wing color. Lipid and transcriptome analyses reveal physiological differences associated with the ALHS. Together, these findings characterize a mechanism for a female-limited ALHS.

A matter of timing.

A genetic pathway that times development works together with the sex-determination pathway to control the timing of sexually dimorphic neural development in C. elegans.

Genetic Basis of Melanin Pigmentation in Butterfly Wings.

Despite the variety, prominence, and adaptive significance of butterfly wing patterns, surprisingly little is known about the genetic basis of wing color diversity. Even though there is intense interest in wing pattern evolution and development, the technical challenge of genetically manipulating butterflies has slowed efforts to functionally characterize color pattern development genes. To identify candidate wing pigmentation genes, we used RNA sequencing to characterize transcription across multiple stages of butterfly wing development, and between different color pattern elements, in the painted lady butterfly Vanessa cardui This allowed us to pinpoint genes specifically associated with red and black pigment patterns. To test the functions of a subset of genes associated with presumptive melanin pigmentation, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing in four different butterfly genera. pale, Ddc, and yellow knockouts displayed reduction of melanin pigmentation, consistent with previous findings in other insects. Interestingly, however, yellow-d, ebony, and black knockouts revealed that these genes have localized effects on tuning the color of red, brown, and ochre pattern elements. These results point to previously undescribed mechanisms for modulating the color of specific wing pattern elements in butterflies, and provide an expanded portrait of the insect melanin pathway.

Love spots.

A Quick guide to Love Spots: striking male-specific regions of the eye found in some insects that are used for detecting and chasing females.

Enhancer additivity and non-additivity are determined by enhancer strength in the Drosophila embryo.

Metazoan genes are embedded in a rich milieu of regulatory information that often includes multiple enhancers possessing overlapping activities. In this study, we employ quantitative live imaging methods to assess the function of pairs of primary and shadow enhancers in the regulation of key patterning genes-knirps, hunchback, and snail-in developing Drosophila embryos. The knirps enhancers exhibit additive, sometimes even super-additive activities, consistent with classical gene fusion studies. In contrast, the hunchback enhancers function sub-additively in anterior regions containing saturating levels of the Bicoid activator, but function additively in regions where there are diminishing levels of the Bicoid gradient. Strikingly sub-additive behavior is also observed for snail, whereby removal of the proximal enhancer causes a significant increase in gene expression. Quantitative modeling of enhancer-promoter interactions suggests that weakly active enhancers function additively while strong enhancers behave sub-additively due to competition with the target promoter.

Outpatient minimally invasive spine surgery using endoscopy for the treatment of lumbar spinal stenosis among obese patients.

INTRODUCTION: The safety and effectiveness of outpatient endoscopic minimally invasive spinal surgery (MISS) for the treatment of lumbar spinal stenosis among obese patients is not well documented. METHODS: Pain, disability, and surgical variables were examined on 41 adult obese patients that underwent lumbar laminotomy and foraminotomy surgeries. RESULTS: A significant decrease in pain and disability scores between preoperative and postoperative were found (p-value's < 0.001). DISCUSSION: MISS using endoscopy can achieve sufficient decompression that results in a significant reduction in the level of pain and disability among obese patients with minimal blood loss and no complications.

The evolutionary diversity of insect retinal mosaics: common design principles and emerging molecular logic.

Independent evolution has resulted in a vast diversity of eyes. Despite the lack of a common Bauplan or ancestral structure, similar developmental strategies are used. For instance, different classes of photoreceptor cells (PRs) are distributed stochastically and/or localized in different regions of the retina. Here, we focus on recent progress made towards understanding the molecular principles behind patterning retinal mosaics of insects, one of the most diverse groups of animals adapted to life on land, in the air, under water, or on the water surface. Morphological, physiological, and behavioral studies from many species provide detailed descriptions of the vast variation in retinal design and function. By integrating this knowledge with recent progress in the characterization of insect Rhodopsins as well as insight from the model organism Drosophila melanogaster, we seek to identify the molecular logic behind the adaptation of retinal mosaics to the habitat and way of life of an animal.

The Emerging Microbe Project: Developing Clinical Care Plans Based on Pathogen Identification and Clinical Case Studies.

For many students in the health sciences, including doctor of pharmacy (PharmD) students, basic and clinical sciences often appear detached from each other. In the infectious disease field, PharmD students additionally struggle with mastering the diversity of microorganisms and the corresponding therapies. The objective of this study was to design an interdisciplinary project that integrates fundamental microbiology with clinical research and decision-making skills. The Emerging Microbe Project guided students through the identification of a microorganism via genetic sequence analysis. The unknown microbe provided the basis for a patient case that asked the student to design a therapeutic treatment strategy for an infected patient. Outside of lecture, students had two weeks to identify the pathogen using nucleotide sequences, compose a microbiology report on the pathogen, and recommend an appropriate therapeutic treatment plan for the corresponding clinical case. We hypothesized that the students would develop a better understanding of the interplay between basic microbiology and infectious disease clinical practice, and that they would gain confidence and skill in independently selecting appropriate antimicrobial therapies for a new disease state. The exercise was conducted with PharmD students in their second professional year of pharmacy school in a required infectious disease course. Here, we demonstrate that the Emerging Microbe Project significantly improved student learning through two assessment strategies (assignment grades and exam questions), and increased student confidence in clinical infectious disease practice. This exercise could be modified for other health sciences students or undergraduates depending upon the level of clinical focus required of the course.

