Analyzing Chromosomes, Ion Channels and Novel Nucleic Acid Structures by AFM

The atomic force microscope (AFM) is proving to be a powerful tool for analysis of biological samples. We provide three examples of the application of AFM to the study of biological questions. First, polytene chromosomes from Drosophila are imaged and manipulated by the AFM. Second, the localization of calcium channels on the release face of a nerve terminal is described. Finally, analyses of a new form of DNA, the G-wire, is presented. These examples illustrate the wide variety of biological questions to which AFM can contribute

Imaging Biological Samples with the Atomic-Force Microscope

The application of atomic force microscopy (AFM) to biological investigation is attractive for a number of reasons. Foremost among these is the ability of the AFM to image samples, even living cells, under near native conditions and at resolution equal to, or exceeding, that possible by the best light microscopes. Moreover, the ability of the AFM to manipulate samples it images provides a novel and far reaching application of this technology

Engineered neurogenesis in naïve adult rat cortex by Ngn2-mediated neuronal reprogramming of resident oligodendrocyte progenitor cells

Adult tissue stem cells contribute to tissue homeostasis and repair but the long-lived neurons in the human adult cerebral cortex are not replaced, despite evidence for a limited regenerative response. However, the adult cortex contains a population of proliferating oligodendrocyte progenitor cells (OPCs). We examined the capacity of rat cortical OPCs to be re-specified to a neuronal lineage both in vitro and in vivo. Expressing the developmental transcription factor Neurogenin2 (Ngn2) in OPCs isolated from adult rat cortex resulted in their expression of early neuronal lineage markers and genes while downregulating expression of OPC markers and genes. Ngn2 induced progression through a neuronal lineage to express mature neuronal markers and functional activity as glutamatergic neurons. In vivo retroviral gene delivery of Ngn2 to naive adult rat cortex ensured restricted targeting to proliferating OPCs. Ngn2 expression in OPCs resulted in their lineage re-specification and transition through an immature neuronal morphology into mature pyramidal cortical neurons with spiny dendrites, axons, synaptic contacts, and subtype specification matching local cytoarchitecture. Lineage re-specification of rat cortical OPCs occurred without prior injury, demonstrating these glial progenitor cells need not be put into a reactive state to achieve lineage reprogramming. These results show it may be feasible to precisely engineer additional neurons directly in adult cerebral cortex for experimental study or potentially for therapeutic use to modify dysfunctional or damaged circuitry

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Localization of individual calcium channels at the release face of a presynaptic nerve terminal

Studies using biophysical techniques suggest a highly structured organization of calcium channels at the presynaptic transmitter release face (Llinás et al., 1981; Stanley, 1993), but it has not as yet proved possible to localize identified channels at the required nanometer level of resolution. We have used atomic force microscopy on the calyx-type nerve terminal of the chick ciliary ganglion to localize single calcium channels tagged via biotinylated ω-conotoxin GVIA to avidin-coated 30 nm gold particles. Calcium channels were in low (modal value approximately ⩽ 1 per μm2) and high (modal value55 per μm2) density areas and exhibited a prominent interchannel spacing of 40 nm, indicating an intermolecular linkage. Particles were observed in clusters and short linear or parallel linear arrays, groupings that may reflect calcium channel organization at the transmitter release site.This article is from Neuron 13 (1994): 1275, doi: 10.1016/0896-6273(94)90414-6.</p

Analyzing Chromosomes, Ion Channels and Novel Nucleic Acid Structures by AFM

The atomic force microscope (AFM) is proving to be a powerful tool for analysis of biological samples. We provide three examples of the application of AFM to the study of biological questions. First, polytene chromosomes from Drosophila are imaged and manipulated by the AFM. Second, the localization of calcium channels on the release face of a nerve terminal is described. Finally, analyses of a new form of DNA, the G-wire, is presented. These examples illustrate the wide variety of biological questions to which AFM can contribute.This is a proceeding from NATO Advanced Research Workshop: "Scanning Probe Microscopies and Molecular Materials" (1994): 1. </p

Imaging Biological Samples with the Atomic-Force Microscope

The application of atomic force microscopy (AFM) to biological investigation is attractive for a number of reasons. Foremost among these is the ability of the AFM to image samples, even living cells, under near native conditions and at resolution equal to, or exceeding, that possible by the best light microscopes. Moreover, the ability of the AFM to manipulate samples it images provides a novel and far reaching application of this technology.This is a proceeding from 51st Annual Meeting of the Microscopy Society of America (1993): 512.</p

Enhancement of presynaptic calcium current by cysteine string protein

The isolated chick ciliary neuron calyx synapse preparation was used to test cysteine string protein (CSP) action on presynaptic N-type Ca2+ channels. Endogenous CSP was localized primarily to secretory vesicle clusters in the presynaptic nerve terminal. Introduction of recombinant CSP into the voltage clamped terminal resulted in a prominent increase in Ca2+ current amplitude. However, this increase could not be attributed to a change in Ca2+ channel kinetics, voltage dependence, prepulse inactivation, or G protein inhibition but was attributed to the recruitment of dormant channels. Secretory vesicle associated endogenous CSP may play an important role in enhancing Ca2+ channel activity at the transmitter release site