Minimally invasive surgery through endoscopic laminotomy and foraminotomy for the treatment of lumbar spinal stenosis.

BACKGROUND: Lumbar spinal stenosis is a common cause of radicular and generalized back pain among older adults. Endoscopic minimally invasive surgery, in contrast to open decompression, may provide the opportunity for a less invasive surgical intervention. Thus, the purpose of this study is to evaluate the safety (operative complications, estimated blood loss, operative room time) and effectiveness (pre- versus postoperative level of disability and pain severity) of minimally invasive surgery using endoscopic laminotomy and foraminotomy among a large sample of patients with lumbar spinal stenosis. METHODS: This study is composed of 320 consecutive patients with lumbar spinal stenosis who underwent posterior lumbar laminotomy and foraminotomy between 2008 and 2011. Outcome measures consisted of perioperative complications, estimated blood loss, operative room time, level of disability, and pain severity. Pain severity and level of disability were prospectively analyzed to an average of 18 months (12-36 months) post-surgery. RESULTS: There was an average estimated blood loss of 39.3 cc and a mean operative room time of 74 min. Seven patients experienced minor operative complications. All patients were discharged the same day as surgery and reported a significantly lower level of disability (p = 0.00) and pain severity (p = 0.00) postoperative compared to preoperative. CONCLUSIONS: Minimally invasive surgery using endoscopy for the treatment of lumbar spinal stenosis has a short operative time, a low operative complication rate, and minimal estimated blood loss. This study also indicates that MIS for the treatment of LSS can significantly reduce pain and disability level. Thus, minimally invasive surgery using endoscopic laminotomy and foraminotomy appears to be a safe and effective alternative surgical treatment for open decompression surgery in adult patients with lumbar spinal stenosis.

Precision of hunchback expression in the Drosophila embryo.

Activation of the gap gene hunchback (hb) by the maternal Bicoid gradient is one of the most intensively studied gene regulatory interactions in animal development. Most efforts to understand this process have focused on the classical Bicoid target enhancer located immediately upstream of the P2 promoter. However, hb is also regulated by a recently identified distal shadow enhancer as well as a neglected "stripe" enhancer, which mediates expression in both central and posterior regions of cellularizing embryos. Here, we employ BAC transgenesis and quantitative imaging methods to investigate the individual contributions of these different enhancers to the dynamic hb expression pattern. These studies reveal that the stripe enhancer is crucial for establishing the definitive border of the anterior Hb expression pattern, just beyond the initial border delineated by Bicoid. Removal of this enhancer impairs dynamic expansion of hb expression and results in variable cuticular defects in the mesothorax (T2) due to abnormal patterns of segmentation gene expression. The stripe enhancer is subject to extensive regulation by gap repressors, including Kruppel, Knirps, and Hb itself. We propose that this repression helps ensure precision of the anterior Hb border in response to variations in the Bicoid gradient.

Neural-specific elongation of 3' UTRs during Drosophila development.

The 3' termini of eukaryotic mRNAs influence transcript stability, translation efficiency, and subcellular localization. Here we report that a subset of developmental regulatory genes, enriched in critical RNA-processing factors, exhibits synchronous lengthening of their 3' UTRs during embryogenesis. The resulting UTRs are up to 20-fold longer than those found on typical Drosophila mRNAs. The large mRNAs emerge shortly after the onset of zygotic transcription, with several of these genes acquiring additional, phased UTR extensions later in embryogenesis. We show that these extended 3' UTR sequences are selectively expressed in neural tissues and contain putative recognition motifs for the translational repressor, Pumilio, which also exhibits the 3' lengthening phenomenon documented in this study. These findings suggest a previously unknown mode of posttranscriptional regulation that may contribute to the complexity of neurogenesis or neural function.

Multiple enhancers ensure precision of gap gene-expression patterns in the Drosophila embryo.

Segmentation of the Drosophila embryo begins with the establishment of spatially restricted gap gene-expression patterns in response to broad gradients of maternal transcription factors, such as Bicoid. Numerous studies have documented the fidelity of these expression patterns, even when embryos are subjected to genetic or environmental stress, but the underlying mechanisms for this transcriptional precision are uncertain. Here we present evidence that every gap gene contains multiple enhancers with overlapping activities to produce authentic patterns of gene expression. For example, a recently identified hunchback (hb) enhancer (located 5-kb upstream of the classic enhancer) ensures repression at the anterior pole. The combination of intronic and 5' knirps (kni) enhancers produces a faithful expression pattern, even though the intronic enhancer alone directs an abnormally broad expression pattern. We present different models for "enhancer synergy," whereby two enhancers with overlapping activities produce authentic patterns of gene expression.